Saccharomyces cerevisiae and Saccharomyces paradoxus are used as model systems for molecular, cell and evolutionary biology; yet we know comparatively little of their ecology. One niche from which these species have been isolated is oak bark. There are no reports of these species from oak in the Southern Hemisphere. We describe the recovery of both S. cerevisiae and S. paradoxus from oak in New Zealand (NZ), and provide evidence for introgression between the species. Genetic inference shows that the oak S. cerevisiae are closely related to strains isolated from NZ and Australian vineyards, but that the S. paradoxus strains are very closely related to European isolates. This discovery is surprising as the current model of S. paradoxus biogeography suggests that global dispersal is rare. We test one idea to explain how members of the European S. paradoxus population might come to be in NZ: they were transported here along with acorns brought by migrants ∼200 years ago. We show that S. paradoxus is associated with acorns and thus provide a potential mechanism for the unwitting global dispersal of S. paradoxus by humans.
Humans have a long and close association with the microbial agents responsible for the production of wine, beer and bread (Pretorius, 2000; McGovern, 2004; Piskur, 2006; Goddard, 2008). Almost 150 years ago, Pasteur showed that fermentation is a biological process conducted by microorganisms including Saccharomyces cerevisiae, and subsequently, S. cerevisiae's physiology, genetics and molecular biology have been scrutinized. The experimental tractability of S. cerevisiae, and the fact that it is a eukaryote capable of sexual reproduction, positioned it as an ideal model for genetics and cell biology (Dujon, 1996; Landry, 2006). In summary this single-celled fungus has been used to considerably advance our biological understanding in these areas. In addition to the wealth of molecular information, the ease with which S. cerevisiae populations may be stored, propagated and assayed has meant that it is now also an upcoming model used to experimentally test fundamental questions in population biology, ecology and evolution (Zeyl, 2000; Greig, 2007; Replansky, 2008).
Population genetic analyses, including recent studies that have compared whole-genome data for 30–60 strains, show that there are certain lineages of S. cerevisiae that appear to be closely associated with wine and fermentation, but that a general diversity of S. cerevisiae exists in a number of natural populations isolated from other niches (Fay & Benavides, 2005; Legras, 2007; Liti, 2009; Schacherer, 2009). There is a reasonable amount of gene flow between these S. cerevisiae populations on a global scale, which may well be facilitated by interaction with humans, and the transport of vines and other wine-related paraphernalia (Legras, 2007; Liti, 2009; Goddard, 2010). In general, S. cerevisiae is well-adapted to invade fruit niches via ecosystem engineering, a trait that evolved long before humans learned to harness it (Wolfe & Shields, 1997; Piskur, 2006; Goddard, 2008). While S. cerevisiae has been isolated from a range of fermenting environments, the other main niche from which it has been isolated is the bark of, and soil associated with, oak (Quercus sp.) trees (Sniegowski, 2002; Sampaio & Goncalves, 2008).
Saccharomyces paradoxus on the other hand is rarely isolated from fruits/ferments, which is curious, given that it is also capable of fermentation (i.e. it is Crabtree positive). However, S. paradoxus has also been isolated from oak bark, sometimes contemporaneously with S. cerevisiae and other Saccharomyces sensu stricto species (Sniegowski, 2002; Sampaio & Goncalves, 2008). Recent work and whole-genome population genetic analyses show that in contrast to S. cerevisiae, S. paradoxus' population structure appears to be well-described by geographic distance (Koufopanou, 2006; Liti, 2006, 2009). Within continents, S. paradoxus populations appear to be reasonably well-mixed, but there is a strong genetic demarcation between strains from Europe, America and the Far East, implying that global dispersal/gene flow is a much weaker force for S. paradoxus compared with S. cerevisiae (Liti, 2009). However, this biogeography model is not absolute as there is one inference of an ancient S. paradoxus intercontinental dispersal from Eurasia to North America (Kuehne, 2007). In general, S. paradoxus is a species that is perceived not to be associated with humans.
