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1 University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
2 Centraalbureau voor Schimmelcultures, PO Box 85167, 3508 AD Utrecht, The Netherlands
Correspondence
Neza Cadez
neza.cadez{at}bf.uni-lj.si
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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(EF-1), as well as two ribosomal gene regions, D1/D2 domains of the large subunit and both internal transcribed spacers including the 5.8S gene region, were evaluated regarding their usefulness for reconstruction of phylogenetic relationships in the Hanseniaspora–Kloeckera species group. This included analyses of sequence divergence values, heterogeneity of evolutionary rates and the reliability of the inferred trees. Both protein-coding genes showed greater capacities to resolve at the strain level and between the closely related species of Hanseniaspora–Kloeckera, compared with the ribosomal gene regions. However, to obtain a fully resolved and reliable phylogenetic tree that reflected the biological relationships it was necessary to combine three congruent sequence datasets. The novel species Hanseniaspora pseudoguilliermondii sp. nov. (type strain CBS 8772T) is described as a result of the application of various molecular approaches to delimit species. Furthermore, incongruent gene genealogies of genetically divergent strains of Hanseniaspora occidentalis, as determined by amplified fragment length polymorphism analysis and DNA–DNA reassociation measurements, indicated the presence of two novel varieties, H. occidentalis var. occidentalis (type strain CBS 2592T) and H. occidentalis var. citrica var. nov. (type strain CBS 6783T), which could be distinguished by habitat preference.
, translation elongation factor-1; ITS, internal transcribed spacer; LSU, large subunit; RAPD, randomly amplified polymorphic DNA
The GenBank/EMBL/DDBJ accession numbers for the gene sequences determined in this study are listed in Table 1
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Figures showing saturation plots and phylogenetic analyses of sequences of actin and EF-1 genes and the D1/D2 regions of the LSU rDNA and ITS regions of Hanseniaspora–Kloeckera strains, and tables comparing dataset characteristics and characteristics inferred from the datasets using maximum-parsimony analyses and DNA–DNA relatedness values among strains of Hanseniaspora occidentalis are available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, although their common presence in fruit juices and fermenting musts had already led to the description of the apiculate species Saccharomyces apiculatus (Reess, 1870). The unstable ability of these yeasts to form ascospores contributed to several changes in nomenclature and definition of the genus (Lodder & Kreger-van Rij, 1952). With the introduction of molecular taxonomy, relationships between the species of Hanseniaspora and of the anamorphic genus Kloeckera were established on the basis of DNA base composition (Nakase & Komagata, 1970) and DNA–DNA relatedness (Meyer et al., 1978). The latter study represents the basis for the current classification, which was confirmed by several phylogenetic studies based on different parts of the ribosomal gene and two protein-coding genes (Yamada et al., 1992a; Boekhout et al., 1994; Esteve-Zarzoso et al., 2001; Kurtzman & Robnett, 2003). The genus Hanseniaspora currently comprises ten species, four of which (Hanseniaspora meyeri, Hanseniaspora clermontiae, Hanseniaspora lachancei and Hanseniaspora opuntiae) were recently circumscribed on the basis of DNA–DNA reassociation data (Smith, 1998; Cadez et al., 2003).
Phylogenetic placement of members of Hanseniaspora within the Saccharomyces clade revealed that the genus is divided into two clusters (Kurtzman & Robnett, 1998, 2003). The first cluster contains the species Hanseniaspora vineae, Hanseniaspora osmophila and Hanseniaspora occidentalis. Strains belonging to the latter species were found to be heterogeneous with respect to their randomly amplified polymorphic DNA (RAPD) profiles, electrophoretic karyotypes and restriction patterns of the internal transcribed spacer (ITS) regions (Cadez et al., 2002). The second cluster comprises Hanseniaspora valbyensis and four recently described species that are closely related to Hanseniaspora uvarum and Hanseniaspora guilliermondii (Cadez et al., 2003). The poorly resolved rDNA-based phylogenetic tree suggested that there were very close relationships among species of the H. uvarum–H. guilliermondii complex, which was not in agreement with the genetic relatedness determined by DNA–DNA reassociation analyses (Cadez et al., 2003). This supports the observation that the relationships between recently diverged species complexes are difficult to resolve on the basis of a single gene only (Rokas et al., 2003).
