R E S E A R C H A R T I C L E

Saccharomycesparadoxus andSaccharomyces cerevisiae resideonoak trees inNewZealand: evidence formigration fromEuropeand interspecies hybridsHanyao Zhang, Aaron Skelton, Richard C. Gardner & Matthew R. Goddard

School of Biological Sciences, University of Auckland, Auckland, New Zealand

Correspondence: Matthew R. Goddard,

School of Biological Sciences, University of

Auckland, Private Bag 92019, Auckland

1142, New Zealand. Tel.: 164 9 3737 599;

fax: 164 9 3737 416; e-mail:

m.goddard@auckland.ac.nz

Received 10 June 2010; revised 11 July 2010;

accepted 27 August 2010.

Final version published online 24 September

2010.

DOI:10.1111/j.1567-1364.2010.00681.x

Editor: Teun Boekhout

Keywords

yeast; ecology; dispersal.

Abstract

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.

Introduction

Humans have a long and close association with the micro-

bial agents responsible for the production of wine, beer and

bread (Pretorius, 2000; McGovern et al., 2004; Piskur et al.,

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 mole-

cular 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

et al., 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 et al., 2008).

Saccharomyces cerevisiae was originally thought to be a

purely ‘domesticated’ species not found outside of a close

association with humans (Mortimer & Polsinelli, 1999; Fay

& Benavides, 2005). Thus, one potential criticism is that

S. cerevisiae might not be a good model to provide insights

into the processes operating in natural populations (John-

son et al., 2004). In an attempt to circumvent this issue,

research was focused on S. cerevisiae’s closest relative, which

has been isolated from oak trees, Saccharomyces paradoxus.

Saccharomyces cerevisiae is now known to exist in natural

populations along with S. paradoxus (Sniegowski et al.,

2002; Sampaio & Goncalves, 2008), and together, these

species are being used increasingly to test more general

ecological and evolutionary hypotheses (Greig et al., 1998;

Zeyl, 2000; Greig & Travisano, 2004; Goddard et al., 2005;

Goddard, 2008) and estimate parameters in natural popula-

tions (Johnson et al., 2004; Sweeney et al., 2004; Koufo-

panou et al., 2006; Ruderfer et al., 2006; Kuehne et al., 2007;

Legras et al., 2007; Reuter et al., 2007; Replansky et al., 2008;

Tsai et al., 2008; Liti et al., 2009; Schacherer et al., 2009;

Goddard et al., 2010). Although there are a growing number

FEMS Yeast Res 10 (2010) 941–947 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

YEA

ST R

ESEA

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of studies in this area, the ecology of Saccharomyces species is

still relatively poorly understood compared with our mole-

cular understanding of these species.

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 et al., 2007; Liti et al., 2009; Schacherer et al.,

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 et al., 2007; Liti et al., 2009; Goddard et al., 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

et al., 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

et al., 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 et al., 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 et al.,

2006; Liti et al., 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 et al., 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 et al., 2007). In general, S. paradoxus is a

species that is perceived not to be associated with humans.

Goddard et al. (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 hemi-

sphere. Oak trees are not native to NZ, but were brought

here by European settlers �200 years ago and thus poten-

tially 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 36150059.5700S

174146015.9000E). 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 et al., 2008 for details); it must be noted that the 30 1C

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 inter-

spersed 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 1C.

Where present, up to six yeast-like colonies were selected from

each sample and stored at � 80 1C 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 under-

taken 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 et al. (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-a 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 et al. (2009) in order

to ascertain their relatedness to other S. cerevisiae isolates

from NZ and abroad.

FEMS Yeast Res 10 (2010) 941–947c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

942 H. Zhang et al.

The relevant sequences for the international S. paradoxus

strains generated by Liti et al. (2009) were obtained from

http://www.sanger.ac.uk/research/projects/genomeinformatics/

sgrp.html. DNA sequences were aligned and analysed within

GENEIOUS (Drummond et al., 2009); the distance, parsimony

and maximum likelihood consensus phylogenies were recon-

structed from 100 to 500 bootstrap replicates. SPLITSTREE

(Huson & Bryant, 2006) was used to reconstruct the networks.