Goddard (2010) recently described a genetically distinct natural population of S. cerevisiae in New Zealand (NZ) that appears to be dispersed on local scales at least by insects. There is also evidence that NZ contains migrant S. cerevisiae from Europe vectored unwittingly by humans in oak barrels. Thus far, there are no reports of S. cerevisiae or S. paradoxus associated with oak in the Southern hemisphere. Oak trees are not native to NZ, but were brought here by European settlers ∼200 years ago and thus potentially provide a niche in which Saccharomyces yeasts may reside. Here, we test for the presence of Saccharomyces species on oak bark in NZ.
Materials and methods
A total of 42 Quercus robur and one Quercus palustris trees were sampled during December 2008 in the grounds of the University of Auckland and the adjacent Alten reserve in central Auckland (centred around 36°50′59.57″S 174°46′15.90″E). Each tree was sampled on the North and South side at 0.5 and 1.5 m from the ground for a total of 172 samples. Roughly 1 cm3 of bark and cambium were sampled by hammering a sterile cork borer into the bark at each position. In addition, surface bark samples were taken for visualization by scanning electron microscopy (SEM). A total of 18 acorns from three of these Q. robur trees were also sampled. Samples were transferred to the laboratory, where a selection procedure (comprising SelMed: 1% yeast extract, 2% peptone, 10% glucose and 10% ethanol) designed to enrich for Saccharomyces yeasts was applied to each sample (see Serjeant, 2008 for details); it must be noted that the 30 °C incubation step will likely select against the more cryotolerant Saccharomyces species (Saccharomyces bayanus, Saccharomyces kudriavzevii and Saccharomyces arboricolus) (Sampaio & Goncalves, 2008). After a 16-day incubation period interspersed by one transfer, the samples were removed and 10−2 and 10−3 dilutions were spread onto YPD (1% yeast extract, 2% peptone, 2% glucose) and incubated for 3 days at 30 °C. Where present, up to six yeast-like colonies were selected from each sample and stored at −80 °C in 15% v/v glycerol.
DNA was extracted from cultures of each isolate using a procedure that used a 5% Chelex solution. Following Goddard (2008), the ITS1–5.8S-ITS2 (internal transcribed spacer, ITS) regions of these colonies were initially analysed by restriction fragment length polymorphism (RFLP) (with the restriction endonucleases HaeIII and a more detailed analysis was undertaken for Saccharomyces sensu stricto colonies using MspI) and then by Sanger sequencing in order to identify the isolates to the species level. The divergent domain 1 and 2 (D1/D2) of the 26S rRNA gene was also sequenced for one representative from each class indicated by the RFLP analyses.
Six other loci were amplified and sequenced to further characterize the isolates. Four pairs of primers reported in Johnson (2004) were used, three of which specifically amplify the S. paradoxus MF-a, STE2 and SAG1 loci, while one primer pair amplifies both the S. paradoxus and the S. cerevisiae MF-α locus. Two other primer pairs were specific to the S. cerevisiae PAD1 and IAH1 loci. In addition, those isolates that were diagnosed as S. cerevisiae (in whole or part by our analyses) were typed at 10 variable repeat regions and the MAT locus as described in Richards (2009) in order to ascertain their relatedness to other S. cerevisiae isolates from NZ and abroad.
Figure 1 shows the representative SEM images of Quercus bark samples. While this method clearly does not allow any objective statements to be made about the presence or absence of organisms in this niche, it does show the presence of structures that are of the same size and shape (∼5 μm oval diameter) as yeast cells and spores. We show these images for the reader's interest because of our subsequent findings.
Images taken under SEM. The top two images and the lower left image show potential yeast candidates. The lower right image shows a potential Saccharomyces tetrad (sporulation structure).
