FEMS Yeast Research 4 (2004) 521^525
www.fems-microbiology.org
Sympatric natural Saccharomyces cerevisiae and S. paradoxus
populations have di¡erent thermal growth pro¢les
Joseph Y. Sweeney, Heidi A. Kuehne, Paul D. Sniegowski
Department of Biology, University of Pennsylvania, 415 S. University Avenue, Philadelphia, PA 19104, USA
Received 19 June 2003; received in revised form 30 July 2003; accepted 31 July 2003
First published online 5 September 2003
Abstract
Saccharomyces cerevisiae and its close congener S. paradoxus are typically indistinguishable by the phenotypic criteria of classical yeast
taxonomy, but they are evolutionarily distinct as indicated by hybrid spore inviability and genomic sequence divergence. Previous work
has shown that these two species coexist in oak-associated microhabitats at natural woodland sites in North America. Here, we show that
sympatric populations of S. cerevisiae and S. paradoxus from a single natural site are phenotypically differentiated in their growth rate
responses to temperature. Our main finding is that the S. cerevisiae population exhibits a markedly higher growth rate at 37‡C than the
S. paradoxus population; we also find possible differences in growth rate between these populations at two lower temperatures. We
discuss the implications of our results for the coexistence of these yeasts in natural environments, and we suggest that thermal growth
response may be an evolutionarily labile feature of these organisms that could be analyzed using genomic approaches.
6 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : Thermal growth pro¢les; Natural Saccharomyces populations ; Saccharomyces cerevisiae ; Saccharomyces paradoxus
1. Introduction
Saccharomyces cerevisiae has had a long association
with humans as a fermenting agent and continues to be
one of the most important industrial microorganisms [1].
S. cerevisiae is also a prominent laboratory research organism ; after many decades of work, biologists can access
a wealth of S. cerevisiae cell biology, genetic information,
and genomic resources, including the ¢rst fully sequenced
and annotated eukaryote genome [2]. Our understanding
of the ecology and evolution of S. cerevisiae in nature
remains very limited, but a growing number of studies
demonstrate that the species is common in habitats that
are undisturbed by human activity [3^6] and in vineyards
[7^10]. These studies suggest that the ecology and evolution of this important organism can be investigated in
natural populations (but see [11,12]).
Recent studies have shown that S. cerevisiae and its
congener S. paradoxus occupy the same microhabitat
type (oak exudates, oak bark, and oak-associated soils)
* Corresponding author. Tel. : +1 (215) 573 4085;
Fax : +1 (215) 898 8780.
E-mail address : [email protected] (P.D. Sniegowski).
in widely separated woodland sites in eastern North America [3,5]. S. cerevisiae and S. paradoxus are reproductively
isolated by low hybrid spore viability [13] and exhibit substantial genomic sequence divergence [14,15]. However,
these species are typically indistinguishable by the criteria
of classical yeast taxonomy, which include phenotypic
characters such as cell, spore, and ascus morphology, as
well as features of obvious ecological importance such as
pro¢les of assimilation and fermentation of organic compounds [11]. This close phenotypic similarity raises the
question of whether sympatric populations of these two
species nonetheless di¡er in some ecologically signi¢cant
way, as might be predicted by classical ecological theory
[16,17]. Some previous reports [11,18] have suggested that
S. cerevisiae and S. paradoxus have di¡erent thermal
growth pro¢les, but those reports were based on allopatric
isolates. Here, we compare the pro¢les of growth rate vs.
temperature in S. cerevisiae and S. paradoxus populations
sampled from a single natural site. Our results indicate
that these two species do indeed have di¡erent thermal
growth pro¢les in sympatry; however, the growth rate
pro¢les for our populations di¡er somewhat from those
suggested by earlier studies. We discuss the possible implications of our results for the coexistence of S. cerevisiae
and S. paradoxus in nature, and we speculate that vari-
1567-1356 / 03 / $22.00 6 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S1567-1356(03)00171-5
FEMSYR 1603 18-12-03
522
J.Y. Sweeney et al. / FEMS Yeast Research 4 (2004) 521^525
ability in thermal growth pro¢les might be observable
among natural populations of these yeasts isolated from
divergent thermal environments.
the 23, 30, and 37‡C temperature levels; because of slow
growth, longer time intervals were used between measurements for the 10 and 16‡C levels. Cultures were vortexed
thoroughly to suspend the cells before each measurement.
