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.