Several studies have demonstrated the phylogenetic usefulness of protein-coding genes for inferring relationships among different taxonomic units (Baldauf et al., 2000; Daniel et al., 2001; Belloch et al., 2000; O'Donnell et al., 1998). Housekeeping genes such as translation elongation factor-1 (EF-1) and actin are frequently used as independently evolving markers because they do not appear to have undergone horizontal transfer or gene duplication. Moreover, the rates of sequence substitutions in exons are constrained by codon positions, which may determine the taxonomic level of analysis (Baldauf & Palmer, 1993; Kretzer & Bruns, 1999).
Because the species of Hanseniaspora–Kloeckera fall into several groups of closely related taxa separated by large phylogenetic distances (Cadez et al., 2003), the phylogenetic marker for resolving Hanseniaspora–Kloeckera species should have both the capacity to resolve the sister species and the potential to realistically reconstruct the phylogeny between the divergent lineages. Consequently, the aim of the study was to assess the phylogenetic usefulness of four phylogenetic markers by describing sequence divergence and by comparing the inter- and intraspecific phylogenetic relationships with those derived from genomic DNA–DNA relatedness and physiological properties.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Fermentation ability was determined after 3 and 7 days of incubation. Utilization of carbon compounds was tested in liquid media in tubes, with reciprocal shaking at 30 r.p.m. at 25 °C for 3 weeks. Assimilation of nitrogen compounds was examined by using the auxoanographic method after 1 week (Barnett et al., 2000).
DNA extraction, amplification and sequencing.
DNA was isolated from cultures grown on yeast-malt agar (0.3 % yeast extract, 0.3 % malt extract, 0.5 % peptone, 1 % glucose, 2 % agar), according to the method of Möller et al. (1992).
PCR primers for the amplification of actin and EF-1 protein-coding regions were designed based on sequences of Saccharomyces cerevisiae (GenBank accession numbers NC_001138 and U51033, respectively). Additional primers were designed from generated sequences of the actin gene of H. vineae (Act-HV) and the EF-1 gene of H. meyeri (EF1-HM). Primer sequences, annealing temperatures and annealing sites and references are given in Table 2.
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), 2 min at 72 °C and termination with a final extension step of 7 min at 72 °C. The PCR products were purified using GenElute PCR Clean-up (Sigma–Aldrich), according to the manufacturer's instructions. The pGEM-T vector system was used to clone the PCR products of the protein-coding genes, following the procedure described in the manual (Promega). Plasmid DNA was isolated and purified for subsequent sequencing by using GenElute Plasmid Minipreps (Sigma–Aldrich). Nucleotide sequences were determined in both directions by using a Thermo Sequenase Primer Cycle sequencing kit (Amersham Biosciences) and labelled universal primers M13(–10) or M14R with an ALFexpress II Automatic DNA Sequencer (Amersham Pharmacia Biotech). The D1/D2 regions of the large subunit (LSU) rDNA and ITS regions were sequenced as described previously (Cadez et al., 2003). Sequences of both strands were aligned and edited with the ALFwin Sequence Analyser 2.10.06 software (Amersham Pharmacia Biotech).
Phylogenetic analyses.
The number of variable and parsimony-informative sites, the genomic DNA G+C contents and the mean uncorrected sequence divergences between the groups of taxa were calculated using the computer program MEGA 2.1 (Kumar et al., 2001).
Sequences were aligned automatically using the multiple sequence alignment program CLUSTAL X version 1.83 (Thompson et al., 1997). Positions where gaps existed in one of the sequences were manually excluded using BioEdit (Hall, 1999). All subsequent phylogenetic analyses employed the PAUP* 4.0b10 software package (Swofford, 2002). Most-parsimonious trees were generated by a heuristic search procedure with 1000 random addition replicates and tree bisection–reconnection branch swapping. Nucleotide sites were equally weighted. The stability of the branches was assessed by bootstrap analysis (Felsenstein, 1985) in which 1000 replicates were used. The homogeneity of the signal from the four datasets was tested with partition-homogeneity tests (Farris et al., 1995) implemented in PAUP* 4.0b10 with 1000 heuristic replicate searches and exclusion of the invariant characters.