2. Materials and methods
2.3. Statistical analysis
2.1. Yeast isolates
The response variable used in the analysis of growth
rates at each temperature was the maximum change in
absorbance observed between successive measurements
during the growth of each culture to stationary phase,
which is a valid proxy for growth rate because the in£ection point of the growth curve is in all likelihood contained within that time interval. The data were analyzed
as a two-way, mixed-model ANOVA in which nine isolates were nested within each of the two species across the
¢ve temperature levels. The three-fold replication of each
isolate/temperature combination yielded 270 experimentwide observations. Species, temperature, and their interaction were analyzed as ¢xed e¡ects; isolate and its interaction with temperature were analyzed as random e¡ects. All
analyses were carried out using PROC GLM in SAS (SAS
Institute, Cary, NC, USA).
Nine diploid homothallic isolates of both S. cerevisiae
and S. paradoxus were studied. The S. cerevisiae isolates
were strains YPS128, YPS129, YPS133, YPS134, YPS139,
YPS141, YPS142, YPS143 and YPS154; the S. paradoxus
isolates were strains YPS125, YPS126, YPS138, YPS145,
YPS150, YPS151, YPS152, YPS155 and YPS158. Methods employed in the collection and identi¢cation of these
isolates have been described previously [5]. Brie£y, all were
collected during a 2-week interval in July 1999 at Tyler
Arboretum, near Media, Pennsylvania in eastern North
America. The arboretum includes a mature, secondgrowth, deciduous woodland of approximately 182 ha
that is contiguous with 1052 ha of similar habitat at Ridley Creek State Park. Samples of exudate, bark, and soil
taken from on or around multiple red and black oaks
(Quercus spp.) were subjected to enrichment culturing in
the laboratory. S. cerevisiae and S. paradoxus isolates were
identi¢ed by test matings in the laboratory. All isolates are
currently stored in the corresponding author’s laboratory
at the University of Pennsylvania.
2.2. Growth rate measurements
We estimated the growth rate of each isolate in laboratory culture in yeast extract^peptone^dextrose (YPD)
broth [19] at ¢ve temperatures chosen to span most of
the growth-permissive range beneath the forest canopy at
our collection site: 10, 16, 23, 30, and 37‡C. Prior to each
growth rate assay, isolates were conditioned by inoculating
them from freezer storage into 5 ml of YPD broth in
loosely capped 50-ml Erlenmeyer £asks and growing
them to stationary phase at 30‡C with aeration. For the
growth rate assays, three replicate cultures of each isolate
for each temperature were started by inoculating 100-Wl
aliquots from the stationary phase cultures into tightly
capped 15-ml screwcap glass tubes containing 5 ml of
YPD. The tubes were positioned in a rack fastened to a
rocking platform set at 100 rpm (VWR Scienti¢c Model
100) and placed on the center shelf of a temperature-controlled incubator (Percival VL36, Boone, IA, USA). Culture turbidity during growth to stationary phase was measured as absorbance at 600 nm on a Spectronic 20+ Series
unit (Spectronic, Rochester, NY, USA). The spectrophotometer was frequently recalibrated to zero absorbance
during sets of readings using a control tube containing
only YPD. Tubes were removed for spectrophotometric
measurements of culture turbidity at hourly intervals for
3. Results
Fig. 1 illustrates pro¢les of average growth rate vs. temperature observed in both species. These growth rate pro¢les were signi¢cantly di¡erent across the whole experiment (F1;16 = 15.43; P = 0.0012), and the interaction
between species and temperature was highly signi¢cant
(F4;64 = 14.02; P 6 0.0001). Overall, no signi¢cant e¡ect
Fig. 1. Average growth rates ( R S.E.M.) of sympatric natural S. cerevisiae (solid line) and S. paradoxus (dashed line) isolates at ¢ve temperatures. Growth rate values, shown as vODmax , are the maximal hourly
change in A600 observed during culture growth.