Distribution of isolates
In total, 106 of the 172 samples (61.6%), deriving from 40 of the 43 trees, yielded colonies on YPD agar after the enrichment procedure. We tested whether there was a significant propensity for isolates to be more likely recovered from any of the high/low or North/South sites using the binomial distribution assuming an equal probability of isolation from any site, and found there was not (P∼0.4).
One to three colonies were randomly selected for identification from each bark sample that yielded colonies and subjected to ITS RFLP analyses (a total of 158 isolates deriving from 106 bark samples). Representatives from the 10 RFLP cohorts identified had their ITS and D1/D2 26S rRNA gene regions two-way sequenced. These sequences (accession numbers in Table 1) were compared with those present in the NCBI database using the nucleotide blast-nr search tool and a total of 10 species were revealed; the species and their percent identity to deposits in the NCBI nucleotide database are shown in Table 1. The presence/absence incidence of these species among samples are also tabled: as more than one colony may have been selected from a sample, isolates recovered from the same sample site were not independent; thus, the isolate count in Table 1 was collapsed to one when more than one colony of the same species were recovered from a given bark sample. These data should be treated with caution as many isolates were identified solely according to their ITS RFLP – we determined the sequence of only a few examples from each ITS RFLP cohort.
↵* The % sequence match of the divergent domains 1 and 2 of the diagnostic 26S rRNA gene to numerous deposits in GenBank.
↵† The incidence of species among bark samples is based on the presence/absence data of each species in each sample after enrichment (i.e. if two colonies from one sample were identified as being the same species, this would simply result in a count of one for that sample).
Recalling the selective method used, the results show that at least 10 species are present on NZ oak bark, nine of which are Ascomycetes: Saccharomycetes (phylum: class) yeasts. Along with S. cerevisiae and S. paradoxus, Lachancea sp. and Torulaspora sp. have been recovered from oak bark in the Northern hemisphere (Sampaio & Goncalves, 2008). While both S. cerevisiae and S. paradoxus were isolated, S. paradoxus was the more numerous of the two species in this niche. As far as we are aware, this is the first report of S. cerevisiae and S. paradoxus from exotic oaks in the Southern hemisphere; note: the only other S. paradoxus isolates from the Southern hemisphere derived from Drosophila around Rio de Jenerio (Liti, 2009) and soil in South Africa (Naumov, 1993). The per-bark sample recovery rate of S. paradoxus of 24% from Q. robur bark in NZ is higher than that reported by Sampaio & Goncalves (2008) from Canada and Germany (∼8%) and by Johnson (2004) from the United Kingdom (also 8%), but note that these other studies used different enrichment protocols. Higher rates of recovery between 23% and 80% have been achieved from other Quercus species, though (Sampaio & Goncalves, 2008).
To confirm the identity of the S. paradoxus and S. cerevisiae isolates, six other loci were amplified and sequenced in six randomly selected isolates. We noticed some species identification conflict between the ITS, 26S D1/D2, MF-a, MF-α, STE and SAG loci for two NZ oak isolates. For example, the HZ140 isolate has ITS, 26S D1/D2 and MAT sequences that match 100% to S. cerevisiae, and also amplified fragments from all 10 S. cerevisiae-specific microsatellite loci. However, the STE2, SAG1, MF-a and MF-α sequences from this same isolate were closely related to the respective S. paradoxus sequences. Another isolate, HZ101, has ITS and 26S D1/D2 sequences that are 100% identical to S. paradoxus, but an MF-α allele that is homologous to S. cerevisiae. In addition, the IAH1 and PAD1 primers amplify alleles from HZ101 that are homologous to S. cerevisiae, and the S. paradoxus-specific primers fail to amplify the MF-a, STE2 and SAG1 loci.