For each dataset, a saturation plot was drawn to evaluate the degree of homoplasy and the heterogeneity of evolutionary rates and to choose an optimal outgroup species (Bush & Everett, 2001; Misof et al., 2001). The corrected evolutionary rates of nucleotide substitutions per site were plotted against the proportion of nucleotide changes per site (p distance). The corrected distances were calculated under the optimality criteria of maximum-likelihood using PAUP* 4.0b10 with corrections for multiple substitutions at the same site, substitutional rate biases and differences in evolutionary rates among sites for each dataset. These corrections were included in substitution models selected using the likelihood-ratio test implemented in MODELTEST 3.5 (Posada & Crandall, 1998). The general time reversible model with a gamma distribution parameter for among-site rate variations (GTR+
) was found to be of adequate complexity for the actin gene dataset. The Tamura–Nei model was chosen for the EF-1 and D1/D2 datasets, with only the gamma distribution parameter (TN+) for the former dataset and the addition of the estimate of invariant sites (TN+I+) for the latter. The Hasegawa–Kishino–Yano 85 model with the gamma distribution parameter and the estimate of invariant sites (HKY+I+) was proposed for the ITS dataset. When sequences of closely related taxa are compared, a strong correlation between phyletic and p distances exists. With increasing distances between taxa, the degree of sequence divergence is underestimated due to parallel and backward substitutions and the regression curve starts to decline. When the slope of the curve approaches zero, mutational saturation is reached and the phylogenetic reconstruction is more likely to suffer from artefacts such as long-branch attraction (Felsenstein, 1978).
S. cerevisiae, Lachancea kluyveri and Pichia anomala were tested as outgroup species using the saturation plots. Because the levels of sequence divergence between S. cerevisiae, L. kluyveri and ingroup species overlapped, P. anomala was chosen as the outgroup.
AFLP assay.
Amplified fragment length polymorphism (AFLP) analysis was performed according to the procedure described by Vos et al. (1995) and adapted for fluorescence-based detection by Bandelj et al. (2004). Genomic DNA was digested with the restriction enzymes MseI (1 U) and EcoRI (5 U) and linked to MseI and EcoRI adapters (50 and 5 pmol, respectively). Restricted and ligated DNA was pre-amplified using MseI-core and EcoRI-core primers. The PCR conditions for the pre-amplification step were 2 min at 72 °C, followed by 20 cycles of 30 s at 94 °C, 30 s at 56 °C and 2 min at 72 °C. Selective amplifications were performed with diluted (1 : 10, by vol.) pre-amplification mixture using three selective primer combinations (MseC/EcoAC, MseC/EcoCG and MseAC/EcoCG). The EcoAC and EcoCG primers were labelled for fluorescence detection with Cy5 dye. The PCR conditions were 2 min at 94 °C, 10 cycles of 30 s at 94 °C, 30 s at 66 °C (decreasing by 1 °C each step of the cycle) and 2 min at 72 °C, followed by 20 cycles of 30 s at 94 °C, 30 s at 56 °C and 2 min at 72 °C. The PCR products were prepared for electrophoresis by addition of an equal volume of formamide loading dye (98 % formamide, 10 mg blue dextran ml–1, 50 mM EDTA) and denatured by heating at 94 °C for 3 min. AFLP fragments were separated on 7.5 % polyacrylamide gels containing 7 M urea on ALF Express II (Amersham Pharmacia Biotech). ALF-Express sizer 50-500 (Amersham Pharmacia Biotech) was used as an external standard.
The AFLP profiles were combined in composite fingerprints using the Bionumerics software package (version 4.0; Applied Maths). Similarities between profiles were calculated using Pearson's correlation coefficient and cluster analysis was performed using the UPGMA algorithm.
Genomic DNA analysis.
