Can Interspecies Hybrid Zygosaccharomyces rouxii Produce anAllohaploid Gamete?

Jun Watanabe,a Kenji Uehara,b Yuichiro Tsukiokaa

aManufacturing Division, Yamasa Corporation, Choshi, Chiba, JapanbAkita Research Institute of Food and Brewing (ARIF), Arayamachi, Akita, Japan

ABSTRACT In soy sauce manufacturing, Candida versatilis plays a role in the pro-duction of volatile flavor compounds, such as volatile phenols, but limited accessibleinformation on its genome has prevented further investigation regarding aroma pro-duction and breeding. Although the draft genome sequence data of two strains ofC. versatilis have recently been reported, these strains are not similar to each other.Here, we reassess the draft genome sequence data for strain t-1, which was origi-nally reported to be C. versatilis, and conclude that strain t-1 is most probably not C.versatilis but a gamete of hybrid Zygosaccharomyces rouxii. Phylogenetic analysis ofthe D1/D2 region of the 26S ribosomal DNA (rDNA) sequence indicated that straint-1 is more similar to the genus Zygosaccharomyces than to C. versatilis. Moreover,we found that the genome of strain t-1 is composed of haploid genome contentand divided into two regions that show approximately 100% identity with the T or Psubgenome derived from the natural hybrid Zygosaccharomyces rouxii, such asNBRC110957 and NBRC1876. We also found a chromosome crossing-over signaturein the scaffolds of strain t-1. These results suggest that strain t-1 is a gamete of thehybrid Z. rouxii, generated by either meiosis or chromosome loss following recipro-cal translocation between the T and P subgenomes. Although it is unclear whystrain t-1 was misidentified as C. versatilis, the genome of strain t-1 has broad impli-cations for considering the evolutionary fate of an allodiploid.

IMPORTANCE In yeast, crossing between different species sometimes leads to inter-species hybrids. The hybrid generally cannot produce viable spores because dissimi-larity of parental genomes prevents normal chromosome segregation during meioticdivision, leading to a dead end. Thus, only a few natural cases of homoploid hybridspeciation, which requires mating between 1n gametes of hybrids, have been de-scribed. However, a recent study provided strong evidence that homoploid hybridspeciation is initiated in natural populations of the budding yeast, suggesting thepotential presence of viable 1n gametes of hybrids. The significance of our study isfinding that the strain t-1, which had been misidentified as Candida versatilis, is a vi-able 1n gamete derived from hybrid Zygosaccharomyces rouxii.

KEYWORDS genome sequence, Candida versatilis, hybrid, genus Zygosaccharomyces,speciation

Interspecies hybridization is often observed in yeast. The increased genome size andcomplexity due to the hybridization can confer a selective advantage called “hetero-

sis,” but the hybrids are generally sterile (unable to produce viable spores) or infertile(unable to sporulate). The hybrid sterility (or infertility) can be explained by incompat-ibility between genes from different species (1), which is known as Dobzhansky-Muller(DM) incompatibility (2, 3), or dissimilarity between chromosomes from different spe-cies, which prevents precise chromosome pairing essential for meiosis (4–7). Althoughviable allohaploids (haploid gametes of hybrids generated by mating between two

Received 22 August 2017 Accepted 17October 2017

Accepted manuscript posted online 27October 2017

Citation Watanabe J, Uehara K, Tsukioka Y.2018. Can interspecies hybridZygosaccharomyces rouxii produce anallohaploid gamete? Appl Environ Microbiol84:e01845-17. https://doi.org/10.1128/AEM.01845-17.

Editor Marie A. Elliot, McMaster University

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Jun Watanabe,j_watanabe@yamasa.com.

EVOLUTIONARY AND GENOMIC MICROBIOLOGY

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different species) can be generated experimentally (8), it is less understood whether theviable haploid gametes derived from hybrids exist in the natural environment. Toaddress this question, accumulation of genome data in various yeasts is essentialbecause it is difficult to discriminate between parental haploids and allohaploidsderived from hybrids, which are generated by the fusion of two close relatives. Recentadvances in next-generation sequencing technology and its outcomes offer an oppor-tunity to explore allohaploids from the international nucleotide sequence database.

Candida versatilis (also previously called Torulopsis versatilis) is a yeast strain with arecently uncovered genome. C. versatilis is a highly halotolerant yeast used in theproduction of soy sauce and soybean paste, similar to Zygosaccharomyces rouxii. In soysauce manufacturing, C. versatilis produces volatile phenols, such as 4-ethylguaiacol(4-EG) and 4-ethylphenol (4-EP), that confer the characteristic aroma in soy sauce (9).Although excess quantities of volatile phenols in soy sauce normally impart an un-pleasant aroma, the presence of 1 to 2 mg/liter of 4-EG gives a better flavor quality tothe soy sauce as deemed by subjective evaluations (9).