These discrepancies were rechecked and confirmed; the best explanation for the conflicting sequence homology within these isolates is that they are, or have ancestors that were, hybrids and thus contain genetic material from both S. cerevisiae and S. paradoxus. While hybrids between these species have been made in the laboratory (Greig, 2002), there are only a few previous reports of natural hybrids between S. cerevisiae and S. paradoxus (Naumov, 1987, 1996; Liti & Louis, 2005; Liti, 2006). Our observations add to the few inferences of natural hybrids, and thus possibly introgression between these species. We did not ascertain the spore viability of hybrids nor quantify the incidence of hybrids among the remaining isolates.
Given the availability of sequence data for overseas isolates of S. paradoxus (Liti, 2009) and microsatellite profiles for NZ and overseas isolates of S. cerevisiae (Goddard, 2010), we were interested in testing the global relationships of the NZ oak Saccharomyces.
While Goddard (2010) showed that NZ harbours a distinct population of S. cerevisiae, their data also provided tentative evidence for the presence of some migrants from Europe. Globally, the data for S. cerevisiae show no strong correlation with geographic distance, but are more structured around the niche of isolation (Liti, 2009). The microsatellite profile of these NZ oak S. cerevisiae isolates was compared with our database of microsatellite profiles for NZ and international isolates (Richards, 2009; Goddard, 2010). The genotype of the oak S. cerevisiae strain is unique within our database, but showed a very close match (1 bp different at two loci) to DBVPG1106, which was isolated in Australia from wine grapes. It is worth noting that DBVPG1106 clusters with the ‘European wine’ group according to Liti 's (2009) analyses. The S. cerevisiae NZ oak isolate also clusters with NZ S. cerevisiae strains isolated from the soil of an Auckland vineyard approximately 40 km away. It appears this S. cerevisiae is closely related to other strains residing in the local area and the Australasian continent, but not other oak strains.
The existing data indicate that S. paradoxus' population structure is strongly described by geographic distance (Koufopanou, 2006; Liti, 2006, 2009), with discrete populations at the continental scale. There is only one previous inference of the intercontinental dispersal of S. paradoxus from Eurasia to North America (Kuehne, 2007). We determined the sequence at the MF-α locus for 11 of the NZ S. paradoxus strains and these were all identical. We then determined the sequences at MF-a, STE2 and SAG1 for a subset of isolates: these sequences were also identical. The lack of polymorphism seen in these NZ isolates suggests that these S. paradoxus are relatively clonal in nature on local scales and this concurs with inferences from the European population (Koufopanou, 2006). We thus obtained a single sequence at each of these four loci that is our best estimate for alleles in the NZ oak S. paradoxus population.
We then tested the hypothesis that S. paradoxus global diversity is principally defined by geographic distance by reconstructing a phylogeny for the NZ and the 35 international S. paradoxus strains described by Liti (2009). The concatenated sequence data comprising the four loci totalled 5110 bp with ∼20% polymorphic sites and 243 parsimony-informative characters. NZ is approximately 7000 km from the Hawaiian, 10 000 km from the Far Eastern, 12 000 km from the American and 19 000 km from the European sample sites and thus from the S. paradoxus populations they contain. The phylogenetic signal from all four loci, independently and combined, places the NZ isolates with the European group with >98% bootstrap support (by distance, parsimony and likelihood methods): this shows that the NZ isolates derive from the European population, which is the furthest away from NZ. This result is unsurprising, given that three (MF-α, SAG and STE) of the NZ isolates' four alleles are identical to alleles in the European population. There is one unique single-nucleotide polymorphism in the NZ isolates' MF-a allele (thus minimizing the possibility that these are contaminants from our strain collection, accession number HM640252), but the remainder of the sequence is identical to an allele in the European population. The lack of recombination within these multilocus data on continental scales is shown in Fig. 2, which is a network generated by the neighbour net algorithm in splitstree (Huson & Bryant, 2006). The clean signal in the sequence data is apparent, as the network appears tree-like, with the NZ isolates clustering strongly with the European population. A closer examination of the NZ strains' relationship with the European population shows that the NZ strains contain a unique combination of alleles at these four loci (i.e. they represent a unique genotype), again minimizing the likelihood that these represent contaminants.