For DNA extraction, the strains were grown for 2 days at 25 °C on a rotary shaker at 125 r.p.m. in 0.5 l yeast extract/malt extract broth (YM; Difco) using 1 l flat-bottom flasks. Isolation and purification of the DNA, determination of DNA base composition and DNA–DNA reassociation were performed according to procedures described previously (Golubev et al., 1989). Strains used for DNA–DNA reassociation are indicated in Table 1.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, were generated and analysed phylogenetically for 26 strains of Hanseniaspora–Kloeckera species. The newly generated sequences were compared with the D1/D2 sequences of the LSU rDNA and ITS1–5.8S rDNA–ITS2 sequences. Sequences for the two regions of the ribosomal gene complex were obtained from previous studies in which they were used as phylogenetic markers for inferring relationships between Hanseniaspora–Kloeckera species (Kurtzman & Robnett, 1998; Cadez et al., 2003). We compared sequence divergence values, the heterogeneity of evolutionary rates and the reliability of the inferred trees from the four datasets of Hanseniaspora–Kloeckera strains. The results are presented in Supplementary Table S1 and Fig. S1 in IJSEM Online. The four datasets are influenced by functional and structural constraints and therefore differed in the amount and form of variation. For example, the ITS regions are under more relaxed structural constraints, which was evident from the highest mean sequence divergence between Hanseniaspora–Kloeckera species (0.253; Supplementary Table S1). On the other hand, the protein-coding datasets resolved better at the strain level (0.005; Supplementary Table S1) and between the closely related species of Hanseniaspora–Kloeckera (0.03; Supplementary Table S1). These results are consistent with the observations of Daniel & Meyer (2003) that synonymous substitutions in codons account for the high degree of sequence divergence between closely related taxa. Furthermore, the saturation plots (Supplementary Fig. S1 in IJSEM Online) indicated heterogeneity in the evolutionary rates for the ITS dataset (i.e. mean p distance, 0.12; SD=0.11; Supplementary Fig. S1, ITS, point 3). This had been already predicted from the fact that the number of substitutions in the ITS regions was incongruent with the DNA–DNA reassociation values for closely related species of Hanseniaspora (Cadez et al., 2003).
For all four datasets, we detected an overlap in the levels of sequence divergence between the distantly related Hanseniaspora–Kloeckera species and the levels of sequence divergence between the Hanseniaspora–Kloeckera species and the outgroup species of the Saccharomycetaceae (Supplementary Fig. S1 in IJSEM Online). Kurtzman (2003) observed a similar overlap of intra- and intergeneric distances for Hanseniaspora and four related genera calculated from a dataset of six genes. In view of these results, the reinstatement of the genus Kloeckeraspora to accommodate H. occidentalis, H. osmophila and H. vineae, as proposed by Yamada et al. (1992b), could be justified. Nevertheless, we advocate the argument of Boekhout et al. (1994) against splitting the genus on the basis of genetic divergence only, because the two species groups share many similarities in morphology, physiology and ecology.
The phylogenetic usefulness of each dataset could only be determined after cladistic analyses (Damgaard & Cognato, 2003). Most-parsimonious trees based on single datasets and their characteristics are available as Supplementary Fig. S2 and in Supplementary Table S1, respectively, in IJSEM Online. In general, the trees showed some degree of congruence. However, the lack of divergence between the recently diverged H. uvarum–H. meyeri–H. clermontiae and H. guilliermondii–H. lachancei–H. opuntiae–Hanseniaspora sp. CBS 8772T species complexes resulted in a lack of statistical support for these clades in single genealogies. Therefore, we concatenated the datasets in order to increase the phylogenetic information. Prior to concatenation, we tested the congruence of the datasets with the partition homogeneity test (Farris et al., 1995). When all four datasets were combined, significant incongruence (P=0.016) was found and this resulted in a weakly supported phylogenetic tree. Therefore we tested different combinations of the concatenated datasets that resulted in the highest P values for the combined dataset without either ITS or EF-1 sequence data (P=0.35 and 0.24, respectively). Finally, parsimony analysis of the combined dataset of actin, D1/D2 and ITS sequences without EF-1 data produced a completely resolved tree with high statistical support (Fig. 1).