Recently, the draft genome sequence data of C. versatilis became available; thegenome data for strain t-1 (originally reported to be C. versatilis) and strain JCM5958were submitted by Hou et al. (10) and by the RIKEN Center for Life Science Technologiesof Japan, respectively. Hou et al. did not clearly describe the strain name and accessionnumber in their paper (10), but they certainly described strain t-1 because the NCBInucleotide database entries for strain t-1 (accession numbers KV452433 to KV452453)directly cite the paper by Hou et al. as their source.

More recently, the genome sequence analyses of hybrid Z. rouxii NBRC110957 andNBRC1876 have been reported (11, 12). These hybrids contain two subgenomes; one isderived from one parent similar to haploid Z. rouxii CBS732T (referred to as the Tsubgenome) and another may be derived from one parent similar to NCYC3042,informally called Z. pseudorouxii (referred to as the P subgenome). However, genomesequence analysis of NCYC3042 has not yet been performed, so it remains to beexamined in detail whether one parent of hybrid Z. rouxii (e.g., NBRC110957 andNBRC1876) is indeed Z. pseudorouxii. Interestingly, we noticed that the genome se-quence data of strain t-1 are not similar to those of strain JCM5958 and seemed to besimilar to those of hybrid Z. rouxii. Unlike hybrid Z. rouxii, strain t-1 has haploid genomecontent; alternatively, it has a mosaic scaffold sequence similar to both the T and Psubgenomes of hybrid Z. rouxii.

In this study, we conducted a comparative analysis between C. versatilis and Z. rouxiito clarify the taxonomic status and the origin of strain t-1 and revealed that strain t-1is most probably not C. versatilis but a gamete of hybrid Z. rouxii. Our study providesthe evidence implicating isolation of an allohaploid derived from hybrid Z. rouxii in anatural environment.

RESULTS AND DISCUSSIONDissimilarity between strains t-1 and JCM5958. We first focused on the diver-

gence of G�C content between the genomes of strains t-1 and JCM5958 in the NCBIgenome database (https://www.ncbi.nlm.nih.gov/genome/genomes/44240?). The G�Ccontent of the strain t-1 genome is 40.1% (39.74% in reference 10) and that of theJCM5958 genome is 44.8%, suggesting that these strains are phylogenetically diver-gent. To confirm the presence of the genes that have been previously cloned from C.versatilis (13–15), we conducted a BLASTN search using these genes as a query againstthe strain t-1 and JCM5958 genomes (Table 1). The genes encoding Cagpd1 (13),Cagpd2 (14), CvGPD1 (15), PLB1, and PLB2 were detected with high sequence identityin the JCM5958 genome, while they were not detected in the strain t-1 genome (Table1). We found the D1/D2 region of the 26S ribosomal DNA (rDNA) sequence that can beamplified by the primer sets NL1 and NL4 (16) in the scaffold00031 (bp 10,704 to11,327) of the strain t-1 genome. The analysis of the sequence in this region revealedthat the D1/D2 region of the 26S rDNA sequence in strain t-1 shares 100% identity withthe corresponding region of Z. rouxii CBS732T with 100% query cover (see Fig. S1 in the

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supplemental material). Alternatively, the same region of JCM5958 shares 99% identitywith the corresponding region of C. versatilis CBS1752T with 100% query cover (Fig. S1).Phylogenetic analysis of the D1/D2 region of the 26S rDNA sequence indicated thatstrain t-1 is more similar to the genus Zygosaccharomyces than to C. versatilis (Fig. 1).Ribosomal DNA arrays in the genome of the Z. rouxii complex species consist of Z.rouxii-, Z. sapae-, and Z. mellis-like sequences (17). This indicates the possibility thatstrain t-1 may also contain a Z. rouxii type D1/D2 sequence and other Zygosaccharo-myces species type internal transcribed spacers (ITS) sequences, so we attempted thephylogenetic analysis of the D1/D2 region and compared it to the ITS sequence in orderto confirm whether strain t-1 has a mosaic rDNA array such as those seen in Z. rouxiicomplex species. However, we cannot fill several sequence gaps in the scaffold00031 ofstrain t-1 because of the difficulty in obtaining strain t-1, which is not deposited in thepublic culture collection, preventing the phylogenetic analysis of the ITS sequence. In

TABLE 1 Genes previously cloned from C. versatilis

Gene encoding: Function Accession no. Strain name

BLASTn resulta

ReferenceJCM5958 t-1

Cagpd1 Glycerol 3-phosphate dehydrogenase LC015796 SN-18 1,114/1,137 (98) NDb 13Cagpd2 Glycerol 3-phosphate dehydrogenase LC015797 SN-18 1,118/1,137 (98) ND 13CvNHA1 Na(�)/H(�) antiporter AB255166 NBRC 10650 ND ND 14CvGPD1 Glycerol 3-phosphate dehydrogenase AB296385 NBRC 10650 1,323/1,337 (99) ND 15PLB2 Phospholipase AB479988 Unknown 1,879/1,943 (97) NDPLB1 Phospholipase AB479989 Unknown 1,996/2,021 (99) NDaResults show the no. of bases in the hit sequence/no. of bases in the query sequence (% identity).bND, not detected.

