Discussion
In this study, we applied comparative phylogenetic methods for exploring
the evolution of fermentative capacity of yeasts, using compiled traits.
To address this problem, we applied the lasso-OU algorithm, which is
designed to detect adaptive shifts in phenotypic values provided a
phylogeny and a dataset. According to this procedure, the three proxies
of fermentative capacity that we considered (i.e., glycerol production,
ethanol yield and respiratory quotient, collectively “physiological
traits” hereafter) are consistent with a single evolutionary episode; a
whole-genomic duplication (WGD) that occurred in the evolution of yeasts
ca. 150 million years ago (Dashko et al., 2014). Thus, our results
partially support this idea but it indicates that the differentiation
between lineages occurred perhaps later, according to the combination of
calibrated phylogenies and traits (i.e., phenograms, Fig 4) 75 million
years ago. Our results are then different of what was obtained
originally by the authors (Hagman et al., 2003). This is probably due to
the phylogenetic comparison, which permits to account for the
evolutionary distances among species when comparing trait values.
Gene duplications represent a typical way for increasing phenotypic
capacities (Zhang, 2003). For the Saccharomycotina clade, recent
evidence suggests that the mechanism of genomic duplication was
interspecies hybridization, an episode that provided stability to the
recently formed allopolyploid (Marcet-Houben & Gabaldon, 2015). In
fact, it is accepted that the yeast WGD likely involved mating between
two different ancestral species followed by a doubling of the genome to
restore fertility. Then, the duplicated genes were retained either
through neofunctionalization or sub functionalization in many genomes,
increasing performance under nutrient competitive conditions (Scannell
et al., 2007, Chen et al., 2008). In fact, compared with other genes,
paralogs that were generated after the WGD in yeasts have long-lasting
regulatory effects (Thompson et al., 2013, Chen et al., 2008). In
addition, genome content doubling has been recurrently observed in
laboratory evolution assays using haploid lines (Fisher et al., 2018,
Gallone et al., 2016, Voordeckers et al., 2015). For example, it was
demonstrated that WGD in haploids provides an immediate fitness gain at
the expense of slowing subsequent adaptation in autodiploids, however
this positive effect can be condition dependent (Fisher et al., 2018,
Chen et al., 2008). In this context, in wine fermentation, the selective
environment of several domesticated yeasts, the greater dosage of genes
permits a rapid consumption of nutrients and a competitive displacement
of other microorganisms (Querol et al., 2003, Gutierrez et al., 2016).
Hence, results suggesting that yeasts’ phenotypic diversification in
ethanol yield, ethanol production, glycerol production, and
CO2 production was modulated by the WGD is interesting
(Piskur, 2001, Conant & Wolfe, 2007). The WGD should have facilitated
the specialization on the fermentative niche through gene duplication
and retention, including post-transcriptional regulation, finally
producing lineages with a selective increase of useful genes for
fermentation and eliminating others by purifying selection (Wolfe &
Shields, 1997). Furthermore,
paralog duplicated genes tend to have a wider gene expression variation
pattern than singleton genes, likely explained by cis -effects as
a key adaptation for the organism to respond and adapt to fluctuating
environment (Dong et al., 2011).
One of the most important adaptive features of post WGD species is the
capacity to consume glucose rapidly, then depleting media from
nutrients, and hampering respiration in other non-fermentative cells
(Gutierrez et al., 2016, Hagman & Piskur, 2015). Glucose uptake rate
and metabolism directly impacts CO2 production levels,
which is determined by glucose hexose transporters (encoded byHXT genes) (Luyten et al., 2002). The HXT genes have been
extensively amplified in fungal lineages that have independently evolved
aerobic fermentation (such as S. cerevisiae and C.
glabrata ), while a reduction of the number of HXT genes has been
reported in aerobic respiratory species (such as K. lactis ) (Lin
& Li, 2011), in agreement with our results. Interestingly, there is a
cost: since the fermentation process allows cells to rapidly convert
sugars to ethanol, this goes at the expense of decreasing biomass
production (Dashko et al., 2014). However, we did not detect such costs
on growth rate measurements (here measured as dry mass growth rate),
which appeared undifferentiated across lineages.