FEMSYR 1603 18-12-03
J.Y. Sweeney et al. / FEMS Yeast Research 4 (2004) 521^525
of isolates within species was detected (F16;64 = 0.88;
P = 0.596).
Growth rates of the two species appeared to di¡er at
three temperatures : 16, 30, and 37‡C. Before correction
for an experiment-wide signi¢cance level of 0.05, S. cerevisiae exhibited a higher growth rate at 16‡C (non-orthogonal unplanned comparison: F1;64 = 5.69; P = 0.0201),
S. paradoxus exhibited a higher growth rate at 30‡C
(F1;64 = 4.55; P = 0.037), and S. cerevisiae exhibited a higher growth rate at 37‡C (F1;64 = 59.28; P 6 0.0001). When
the sequential Bonferroni correction [20] was applied, the
di¡erences observed at 16 and 30‡C became marginally
non-signi¢cant, whereas the di¡erence at 37‡C remained
highly signi¢cant.
Unplanned comparisons indicated that all di¡erences in
growth rates between temperatures within each species
were signi¢cant except the di¡erence between S. paradoxus
at 30‡C and S. paradoxus at 37‡C.
4. Discussion
We have shown that S. cerevisiae and S. paradoxus
populations sampled in sympatry from a Pennsylvania
woodland have di¡erent thermal growth rate pro¢les. To
our knowledge, our study is the ¢rst to compare ecologically important phenotypic characters in sympatric natural
Saccharomyces populations, although previous work has
considered thermal growth pro¢le and other phenotypic
di¡erences between S. cerevisiae and S. bayanus strains
isolated during wine making [21^23]. The possible in£uence of growth temperature relations in determining yeast
species distribution has also recently been addressed by
Lachance et al. [24], who conclude that maximum growth
temperature may be a critical property of the fundamental
niche of the Metschnikowia and Candida species associated
with morning glory £owers in Hawai’i.
As mentioned previously, S. cerevisiae and S. paradoxus
are typically indistinguishable by the standard phenotypic
criteria of yeast taxonomy, which include fermentation
and assimilation pro¢les for compounds likely to be available in natural habitats. It was therefore of interest to ask
whether sympatric isolates of these species would nonetheless exhibit some consistent phenotypic di¡erence, as
might be predicted from the classical ecological theory
that niche di¡erentiation mediates species coexistence
[16,17]. We focused upon the growth rate response to
temperature because previous studies had suggested that
S. cerevisiae and S. paradoxus have di¡erent optimal
growth temperatures [11] ; although the phenotypes of
our study populations may also di¡er in other respects,
we did not investigate this possibility.
Whether the thermal growth pro¢le di¡erence that we
have observed between S. cerevisiae and S. paradoxus affects co-occurrence of these species at our study site is
uncertain. Although S. cerevisiae grows markedly faster
523
than S. paradoxus at the top end of the thermal range
investigated (37‡C), sustained temperatures as high as
37‡C are probably uncommon at our study site. Our
data also hint at di¡erences in growth rate at lower temperatures, with S. cerevisiae growing faster at 16‡C and
S. paradoxus growing faster at 30‡C. Assuming for the
moment that these di¡erences at lower temperatures are
real, a speculative ecological hypothesis might be that
growth rate advantage alternates across the £uctuating
daily or seasonal temperature regime in our natural site
in a manner that does not consistently favor one or the
other species.