Results and discussion

Bark SEM

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 (�5mm oval

diameter) as yeast cells and spores. We show these images for

the reader’s interest because of our subsequent findings.

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 enrich-

ment 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).

Genetic identification

One to three colonies were randomly selected for identifica-

tion 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

Fig. 1. 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).

FEMS Yeast Res 10 (2010) 941–947 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

943New Zealand Saccharomyces ecology

cohorts identified had their ITS and D1/D2 26S rRNA gene

regions two-way sequenced. These sequences (accession num-

bers 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.

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 Droso-

phila around Rio de Jenerio (Liti et al., 2009) and soil in

South Africa (Naumov et al., 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

et al. (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).

Hybrids

To confirm the identity of the S. paradoxus and S. cerevisiae

isolates, six other loci were amplified and sequenced in six

Fig. 2. 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.

Table 1. The diversity of species discovered from selective enrichment of

oak (Quercus) bark samples from Auckland, NZ

Species

Accession

number

Match�

(%) Incidencew

Saccharomyces cerevisiae GU213449.1 100 1

Saccharomyces paradoxus GU213453.1 100 43

Pichia pastoris GU213447.1 100 15

Debaromyces polymorphus GU213452.1 100 7

Lachancea kluyveri GU213454.1 99.8 15

Torulaspora delbrueckii GU213448.1 100 3

Pichia membranifaciens GU213450.1 100 26

Candida sp.? NRRL Y-27159 GU213446.1 99 1

Hanseniaspora osmophila GU213455.1 100 1

Rhodotorula mucilaginosa

(Basidomycete)

GU213451.1 99.7 1

�The % sequence match of the divergent domains 1 and 2 of the

diagnostic 26S rRNA gene to numerous deposits in GenBank.wThe 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).

FEMS Yeast Res 10 (2010) 941–947c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

944 H. Zhang et al.

randomly selected isolates. We noticed some species identi-

fication conflict between the ITS, 26S D1/D2, MF-a, MF-a,

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-a 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-

a 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 et al., 2002),

there are only a few previous reports of natural hybrids

between S. cerevisiae and S. paradoxus (Naumov, 1987, 1996;

Liti & Louis, 2005; Liti et al., 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.

Biogeography

Given the availability of sequence data for overseas isolates

of S. paradoxus (Liti et al., 2009) and microsatellite profiles

for NZ and overseas isolates of S. cerevisiae (Goddard et al.,

2010), we were interested in testing the global relationships

of the NZ oak Saccharomyces.

While Goddard et al. (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 struc-

tured around the niche of isolation (Liti et al., 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 et al., 2009;

Goddard et al., 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 et al.’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 (Kou-

fopanou et al., 2006; Liti et al., 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 et al.,

2007). We determined the sequence at the MF-a 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 et al., 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 interna-

tional S. paradoxus strains described by Liti et al. (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 East-

ern, 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 4 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-a, 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 mini-

mizing 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.

FEMS Yeast Res 10 (2010) 941–947 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

945New Zealand Saccharomyces ecology

Mode of global dispersal for S. paradoxus

How might members of the European S. paradoxus popula-

tion come to be on trees 19 000 km away? There are clearly

many possibilities, but a very recent study by Isaeva et al.

(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 et al., 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-a, 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.

Conclusions

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 et al., 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 et al., 2005, 2006; Muller & McCusker, 2009).

The current understanding is that S. cerevisiae’s popula-

tion is generally ecologically partitioned, but that S. para-

doxus 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 popula-

tions, 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 et al.,

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. cerevisiae Liti et al. (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.

Acknowledgements

The authors thank A. Turner and C. Hobbis for help with

SEM imagery.

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947New Zealand Saccharomyces ecology