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; Cadez et al., 2003). Fig. 1 shows that clustering of the species is consistent with the current taxonomy. Clusters of closely related species (H. vineae–H. osmophila, H. guilliermondii–H. lachancei–H. opuntiae–Hanseniaspora sp. CBS 8772T, H. uvarum–H. meyeri–H. clermontiae and H. valbyensis–Kloeckera lindneri) generally share the same nutritional characteristics with two exceptions, H. lachancei and H. clermontiae. Furthermore, the congruence between the relationships determined from the concatenated datasets and DNA–DNA reassociation improved, in particular for H. lachancei, H. opuntiae and Hanseniaspora sp. CBS 8772T, which were placed inconsistently in the single genealogies (bootstrap support <50 %; Supplementary Fig. S2 in IJSEM Online). The latter species was not named in our previous study (Cadez et al., 2003) because its description would rest on a single isolate. However, based on additional molecular evidence of species delimitation we propose that strain CBS 8772T represents a novel species with the name Hanseniaspora pseudoguilliermondii sp. nov.
Conflicting gene genealogies within H. occidentalis
A comparison of multiple gene genealogies can be used to detect reproductive or geographical isolation between distinct populations. Incongruences between different gene genealogies may be indicative of genetic exchange among individuals within a species (Taylor et al., 2000). In order to clarify the species concept for H. occidentalis, we examined the consistency of phylogenetic relationships among H. occidentalis strains using three phylogenetic markers. In a previous study (Cadez et al., 2002), the six strains of H. occidentalis were found to be genetically heterogeneous, as shown by their chromosomal make-up, RAPD-PCR profiles and restriction patterns of the ITS regions.
First, we determined the genetic variability of 14 H. occidentalis strains by cluster analysis of the combined AFLP fingerprints. Fig. 2 shows that the strains segregated into two major groups at a similarity level of 32 %. Interestingly, this grouping coincided with the source of isolation: the strains from group I were isolated from soil whereas the strains from group II were isolated from oranges or orange juices. An exception was strain CBS 2569, which was isolated from a fruit fly. Nevertheless, this strain showed a moderate AFLP similarity (42 %) to the rest of the strains in group I.
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). However, the interpretation of DNA–DNA reassociation values of 60–78 % determined between the major groups is ambiguous without further evidence of genetic isolation between the strains.
Phylogenetic trees inferred from the sequences of the actin gene, the EF-1 gene and the ITS regions of H. occidentalis strains are presented in Supplementary Fig. S3 in IJSEM Online. Only the topology of the actin tree (Supplementary Fig. S3a) recovered the relationships obtained from the AFLP fingerprints (Fig. 2), whereas the positions of strains CBS 2569 and CBS 9921 in the EF-1 and ITS trees were incongruent. For example, strain CBS 9921, a member of group II in the actin and ITS trees (Supplementary Fig. S3a, c), was placed in group I in the EF-1 tree (Supplementary Fig. S3b). On the other hand, strain CBS 2569 was basal to group I in the actin tree but was basal to group II in the EF-1 and ITS trees. The sequence divergence in the D1/D2 regions was too low (0–1 substitutions) for phylogenetic reconstruction.
From the results presented, we might predict that the two groups of H. occidentalis are isolated by habitat preference and may present an example of an ongoing speciation event. Therefore, we propose that the two divergent groups within H. occidentalis should be recognized as varieties, with the names H. occidentalis var. occidentalis for group I and H. occidentalis var. citrica for group II.
Taxonomy
Latin diagnosis of Hanseniaspora pseudoguilliermondii N. Cadez, P. Raspor et M. Th. Smith sp. nov.