In Crabtree positive species, pyruvate is preferentially converted into
acetaldehyde and subsequently, ethanol. In these species glycerol is
synthesized by the reduction of dihydroxyacetone phosphate followed by
dephosphorylation catalysed by glycerol-3-phosphate dehydrogenase
(GPD1 ) and glycerol-3-phosphatase (GPP1 ) (Albertyn et al.,
1994). These two enzymes have duplicated genes, GPD2 andGPP2 , originated from gene retention and adaptive
sub-functionalization after the WGD (Wolfe & Shields, 1997). Moreover,
functional divergence of ADH1 and ADH2 , the latest only
present in Crabtree positive yeasts, allowed increasing ethanol
production and converting it to acetyl CoA for subsequent utilization in
the TCA cycle (Thomson et al., 2005, Zhou et al., 2017). Then, the
enhanced glycerol production we also observed in fermentative yeasts
(Fig 3d) represents a secondary adaptation for osmotolerance, as a mean
to compensate for the increased external osmotic pressure of the
fermentative environment.
Unicellular and multicellular organisms share essential aspects of their
design and function, because of the methods for characterizing them many
conceptual issues developed in one realm, maybe do not apply to the
other (see a critical discussion in Goddard & Grieg, 2015). Here we
considered the application of comparative phylogenetic methods (a family
of methods developed for multicellular organisms) to characterize
phenotypic evolution in unicellular organisms. We found that the
analysis produced informative results, suggesting that (above the
reasonable doubt) the WGD has visible effects on the phenotypic
diversification of fermentative yeasts, more than other genomic
rearrangements that were not identified by this analysis. Although
literature is scant regarding comparative analyses in microorganisms, a
handful of authors have tested adaptive hypotheses considering
phylogenetic relationships (Gubry-Rangin et al., 2015, Nakov et al.,
2014, Starmer et al., 2003, Ernst et al., 2003, Ravot et al., 1996). For
instance, Ravot et al. (1996) inferred adaptive patterns of
hyperthermophilic bacteria, based on the production of L-alanine in some
clades. Also in bacteria, Ernst et al. (2003) analyzed (putative)
adaptive radiations of picocyanobacteria supposedly associate with the
presence of major accessory pigments as key innovations. Working with
fermentative yeasts, Starmer et al. (2003) concluded (qualitatively)
convergent adaptive features for the cactus-yeast community. In a
comprehensive analysis, Gubry-Rangin et al. (2015) associated the high
rates of diversification observed in terrestrial Thaumarchaeota
(Archaea) to acidic adaptation of their ancestor. Although these authors
did not exactly apply trait-based comparative analyses, they were the
firsts to link evolutionary diversification to environmental adaptation
in a prokaryotic phylum. Here we show that laboratory experiments
combined with a comparative approach, could give important results for
testing a given evolutionary hypothesis in microorganisms. We encourage
authors to explore this possibility for testing evolutionary hypotheses
in other lineages.
Acknowledgements . This study was funded by Fondo Basal CAPES
0002-2014 to
Francisco Bozinovic. J.F.Q.-G. (N° 21160901) and J.J.S-I (Nº 21160530)
thanks a CONICYT-fellowship. Doctorado Nacional Chile /2016. R.F.N. and
F.C. are funded by MIISSB Iniciativa Científica Milenio-MINECON. Roberto
Nespolo also thanks a FONDECYT grant number 1180917. We also thank
Cletus Kurtzman for kindly providing us with advice for the
reconstruction of the phylogenies.
Table 1. Traits, units and meaning of the measured variables. All
species were grown at similar conditions (batch cultivation) of media
and temperature (25ºC), and traits are presented in standardized units
to biomass. Variables were measured at the moment of maximum growth
rate. Extended and detailed methods, as well as the descriptive
statistics of all the variables are provided in the original reference
(Hagman et al., 2013).