FIG 1 Phylogenetic tree showing the relationship among the D1/D2 region of the 26S rDNA sequence. Thedendrogram was constructed using the neighbor-joining method. Bootstrap values were calculated from1,000 replications and expressed as percentages. The scale bar represents 0.02 substitutions per nucleotideposition. Multiple-sequence alignment was used to construct the phylogenetic tree shown in Fig. S1.Sequence data were downloaded from the DNA data bank of Japan and National Institute of Technology andEvaluation Biological Resource Center (http://www.nbrc.nite.go.jp/NBRC2/NBRCDispSearchServlet?lang�en).Zr, Zygosaccharomyces rouxii; HyZr, Hybrid Zygosaccharomyces rouxii; Zm, Zygosaccharomyces mellis; Zb,Zygosaccharomyces bailli; Cv, Candida versatilis; Ce, Candida etchellsii.

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any case, our partial phylogenetic analysis based on the D1/D2 region of the 26S rDNAsequence suggests that strain t-1 might be misidentified as C. versatilis.

To test this possibility, we examined the genome-wide synteny and identity be-tween C. versatilis and Z. rouxii by dot plot analysis using YASS. YASS is a genomicalignment search tool that uses a new spaced-seed model called transition-constrainedseeds that takes advantage of statistical properties of real genomic sequences toachieve high sensitivity on the nucleic sequences being compared (18). The genomecomparison of strain t-1 and Z. rouxii CBS732T shows that they share significant syntenyand identity (Fig. 2), but the genomes of strains t-1 and JCM5958 or of JCM5958 and Z.rouxii CBS732T showed poor synteny and very low identity (see Fig. S2 in the supple-mental material). This result suggests that strain t-1 is most probably not C. versatilis buta haploid strain related to Z. rouxii. Note that we assume that there are strong, but notcomplete, collinearities among the genomes of strain t-1, CBS732T, and the T and Psubgenomes of hybrid Z. rouxii. There may be some additional chromosome rearrange-ments that are not visible by assembly of short reads in strains t-1 (10), NBRC110957(11), and NBRC1876 (12). For example, a physical linkage between scaffold00009 andscaffold00008 in strain t-1 has not been confirmed, suggesting that unexpectedchromosomal rearrangement may have occurred.

Strain t-1 is an allohaploid derived from hybrid Z. rouxii. Is strain t-1 just a haploidof Z. rouxii? To confirm the phylogenetic status and the origin of strain t-1, we conductedneighbor-joining (NJ)-based phylogenetic analysis of 14 proteins encoded by orthologgenes that are conserved among strains t-1, JCM5958, NBRC110957, and NBRC1876 andcover seven chromosomes of the haploid Z. rouxii CBS732T genome (Fig. 3; see also Fig. S3in the supplemental material). First, to detect ortholog genes, we performed a TBLASTNsearch against the genomes of strains t-1, JCM5958, NBRC110957, and NBRC1876 using thesequences of housekeeping proteins in Z. rouxii as the query. Next, we compared the geneorder around the ortholog genes in each strain to confirm synteny using Yeast GenomeAnnotation Pipeline (19) and analyzed the results with Yeast Gene Order Browser (20).Except for JCM5958, the gene order around the orthologous target genes was highlyconserved among strains t-1, NBRC1876, NBRC110957, and CBS732T, which ensuredthat orthologous target genes analyzed in this study diverged from a common ances-

FIG 2 Dot plot analysis between the genome of Z. rouxii CBS732T and the reconstructed genome of straint-1. The x axis indicates the aligned scaffold of strain t-1, and the y axis indicates the reference genomeof CBS732T. The number shows only the largest 12 scaffolds of strain t-1. The red number shows thereverse complement of the scaffold sequence. Scaffolds 29, 31, 32, 34, 35, and 36 are aligned but are notvisible in this scale (because they are too small). “Other scaffolds” indicates the remaining scaffolds, 37to 69. Dot plots were made using the program YASS (18), and horizontal and vertical lines were addedaccording to the length of chromosomes of CBS732T and scaffold of NBRC110957, respectively.

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tral sequence by speciation. Consistent with a previous study, both allodiploidNBRC110957 and NBRC1876 have two types of proteins: T-type proteins encoded bya T subgenome originated from a donor similar to the Z. rouxii CBS732T and P-typeproteins encoded by the P subgenome originated from donors similar to strainsrelated to Z. rouxii (Fig. 3 and S3). In contrast, strain t-1 has only one type of protein;Pho88, Met16, Atp12, Leu2, Ura3, Ade2, and Arg2 were clustered in a T-type protein,while Ade1, Trp1, Aur1, Bet3, Aro8, His3, and Ump1 were clustered in a P-typeprotein (Fig. 3 and S3). These results suggest that the haploid genome of strain t-1can be comprised of both T- and P-subgenome-derived sequences.