Network of relationships between the Saccharomyces paradoxus strains ascertained from the concatenated sequence data. The tree-like structure symbolizes the lack of recombination between the main groups. The NZ oak strains cluster strongly with the European population. The numbers on branches are maximum likelihood bootstrap consensus proportions.
Mode of global dispersal for S. paradoxus
How might members of the European S. paradoxus population come to be on trees 19 000 km away? There are clearly many possibilities, but a very recent study by Isaeva (2009) reports that Candida railenensis (which is in the Class Saccharomycetes) is associated with acorns of Q. robur. Thus, it is feasible that S. paradoxus was present on/in acorns brought here by migrants, possibly from Europe. One report states that Quercus acorns were sent from Sydney (Australia) and were planted during 1841–1842 in the University of Auckland's grounds (Barber, 1885). The origin of at least some Q. robur planted in New South Wales may be traced back to acorns from trees in the Royal Botanical Gardens at Kew in the United Kingdom (Spencer, 1995). It is intriguing to note that the NZ oak strains share alleles with European S. paradoxus that were isolated from oaks in Windsor Great park (Johnson, 2004), which is only 25 km away from Kew.
We collected and sampled acorns from some of the same trees that we initially sampled, and subjected these to the enrichment culture procedure. ITS RFLP and then sequence analyses show that some of the colonies deriving from these acorns were S. paradoxus. We went on to sequence the MF-a, MF-α, SAG and STE alleles in one of these strains and found alleles that were exact matches to the alleles of bark isolates. These data show that the same genotype of S. paradoxus may be found on the bark and acorns derived from a single tree.
Our results demonstrate that S. cerevisiae and S. paradoxus may contemporaneously reside on oak bark in the Southern hemisphere and extend S. paradoxus' known geographic range. Samples of NZ native Nothofagus failed to recover Saccharomyces (Serjeant, 2008), but it would be of interest to sample other trees native to NZ. Our inference of hybrids supports previous data suggesting that there is gene flow between S. cerevisiae and S. paradoxus and show that these species may naturally hybridize and persist to some degree (Liti, 2005, 2006; Muller & McCusker, 2009).
The current understanding is that S. cerevisiae's population is generally ecologically partitioned, but that S. paradoxus is geographically partitioned. Our findings seem the opposite of this: the NZ oak S. cerevisiae are most closely related to other S. cerevisiae isolated locally from vineyards and not other S. cerevisiae from overseas oak bark; the NZ oak S. paradoxus are most closely related to S. paradoxus from Europe – the most distant of the S. paradoxus populations, and not the Hawaiian or Far East populations, which are closer to NZ. Together with a previous report for the intercontinental dispersal of S. paradoxus (Kuehne, 2007), our inference of interhemisphere dispersal shows that global gene flow is not necessarily a minor force for S. paradoxus. The Australian vineyard isolate, to which the NZ oak S. cerevisiae is closely related, clusters strongly with the European S. cerevisiaeLiti (2009); the NZ oak S. paradoxus cluster strongly with European S. paradoxus: together, these suggest that the Australasian Saccharomyces group may have derived from Europe via human dispersal. We show that one possible vector for S. paradoxus dispersal is acorns: because European migrants brought acorns to Australasia, we speculate that this may be the source of the NZ oak S. paradoxus population.
These data show that S. paradoxus may be globally dispersed and this may have been aided by unwitting human intervention. There can be few species on the face of the planet that are not affected by human activities in some way, and so it should not come as a surprise that S. paradoxus' biology may also be influenced by humans, albeit to a lesser degree than S. cerevisiae.
The authors thank A. Turner and C. Hobbis for help with SEM imagery.
(2002) Saccharomyces cerevisiae and Saccharomyces paradoxus coexist in a natural woodland site in North America and display different levels of reproductive isolation form European conspecifics.
FEMS Yeast Res