In medio liquido post 48 horas 25 °C cellulae apiculatae, ovoideae vel elongatae, 2.2–8.7x1.6–4.2 µm, singulae vel binae; gemmatione bipolari repruducentes. Post unum mensem annulus tenuis et sedimentum formantur. In agaro farina Zeae maydis confecto pseudomycelium rudimentarium. In quoque asco 4, ascosporae petasiformes. Glucosum et cellobiosum fermentantur. Glucosum, cellobiosum, salicinum, arbutinum, glucono-
-lactonum, 2-ketogluconatum, acidum gluconicum, ethylaminum, lysinum et cadaverinum assimilantur. Non assimilantur galactosum, L-sorbosum, D-glucosaminum, D-ribosum, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, sucrosum, maltosum, trehalosum, methyl -D-glucosidum, melibiosum, lactosum, raffinosum, melezitosum, amylum solubile, glycerolum, erythritolum, ribitolum, xylitolum, L-arabinitolum, D-glucitolum, D-mannitolum, galactitolum, inositolum, acidum D-glucuronicum, acidum D-galacturonicum, acidum DL-lacticum, acidum succinicum, acidum citricum, methanolum et ethanolum. Maxima temperatura crescentiae 37 °C. Crescit in medio addito 10 % NaCl, 50 % glucoso et 0.1 % cycloheximido. G+C acidi deoxyribonucleati 31.5 mol%. Typus CBS 8772T (=NCAIM Y.741T) in collectione zymotica Centraalbureau voor Schimmelcultures, Trajectum ad Rhenum lyophilus depositus.
Description of Hanseniaspora pseudoguilliermondii N. Cadez, P. Raspor & M. Th. Smith sp. nov.
Etymology: the epithet pseudoguilliermondii is chosen because the species is similar to H. guilliermondii.
In YM liquid medium after 48 h at 25 °C cells are apiculate, ovoid to elongate, 2.2–8.7x1.6–4.2 µm, single or in pairs. Budding is bipolar. Sediment is present. After 1 month, a very thin ring and a sediment are formed. After 1 month at 25 °C a streak culture on malt agar is cream coloured, butyrous, smooth, glossy, flat to slightly raised at the centre, with an entire to slightly undulate margin. On cornmeal agar a rudimentary pseudomycelium is formed. Asci containing four hat-shaped ascospores are observed on 5 % Difco malt extract agar at 25 °C. Glucose and cellobiose are fermented. The following carbon compounds are assimilated: glucose, cellobiose, salicin, arbutin, glucono--lactone, 2-ketogluconate and D-gluconate. The following are not assimilated: galactose, L-sorbose, D-glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose, sucrose, maltose, trehalose, methyl -D-glucoside, melibiose, lactose, raffinose, melezitose, starch, glycerol, erythritol, ribitol, xylitol, L-arabinitol, D-glucitol, D-mannitol, galactitol, myo-inositol, D-glucuronate, D-galacturonate, DL-lactate, succinate, citrate, methanol and ethanol. Assimilation of nitrogen compounds is positive for ethylamine, lysine and cadaverine but negative for sodium nitrate and sodium nitrite. Growth at 37 °C is positive; growth at 40 °C is negative. Growth on YM agar with 10 % NaCl is positive. Growth on 50 % glucose (w/w) yeast extract agar is weak. Growth in the presence of 0.1 % cycloheximide is positive. Diazonium blue B reaction is negative. G+C content of DNA (Tm) is 31.5 mol%.
The type strain, CBS 8772T (=NCAIM Y.741T), was isolated from orange juice concentrate, GA, USA. It has been deposited at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
Hanseniaspora occidentalis Smith var. occidentalis
Antonie van Leeuwenhoek (1974) 40, 441–444.
Type strain: ex-type isolate CBS 2592T (=ATCC 32053T) is a living strain in the CBS yeast collection.
Hanseniaspora occidentalis var. citrica N. Cadez, P. Raspor & M. Th. Smith var. nov.
Etymology: the epithet citrica of Citrus, the genus name of the host plant.
Type strain: ex-type isolate CBS 6783T is a living strain in the CBS yeast collection. Varietas a Hanseniaspora occidentalis var. citrica differt: trehalosum assimilantur (inferme). Typus: CBS 6783T in collectione zymotica Centraalbureau voor Schimmelcultures, Trajectum ad Rhenum lyophilus depositus.
This variety differs from Hanseniaspora occidentalis var. occidentalis by assimilation of trehalose (weak). The type strain, CBS 6783T, was isolated from orange juice from Italy. It has been deposited at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
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ACKNOWLEDGEMENTS |
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