To test this hypothesis, a comprehensive BLAST search between strain t-1 scaffoldsand the Z. rouxii CBS732T genome was performed. As a result, we were able to dividethe scaffolds of strain t-1 into two regions by degree of identity to the Z. rouxii CBS732T;the scaffolds with approximately 98 to 100% identity were considered to be the regionderived from the T subgenome (T-type sequence), and the scaffolds with approximately80 to 90% identity were considered to be the region derived from the P subgenome(P-type sequence) (see Table S1 in the supplemental material). Conserved synteny andhigh sequence identity permit us to map the scaffolds of strain t-1 to the Z. rouxiiCBS732T genome. The reconstructed genome structure of strain t-1 is shown in Fig. 4.We found that strain t-1 has genomic contents that are comparable in size to thegenome of haploid Z. rouxii CBS732T, suggesting that strain t-1 is apparently allohap-loid.

The characteristic chromosomal rearrangement between chromosomes C and F inscaffold00008 as a tandem repeat (a region corresponding to one end of CB732chromosome F occurs twice in the strain t-1 genome) (Fig. 2 and 4) is shared with straint-1 and NBRC1876 (11), suggesting that strain t-1 and NBRC1876 share a allodiploidancestor. This characteristic structural feature as a tandem repeat is assumed to beformed by reciprocal translocation between the HML locus of the T subgenome and theMAT locus of the P subgenome in an ancestor of NBRC1876 (11). The chromosomalrearrangement between chromosomes A and G in scaffold00001 seems to be strain t-1specific and is not shared among other strains of hybrid Z. rouxii. Taken together, thesedata indicate that the chromosomal rearrangement between C and F may haveoccurred in a common allodiploid ancestor of strains t-1 and NBRC1876, the ancestorof strain t-1 would have diverged from the common allodiploid ancestor, experiencingchromosome rearrangement between chromosomes C and F, and allohaploid “straint-1” would have been generated by meiotic division before or after the chromosomalrearrangement between A and G. Note that it is also possible that the chromosomalrearrangement between C and F occurred independently in both strain t-1 and theNBRC1876 lineage because the chromosomal translocation between mating-type-likegenes is sometimes detected in haploid Z. rouxii (21).

FIG 3 Patterns of 14 proteins detected in Z. rouxii CBS732T, allodiploids NBRC110957 and NBRC1876, and strain t-1. Red andblue boxes indicate the T- and P-type proteins, respectively. The sequence type discrimination (T type or P type) is supportedby the phylogenetic analysis (see also Fig. S3).

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We further compared genetic relatedness among strains t-1, Z. rouxii CBS732T,NBRC110957, and NBRC1876 genomes (Fig. 5). The average nucleotide identity (ANI)among the T-type sequence of strain t-1 (the region of filled boxes in Fig. 4), CBS732T,and the T subgenomes of NBRC110957 and NBRC1876 showed approximately 100%identity, similar to the P-type sequence of strain t-1 (the region of angled-striped boxes)and the P subgenomes of NBRC110957 and NBRC1876 (Fig. 5). These results suggestthat strain t-1 is an allohaploid strain derived from hybrid Z. rouxii.

A model for generation of allohaploid strain t-1 from an allodiploid. How is theallohaploid genome structure of strain t-1 generated from an allodiploid? There are twopossible ways to generate an allohaploid genome from an allodiploid: meiotic divisionand chromosome loss (22). We found three traces of chromosome crossing-overbetween the T and P subgenomes in scaffolds 1, 9, and 10 (Fig. 4, filled circle; see alsoFig. S4 to S6 in the supplemental material). Although the chromosome crossing-overcan be mapped to sequence regions of 83 to 240 bp, which are identical in the T andP subgenomes, we could not discriminate whether the trace of chromosome crossing-over was due to meiotic division or chromosome loss following the reciprocal trans-location of chromosomes.