In general, whether niche di¡erentiation is necessary for
species coexistence has stimulated considerable debate
among ecologists [25^31]. Certainly, several conditions
must be met before niche di¡erentiation needs to be invoked to explain the co-occurrence of closely similar species. One such condition is that the populations in question be stably coexisting in sympatry rather than merely
coincident. A number of previous studies have suggested
that oak exudates, bark, and associated soils are indeed a
natural habitat or repository for S. paradoxus in temperate
regions worldwide [3,5,13,32^34], and thus there is good
reason to believe that S. paradoxus is a long-term resident
of our study site. However, it is conceivable that S. cerevisiae has had only a short history at our study site; the
woodland habitat might represent a sink for S. cerevisiae
isolates of human origin, or S. cerevisiae may have recently colonized this habitat or site. Although further
study is needed to address this point, two lines of evidence
support long-term residence of S. cerevisiae in the woodland habitat: (1) the presence of S. cerevisiae isolates in
sympatry with S. paradoxus at numerous woodland sites
in eastern North America (H. Kuehne, unpublished data);
and (2) the fact that an S. cerevisiae isolate from the population studied in this paper shows signi¢cantly higher
freeze tolerance and lower copper tolerance than laboratory and vineyard isolates [35]. (The latter ¢ndings are
consistent with adaptation to the woodland habitat because freezing temperatures are common at our natural
sites, and copper tolerance, which is probably an adaptation to the use of copper sulfate as a fungicide in vineyards, may well be costly to ¢tness under natural conditions.) A second condition that must be met before niche
di¡erentiation can be invoked to explain coexistence is
that the species in question co-occur at the microgeographic scale required for direct interaction; to date, we
have no data that would address this possibility for
S. cerevisiae and S. paradoxus at our study site. Finally,
the density of each species must be su⁄cient to a¡ect
growth of the other. It is possible that extrinsic disturbances such as seasonal mortality limit densities su⁄ciently to
preclude competitive interaction between S. cerevisiae and
S. paradoxus; again, data are lacking on this point.
In studies of animal and plant populations, the evolution of niche di¡erentiation as a result of competition
FEMSYR 1603 18-12-03
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J.Y. Sweeney et al. / FEMS Yeast Research 4 (2004) 521^525
between closely related species may be supported by the
observation that speci¢c characters of ecological importance di¡er in sympatry but not in allopatry (e.g.
[36,37]). Clearly, our study does not meet this criterion:
we have only compared S. cerevisiae and S. paradoxus in
sympatry. Obtaining su⁄cient data to conclude that a given site ever harbors only one or the other of these species
may be very di⁄cult, and this limits the prospect of testing
the ecological importance of the observed thermal growth
pro¢le di¡erence by comparing allopatric populations. On
the other hand, because S. cerevisiae and S. paradoxus are
readily culturable, it may be possible to examine the ecological implications of thermal growth pro¢le di¡erentiation experimentally in the laboratory; in principle, such an
approach could test the hypothesis of £uctuating growth
advantage mentioned above.
Whatever the precise ecological signi¢cance of the thermal growth pro¢le di¡erence between sympatric S. cerevisiae and S. paradoxus populations, our results have implications for future ecological and evolutionary work in
Saccharomyces. Previous work has suggested that S. paradoxus exhibits its highest growth rate at 37‡C or higher
and that S. cerevisiae may exhibit its highest growth rate
at lower temperatures [11]. We ¢nd roughly the opposite
pattern: the S. paradoxus isolates from our natural population apparently reach a growth rate plateau at around
30‡C, whereas the S. cerevisiae isolates exhibit their highest growth rate at 37‡C and may grow even faster at temperatures greater than 37‡C. Our results thus suggest that
thermal growth rate properties in Saccharomyces are evolutionarily labile and might vary with the prevailing environmental temperature regime (see also [38,39]). There is
considerable interest in genomic characterization of ecologically and evolutionarily important variation in S. cerevisiae [35,40,41], and similar approaches are now possible in S. paradoxus with the recent publication of its
genome sequence [42]. In the future it may be possible
to study evolved di¡erences in thermal growth properties
within and between S. cerevisiae and S. paradoxus using
genomic approaches.
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Acknowledgements
We thank Dr. Paul Schmidt for the use of his incubator.
We are grateful to two anonymous reviewers and to E.
Fingerman, C.A. Francis and H.A. Murphy for comments
on the manuscript. This research was supported by a grant
from the University of Pennsylvania Research Foundation
to P.D.S.
[21]
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