INTRODUCTION
Evaluating the genetic diversity
of non-model species is essential for obtaining a thorough understanding
of biodiversity and for analyzing the complexity of genotype-phenotype
relationships in nature (Fournier et al., 2017).
In this sense, microorganisms are
particularly important since they represent a great proportion of the
ecosystem’s biomass and provide several ecosystem services such as
foods, fuel, and nitrogen fixation capacity (Chen et al., 2019; Mirza et
al., 2014). Particularly important are organisms belonging to theSaccharomycotina subphylum (yeasts), many of which have been
domesticated for human benefit, showing great capacity for rapid
evolution. Yeasts have small genome sizes, high genetic variation, and a
wide repertoire of metabolisms and lifestyles, representing an ideal
group of model and non-model organisms for phylogenomics and population
genomics studies (Peter and Schacherer, 2016). However, most
phylogenomic studies focused on Saccharomyces species and to a
lesser extent on other genera like Candida or Lachancea ,
ignoring the overwhelming biodiversity in the Saccharomycotinasubphylum (Almeida et al., 2015; Gallone et al., 2016; Goncalves et al.,
2016; Liti et al., 2009). In this context, different phylogenomic
studies have provided new evidence on the evolutionary history of
non-conventional yeast species and the genetic basis of their phenotypic
diversity (Cadez et al., 2019; Friedrich et al., 2012; Vakirlis et al.,
2018; Vakirlis et al., 2016). The ecology and evolution of species such
as Candida albicans (Ford et al., 2015; Hirakawa et al., 2015),Candida glabrata (Gabaldon and Fairhead, 2019),Cryptococcus neoformans (Passer et al., 2019),Hanseniaspora uvarum (Guaragnella et al., 2019),Brettanomyces bruxellensis (Gounot et al., 2020) andLachancea kluyveri (Friedrich et al., 2015) is becoming known
just recently, demonstrating unique phylogenetic distributions in each
case.
A particularly intriguing group of species are the Lachanceacluster (Porter et al., 2019a). The genus was initially proposed in 2003
by Kurtzman P. (Kurtzman, 2003), which diverged from theSaccharomyces linage prior to the ancestral whole-genome
duplication (WGD), 100 or more MYA (Hranilovic et al., 2017). The WGD
event represents the key evolutionary innovation that diversified
fermentative yeasts, thanks to the capacity of exploiting simple sugars
in the ecological niche, an available hallmark in yeast after the
appearing of angiosperms (Dashko et al., 2014; Nespolo et al., 2020a;
Piskur et al., 2006). Thus, Lachancea yeasts would represent a
basal lineage lacking this enhanced capacity, but still being able to
consume sugars and ferment under aerobic conditions (Hagman et al.,
2014).
Lachancea species harbor eight chromosomes and exhibit high
levels of synteny in the coding regions (Porter et al., 2019b). However,
chromosome sizes and nucleotide diversity differ significantly between
species (Lachance and Kurtzman, 2011) and there is higher genetic
diversity and genetic distance between Lachancea species compared
to Saccharomyces (Vakirlis et al., 2016). Lachanceaspecies have been recovered from a wide variety of ecological niches,
ranging from plants (Esteve-Zarzoso et al., 2001; Gonzalez et al., 2007;
Romano and Suzzi, 1993a, b), tree barks (Nespolo et al., 2020b;
Villarreal et al., 2021), tree exudates (Varela et al., 2020), insects
(Phaff et al., 1956), soil (Lee et al., 2009; Mesquita et al., 2013),
water (Kodama and Kyono, 1974) as well as food and beverages (Magalhães
et al., 2011; Marsh et al., 2014; Nova et al., 2009; Pereira et al.,
2011; Tzanetakis et al., 1998; Wojtatowicz et al., 2001). Despite the
large number of ecosystems where Lachancea spp. are present, we
still lack information concerning the genetic and genomic adaptations
needed to survive in such a range of ecological niches (Porter et al.,
2019b). In this sense, the number of currently availableLachancea genomes is still low, and therefore most species remain
unexplored.
The availability of complete
genomes of many individuals is a prerequisite for exploring the
diversity and genomic structure of a species. Thus far, genomic studies
have been carried out in Lachancea thermotolerans , L.
fermentati, and L. kluyveri , contributing to a better
understanding of the population structure, ecology, and evolution of
these species (Porter et al., 2019b). L. thermotolerans is the
best-studied species of the genus (Banilas et al., 2016; Hranilovic et
al., 2017; Hranilovic et al., 2018), and it has been reported that the
evolution of L. thermotolerans was driven by geographic isolation
and local adaptation (Hranilovic et al., 2017). Furthermore, these
species possess an attractive biotechnological potential to produce new
fermented beverages, where the biotransformation of organic acids
provides a novel sensory profile and mouthfeel in the beverage
(Hranilovic et al., 2018; Morata et al., 2018). Nonetheless, studies
related to the natural population diversity, evolution, and
biotechnological potential of other species of the genus are still
scarce, mainly due to the low number of isolates sampled to date.
Recently, L. cidri was
recovered from cider fermentations in Europe, eucalyptus tree sap in
Australia and from Nothofagus forests in Patagonia. The isolates
exhibit an interesting phenotypic diversity and biotechnological
potential for wine and mead fermentations (Nespolo et al., 2020b;
Villarreal et al., 2021). Patagonian L. cidri isolates showed a
greater fitness in high throughput microcultivation assays for
fermentative-related conditions, such as growth under different carbon
sources (fructose, glucose, and maltose), together with high ethanol
tolerance (6 and 8 % v/v). Interestingly, microfermentations in
different musts demonstrated the potential of L. cidri in mead,
surpassing the fermentative capacity of commercial S. cerevisiaestrains, together with providing a distinctive organoleptic profile to
the final ferment, mostly because of the production of acetic and
succinic acid (Villarreal et al., 2021). However, the phenotypic
diversity (and the underpinning genetic variation) of this species has
not been determined. The broad global distribution of L. cidrirepresents an excellent opportunity to establish the ecological and
geographic determinants of the actual distribution of a non-conventional
yeast (Porter et al., 2019a, b; Villarreal et al., 2021).
In order to address this problem,
we characterized the genomic and phenotypic profiles of 55 L .cidri strains. The isolates were collected from the bark ofNothofagus and Araucaria trees in Chile and from sap
samples of Eucalyptus gunni in Australia, together with a single
isolate from cider fermentations from France. Overall, we demonstrate
that Patagonia represents a natural and unique reservoir of genetic and
phenotypic diversity for the species, mainly due to the broad and varied
environments it provides (both spatially and temporally). Then, our
results increased the number of evolutionary units of Lachancea,extending our current knowledge of the genetics, ecology and
biotechnological potential of this novel natural resource from
Patagonia.