We illustrate a model for allohaploid generation from an allodiploid (Fig. 6). In thecase of allohaploid generation through meiotic division, two haploid cells of differentspecies mate and form an allodiploid. This allodiploid could start meiotic division inresponse to nutrient starvation; in most cases, however, homologous chromosomes(homologues) cannot become tethered to each other due to dissimilarity between theT and P subgenomes, leading to failure of chiasma formation. Because the linkageprovided by chiasmata ensures proper segregation of homologues through the processby which the kinetochores attach to microtubules in such a way that the homologueswill be pulled to the opposite rather than the same side of the spindle at anaphase I (23,

FIG 4 Map of the scaffold of strain t-1 aligned on the chromosome structure of Z. rouxii CBS732T. The blackhorizontal bars indicate the chromosome of CBS732T with tick marks for every 100 kbp. Boxes filled with colorsindicate the T-type sequence that shares approximately 98 to 100% identity with the sequence of CBS732T. Boxesfilled with angled striped colors indicate the P-type sequence that shares approximately 80 to 90% identity withthe sequence of CBS732T. The white (T-type sequence) and black (P-type sequence) hatched boxes connected withthe colored boxes represent the positions corresponding to the genome of CBS732T. Only the colored boxes existin the strain t-1 genome (these hatched boxes connected with the colored boxes do not represent redundantsequence in the strain t-1 genome). Arrows indicate the direction of the segment. The numbers show scaffoldnumbers, and the red number shows the reverse complement of the scaffold sequence. The closed circles indicatethe positions of DNA crossing-over shown in Fig. S4 to S6.

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24), defects of chiasmata cause the missegregation of homologues, leading to a deadend. Alternatively, the rare success of chiasma formation enables proper chromosomesegregation. After the second round of chromosome segregation, gametes with hap-loid genomic content could be produced; however, the majority of gametes cannotgerminate due to incompatibility between two chromosomes derived from differentspecies (25–28). Thus, it would be very rare for a viable allohaploid to be generated bymeiosis.

Chromosome loss is a well-established phenomenon in yeast hybrids. For example,heat stress on Saccharomyces cerevisiae � Saccharomyces uvarum hybrids favored lossof the S. uvarum genome (29), and another type of stress promoted rearrangementbetween the parental species’ chromosomes (30). Thus, it would be possible that theallodiploid returns to a haploid genome content by chromosome loss following recip-rocal translocation of chromosomes.

Conclusion. Our results showed that strain t-1 is most probably not C. versatilis buta gamete of hybrid Z. rouxii. Although it is unclear why strain t-1 was misidentified asC. versatilis, the genome of strain t-1 has broad implications for considering theevolutionary fate of allodiploids. In general, allodiploids are viable, but the sexualgametes of this allodiploid are not (1). This hybrid sterility is one of the postzygoticreproductive isolation mechanisms to evolve between recently diverged species.One of several possible causes of the nonviability of sexual gametes is incompatibilitybetween genes derived from different species (1), which is known as DM incompati-bility (2, 3). The presence of compatible alleles in hybrids can mask the effect ofincompatibility because hybrids contain two complete haploid genomes derived fromeach parent species; however, haploid gametes of hybrids could be exposed torecessive incompatibility, preventing allodiploids from reproducing sexually (1). Indeed,some of the incompatible DM pairs have previously been identified in diverse organ-isms (8, 26–28, 31). Under experimental conditions, haploid gametes of allodiploids can

FIG 5 The average nucleotide identity (ANI) among strains t-1, Z. rouxii CBS732T, allodiploid NBRC110957,and NBRC1876. The ANI among the (sub-) genomes of strains t-1, CBS732T, NBRC110957, and NBRC1876was calculated using an ANI calculator (43) with a default setting. The upper horizontal line of the boxis the 75th percentile; the lower horizontal line of the box is the 25th percentile; the horizontal bar withinbox is the median value; the upper and lower horizontal thick bars outside the box indicate 1.5 times theinterquartile range from the box.

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be generated (8), but it is less well understood whether viable haploid gametes ofallodiploids exist in the natural environment. A recent study provided strong evidencethat homoploid hybrid speciation is present in the natural population of the buddingyeast Saccharomyces paradoxus (32), suggesting the potential presence of viable 1ngametes of hybrids. In this study, we demonstrated that strain t-1, which was isolatedfrom the natural environment in soy sauce mash at Tianjin in China in 2008 (BioSamplenumber SAMN03466599), is most probably not C. versatilis but a viable haploid gameteof hybrid Z. rouxii. Most likely, the strain t-1 could be generated in soy sauce mashby either meiosis or chromosome loss following reciprocal chromosome transloca-tion. At this moment, this is the leading hypothesis, which requires further evidenceto be fully confirmed, because isolating haploid gametes from allodiploids usingNBRC110957 and NBRC1876 remains to be validated experimentally. In addition,genome sequence analysis of NCYC3042, which is one of the putative parents ofhybrid Z. rouxii, is necessary to fully demonstrate that strain t-1 is a haploid gametewith a mosaic genome containing T- and P-subgenomic segments.

