DISCUSSION
Large-scale population genomics studies performed onSaccharomyces spp. allowed us to understand yeast dynamics in nature and their association with anthropogenic environments (Peter and Schacherer, 2016). In this sense, the analysis of complete genomes and the use of molecular markers aided the understanding of the natural genetic diversity in Saccharomyces populations and highlights how they adapt to the environments which they inhabit (Almeida et al., 2015; Almeida et al., 2014; Bendixsen et al., 2021; Parts et al., 2021; Peter and Schacherer, 2016). In the last decade, advances in sequencing technologies have made it possible to extrapolate this type of study to non-model species that have long been overlooked (Bansal and Boucher, 2019; Dujon and Louis, 2017). L. cidri , a non-model species, is an attractive model to study since it’s a Crabtree-positive yeast located phylogenetically before the whole-genome duplication (Porter et al., 2019b; Vakirlis et al., 2016), and which exhibits substantial biotechnological potential for fermentation (Villarreal et al., 2021). In contrast to Saccharomyces spp., which are mostly diploid in nature (Almeida et al., 2014; Nespolo et al., 2020b; Peter et al., 2018), L. cidri isolates are haploids, which might be indicative of a different reproductive cycle. It has been reported that even with identical genomes, ploidy itself is known to have different effects on yeasts. Therefore, understanding how ploidy affects the ecology and evolution of organisms has long been a topic of interest (Gerstein et al., 2011; Gerstein and Otto, 2009). Haploid yeasts have a competitive advantage over diploid yeasts. For example, one of the main differences between the two ploidy levels is cell size, where differences in volume have been shown to directly affect relative fitness in some environments. Under nutrient stress, for example, where a limited concentration of nutrients diffuses across the cell membrane, haploid organisms would have a greater advantage, likely favoring their survival under extreme environmental conditions (Gerstein et al., 2011).
Our results demonstrate that L. cidri is widely-distributed in Patagonia. In fact, L. cidri is found between latitudes 35 °S and 45 °S in Australia and Patagonia, which in general are considered non-Mediterranean (cold regions). However, L. cidri was not found in extreme southern regions such as Tierra del Fuego (54 °S), probably due to southern Patagonia’s extreme cold climatic conditions, where the temperature is below freezing for a large fraction of the year (Ponce JF and M., 2014). Samples obtained in South America were collected from tree species belonging to the genera Nothofagus andAraucaria in National Parks from Chile, covering approximately 1,000 km of territory. Then, the biogeographical history of the South American population of L. cidri correlates well with the history and distribution of the Nothofagus forest, comparable to reports for other species isolated in Patagonia (Saccharomyces and non-Saccharomyces ) (Nespolo et al., 2020b). Our results agree with previous findings, where it has been demonstrated that L. cidri exhibits significant host preference, being more frequently isolated from N. dombeyi in Patagonia (Nespolo et al., 2020b; Villarreal et al., 2021). In this way, this study, together with previous studies in Saccharomyces yeasts from forests ecosystems (Cadez et al., 2021; Langdon et al., 2020; Libkind et al., 2011; Nespolo et al., 2020b) demonstrate the impact of the Nothofagus biome (Woodward et al., 2004) across the southern hemisphere on the current distribution of the Lachancea genus throughout Patagonia.
The phylogenomic analysis of wild L. cidri strains demonstrates the remarkable genetic diversity present in South America. Unlike Australia, where isolates were collected from a smaller territory and showed relatively low genetic variation (Varela et al., 2020), in South America, a broad geographic distribution with varied seasonal and spatial variation generated an enormous genetic differentiation and nucleotide diversity. Therefore, our population structure analysis, developed with different strategies, generated a consistent phylogenetic picture: two populations depending on geographic origin (SoAm and Aus), with the Australian isolates forming a tight cluster with the reference strain from France (CBS2950). The Australian population shows a low genetic diversity (π = 0,00005), indicating a recent common ancestor. Despite the large geographic distance between Australian isolates and the French reference strain, we found a low genetic differentiation between both, which would suggest a recent exchange between Australia and Europe, likely coalescing between 405 to 51 years ago. In contrast, the variability found within the South American population is in accordance with the different localities from which the isolates were obtained. The different environmental conditions and the extensive distribution of the Nothofagus forest in Patagonia likely facilitated the presence of a unique genetic diversity (Cadez et al., 2019; Cubillos et al., 2019; Nespolo et al., 2020b; Villarreal et al., 2021), which is reflected in our study. In the same way, the phenotypic data corroborates the large genetic diversity observed in Patagonia. Although it has been reported that genetic diversity is not always a reflection of diversity at the physiological level (Banilas et al., 2016; Pfliegler et al., 2014), we were able to demonstrate how a greater diversity at the genetic level is determinant when performing phenotypic analyses under natural and stressful conditions.
The divergence between Australian and South America strains is interesting and deserves attention. The split could have originated in the Cretaceous-Early Tertiary interchange when Nothofagusoriginated, and continents were separated (Hill, 1992). The absence of fossil records for yeasts complicates the interpretation, but the geographic origin of our samples, the high association with native forests with a well-documented phylogeographic history (e.g., (Acosta et al., 2014; Hinojosa et al., 2016), and the genetic differences found inL. cidri from South America permit to delineate some hypotheses. The last glacial maximum (LGM) during the Late Pleistocene (~35,000 years ago, see Davies et al. 2020)(Davies et al., 2020) resulted in an ice cover that expanded through the south of South America from latitude 53°S to 38ºS, leaving only few areas free of ice, such as Altos de Lircay (Hewitt, 2000; Hinojosa, 1997). Thus, most areas including Coyhaique were covered by the ice, and present-day plants and animals are young populations that colonized the area either from Argentina or from coastal refuges, during the last ~10,000 years. This large isolation barrier would explain the divergence observed between the South American L. cidri populations, unlike the French and Australian strains that likely migrated because of human movements.
In summary, our results demonstrate the presence of two genetically-different populations in L. cidri . In the same way, it is possible to suggest that the geographic location and the ecological niche (host) where each isolate was found are essential factors in determining the genetic differentiation and nucleotide diversity observed in this species. Our results show a high phylogeographic structure among the localities of South America. The high diversity, species delimitation method and genetic differences between Altos de Lircay populations and southern populations in Patagonia suggest that the former represents a different evolutionary unit. This is a striking result, since the unique conditions of Patagonia during the Late Pleistocene could have contributed to the differentiation of L. cidri populations in South America. Furthermore, we propose that there was a recent exchange between Australia and France due to the low genetic differentiation between strains from both regions. In conclusion, this work provides a valuable insight into the genetic and phenotypic diversity of L. cidri , contributing to a better understanding of phylogeography, population structure, ecology, and divergence time of this underexplored yeast species, with remarkable biotechnological potential.