Hybrid Z. rouxii seems to have generated at least two lineages of new species. Onehas an allodiploid/allotetraploid sexual reproduction status (11) and another has anallohaploid/allodiploid sexual reproduction status (e.g., strain t-1). Allodiploid hybrids Z.rouxii with opposite mating types can mate with each other, and the resulting allotet-raploid can form allodiploid viable spores (11). In addition, it is reasonable to assumethat allohaploid strain t-1 can switch mating type and undergo mother-daughtermating, and the resulting allodiploid can also form allohaploid viable spores. It is anopen question as to whether these lineages are reproductively isolated from the parent

FIG 6 Model for allohaploid generation from allodiploid. For a detailed explanation, see Results and Discussion. Ovalsrepresent yeast cells. Red and blue blocks indicate homologue chromosomes that share 80 to 90% sequence identity toeach other.

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species. To demonstrate that hybrid speciation has occurred, three criteria should besatisfied: (i) reproductive isolation of hybrid lineages from parental species, (ii) evidenceof hybridization in the genome, and (iii) evidence that this reproductive isolation is aconsequence of hybridization (33). The hybrid Z. rouxii and strain t-1 satisfy the secondcriterion (ii), but the first and third criteria (i and iii) are untested.

More physiological study of strain t-1 is needed to clarify its adaptation to a harshenvironment. Hybrid Z. rouxii organisms, such as NBRC1876 and ATCC 42981, arecapable of growing in more-extreme conditions than the haploid Z. rouxii CBS732T

(34–36). Given that polyploidization could be an evolutionary device to cope withstrong selection pressures during times of environmental instability by doubling allgenes (37), the haploid strain t-1 isolated from soy sauce mash would retain fitness ina harsh environment by mixing two parental genomes. The rare appearance of viablehaploid gametes of allodiploids may be a driving force to generate diverse speciesfitting a diverse environment.

MATERIALS AND METHODSOrtholog finding. Ortholog gene findings were performed manually using TBLASTN algorithms or

the Yeast Genome Annotation Pipeline (http://wolfe.ucd.ie/annotation/) (19) and analyzed using theYeast Gene Order Browser (http://ygob.ucd.ie/) (20). Dot plot analysis was performed using YASS (18).

Sequence comparison. Multiple nucleotide and amino acid sequence alignments were performedusing Clustal Omega and Clustal W2 (38) and used for phylogenetic analysis by the neighbor-joining (NJ)method (39) with 1,000 bootstrap replications (40). A phylogenetic tree was illustrated by NJplot (41).Searches for nucleotide and protein sequence homology were performed in the GenBank database withBLAST algorithms (42).

Data availability. The scaffold and contig sequences of strain t-1 are available in DDBJ/ENA/GenBankunder accession numbers KV452433 to KV452453 and LAVI01000001 to LAVI01000543, respectively. Thescaffold sequence of C. versatilis JCM5958 is available in DDBJ/ENA/GenBank under accession numbersBCJV01000001 to BCJV01000019. The scaffold sequences of hybrid Z. rouxii NBRC1876 (DF983528 toDF983589) and hybrid Z. rouxii NBRC110957 (BDGX01000001 to BDGX01000132) and the completegenome sequence of haploid Z. rouxii CBS732T (CU928173 to CU928176, CU928178 to CU928179, andCU928181) were downloaded from NCBI.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01845-17.

SUPPLEMENTAL FILE 1, PDF file, 0.9 MB.

REFERENCES1. Greig D. 2007. A screen for recessive speciation genes expressed in the

gametes of F1 hybrid yeast. PLoS Genet 3:e21. https://doi.org/10.1371/journal.pgen.0030021.

2. Dobzhansky T. 1937. Genetics and the origin of species. ColumbiaUniversity Press, New York, NY.

3. Muller H. 1942. Isolating mechanisms, evolution, and temperature. BiolSymp 6:71–125.

4. Hunter N, Chambers SR, Louis EJ, Borts RH. 1996. The mismatch repairsystem contributes to meiotic sterility in an interspecific yeast hybrid.EMBO J 15:1726 –1733.

5. Delneri D, Colson I, Grammenoudi S, Roberts IN, Louis EJ, Oliver SG. 2003.Engineering evolution to study speciation in yeasts. Nature 422:68 –72.https://doi.org/10.1038/nature01418.

6. Liti G, Barton DB, Louis EJ. 2006. Sequence diversity, reproductive isola-tion and species concepts in Saccharomyces. Genetics 174:839 – 850.https://doi.org/10.1534/genetics.106.062166.

7. Karanyicz E, Antunovics Z, Kallai Z, Sipiczki M. 2017. Non-introgressivegenome chimerisation by malsegregation in autodiploidised allotetrap-loids during meiosis of Saccharomyces kudriavzevii x Saccharomycesuvarum hybrids. Appl Microbiol Biotechnol 101:4617– 4633. https://doi.org/10.1007/s00253-017-8274-9.

8. Zanders SE, Eickbush MT, Yu JS, Kang JW, Fowler KR, Smith GR, MalikHS. 2014. Genome rearrangements and pervasive meiotic drive causehybrid infertility in fission yeast. Elife 3:e02630. https://doi.org/10.7554/eLife.02630.

9. Yokotsuka T, Asano Y, Sasaki T. 1967. Studies of the flavorous substances

in shoyu. Part XXXVII. The production of 4-ethylguaiacol during shoyufermentation, and its role for shoyu flavor. Nippon Nogeikagaku Kaishi41:442– 447. (In Japanese.)

10. Hou L, Guo L, Wang C, Wang C. 2016. Genome sequence of Candidaversatilis and comparative analysis with other yeast. J Ind MicrobiolBiotechnol 43:1131–1138. https://doi.org/10.1007/s10295-016-1764-4.

11. Watanabe J, Uehara K, Mogi Y, Tsukioka Y. 2017. Mechanism for resto-ration of fertility in hybrid Zygosaccharomyces rouxii generated by inter-species hybridization. Appl Environ Microbiol 83:e01187-17. https://doi.org/10.1128/AEM.01187-17.

12. Sato A, Matsushima K, Oshima K, Hattori M, Koyama Y. 2017. Draftgenome sequencing of the highly halotolerant and allopolyploid yeastZygosaccharomyces rouxii NBRC 1876. Genome Announc 5(7):e01610-16.https://doi.org/10.1128/genomeA.01610-16.

13. Mizushima D, Iwata H, Ishimaki Y, Ogihara J, Kato J, Kasumi T. 2016. Twoglycerol 3-phosphate dehydrogenase isogenes from Candida versatilisSN-18 play an important role in glycerol biosynthesis under osmoticstress. J Biosci Bioeng 121:523–529. https://doi.org/10.1016/j.jbiosc.2015.10.002.

14. Watanabe Y, Akita H, Higuchi Y, Tsujimatsu R, Kaneta T, Tamai Y.2008. Heterologous expression of Na�/H� antiporter gene (CvNHA1)from salt-tolerant yeast Candida versatilis in Saccharomyces cerevisiaeNa�-transporter deficient mutants. Biosci Biotechnol Biochem 72:1005–1014.

15. Watanabe Y, Nagayama K, Tamai Y. 2008. Expression of glycerol3-phosphate dehydrogenase gene (CvGPD1) in salt-tolerant yeast Can-

Gamete of Hybrid Zygosaccharomyces rouxii Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01845-17 aem.asm.org 9

on March 9, 2021 by guest

http://aem.asm

.org/D

ownloaded from

dida versatilis is stimulated by high concentrations of NaCl. Yeast 25:107–116. https://doi.org/10.1002/yea.1550.

16. O’Donnell K. 1993. Fusarium and its near relatives, p 225–233. In Reyn-olds DR, Taylar JW (ed), The fungal holomorph: miotic, meiotic andpleomorphic speciation in fungal systematics. CAB International, Wall-ingford, United Kingdom.

17. Chand Dakal T, Giudici P, Solieri L. 2016. Contrasting patterns of rDNAhomogenization within the Zygosaccharomyces rouxii species complex.PLoS One 11:e0160744. https://doi.org/10.1371/journal.pone.0160744.

18. Noé L, Kucherov G. 2005. YASS: enhancing the sensitivity of DNA simi-larity search. Nucleic Acids Res 33:W540 –W543. https://doi.org/10.1093/nar/gki478.

19. Proux-Wéra E, Armisén D, Byrne KP, Wolfe KH. 2012. A pipeline forautomated annotation of yeast genome sequences by a conserved-synteny approach. BMC Bioinformatics 13:237. https://doi.org/10.1186/1471-2105-13-237.

20. Byrne KP, Wolfe KH. 2005. The Yeast Gene Order Browser: combiningcurated homology and syntenic context reveals gene fate in polyploidspecies. Genome Res 15:1456 –1461. https://doi.org/10.1101/gr.3672305.

21. Watanabe J, Uehara K, Mogi Y. 2013. Diversity of mating-type chromo-some structures in the yeast Zygosaccharomyces rouxii caused by ectopicexchanges between MAT-like loci. PLoS One 8:e62121. https://doi.org/10.1371/journal.pone.0062121.

22. Solieri L, Dakal TC, Croce MA, Giudici P. 2013. Unravelling genomicdiversity of Zygosaccharomyces rouxii complex with a link to its life cycle.FEMS Yeast Res 13:245–258. https://doi.org/10.1111/1567-1364.12027.

23. Kurdzo EL, Dawson DS. 2015. Centromere pairing—tethering partnerchromosomes in meiosis I. FEBS J 282:2458 –2470. https://doi.org/10.1111/febs.13280.

24. Bomblies K, Jones G, Franklin C, Zickler D, Kleckner N. 2016. The chal-lenge of evolving stable polyploidy: could an increase in “crossoverinterference distance” play a central role? Chromosoma 125:287–300.https://doi.org/10.1007/s00412-015-0571-4.

25. Scannell DR, Butler G, Wolfe KH. 2007. Yeast genome evolution—theorigin of the species. Yeast 24:929 –942. https://doi.org/10.1002/yea.1515.

26. Brideau NJ, Flores HA, Wang J, Maheshwari S, Wang XU, Barbash DA.2006. Two Dobzhansky-Muller genes interact to cause hybrid lethality inDrosophila. Science 314:1292–1295. https://doi.org/10.1126/science.1133953.

27. Lee H-Y, Chou J-Y, Cheong L, Chang N-H, Yang S-Y, Leu J-Y. 2008.Incompatibility of nuclear and mitochondrial genomes causes hybridsterility between two yeast species. Cell 135:1065–1073. https://doi.org/10.1016/j.cell.2008.10.047.

28. Bayes JJ, Malik HS. 2009. Altered heterochromatin binding by a hybridsterility protein in Drosophila sibling species. Science 326:1538 –1541.https://doi.org/10.1126/science.1181756.

29. Piotrowski JS, Nagarajan S, Kroll E, Stanbery A, Chiotti KE, Kruckeberg AL,Dunn B, Sherlock G, Rosenzweig F. 2012. Different selective pressures

lead to different genomic outcomes as newly-formed hybrid yeastsevolve. BMC Evol Biol 12:46. https://doi.org/10.1186/1471-2148-12-46.

30. Dunn B, Paulish T, Stanbery A, Piotrowski J, Koniges G, Kroll E, Louis EJ,Liti G, Sherlock G, Rosenzweig F. 2013. Recurrent rearrangement duringadaptive evolution in an interspecific yeast hybrid suggests a model forrapid introgression. PLoS Genet 9:e1003366. https://doi.org/10.1371/journal.pgen.1003366.

31. Chou JY, Hung YS, Lin KH, Lee HY, Leu JY. 2010. Multiple molecularmechanisms cause reproductive isolation between three yeast species.PLoS Biol 8:e1000432. https://doi.org/10.1371/journal.pbio.1000432.

32. Leducq JB, Nielly-Thibault L, Charron G, Eberlein C, Verta JP, Samani P,Sylvester K, Hittinger CT, Bell G, Landry CR. 2016. Speciation driven byhybridization and chromosomal plasticity in a wild yeast. Nat Microbiol1:15003. https://doi.org/10.1038/nmicrobiol.2015.3.

33. Schumer M, Rosenthal GG, Andolfatto P. 2014. How common is homop-loid hybrid speciation? Evolution 68:1553–1560. https://doi.org/10.1111/evo.12399.

34. Pribylova L, de Montigny J, Sychrova H. 2007. Osmoresistant yeastZygosaccharomyces rouxii: the two most studied wild-type strains (ATCC2623 and ATCC 42981) differ in osmotolerance and glycerol metabolism.Yeast 24:171–180. https://doi.org/10.1002/yea.1470.

35. Solieri L, Vezzani V, Cassanelli S, Dakal TC, Pazzini J, Giudici P. 2016.Differential hypersaline stress response in Zygosaccharomyces rouxiicomplex yeasts: a physiological and transcriptional study. FEMS YeastRes 16:fow063. https://doi.org/10.1093/femsyr/fow063.

36. Solieri L, Dakal TC, Bicciato S. 2014. Quantitative phenotypic analysis ofmultistress response in Zygosaccharomyces rouxii complex. FEMS YeastRes 14:586 – 600. https://doi.org/10.1111/1567-1364.12146.

37. Gordon JL, Wolfe KH. 2008. Recent allopolyploid origin of Zygosaccha-romyces rouxii strain ATCC 42981. Yeast 25:449 – 456. https://doi.org/10.1002/yea.1598.

38. Li W, Cowley A, Uludag M, Gur T, McWilliam H, Squizzato S, Park YM,Buso N, Lopez R. 2015. The EMBL-EBI bioinformatics web and program-matic tools framework. Nucleic Acids Res 43:W580 –W584. https://doi.org/10.1093/nar/gkv279.

39. Saitou N, Nei M. 1987. The neighbor-joining method: a new method forreconstructing phylogenetic trees. Mol Biol Evol 4:406 – 425.

40. Felsenstein J. 1985. Confidence limits on phylogenies: an approachusing the bootstrap. Evolution 39:783–791.

41. Perrière G, Gouy M. 1996. WWW-Query: an on-line retrieval system forbiological sequence banks. Biochimie 78:364 –369. https://doi.org/10.1016/0300-9084(96)84768-7.

42. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic localalignment search tool. J Mol Biol 215:403– 410. https://doi.org/10.1016/S0022-2836(05)80360-2.

43. Rodriguez-R LM, Konstantinidis KT. 2016. The enveomics collection: atoolbox for specialized analyses of microbial genomes and metag-enomes. Peer J Preprints 4:e1900v1.

Watanabe et al. Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01845-17 aem.asm.org 10

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