**4. Conclusions**

The results sugges<sup>t</sup> that *H. vineae* is clearly better adapted to the fermentation niche compared to what we named as the *Hanseniaspora* fruit clade. These results are in agreemen<sup>t</sup> with a separately evolution divergence between the two clades of the genus *Hanseniaspora* as was proposed previously. Phenotypic behavior of *H. vineae* growth, ethanol tolerance, and fermentation kinetics are in agreemen<sup>t</sup> with the genetic and transcriptomic data provided. The results obtained demonstrate that *H. vineae* and a genetically closely related species, *H. osmophila*, behave similarly. Homologies of glycolytic and alcoholic fermentation enzyme sequences of both species were compared to *S. cerevisiae*, and the similarities observed allowed the di fferentiation of *H. uvarum* from *H. osmophila* and *H. vineae*. High sequence homology in these latter two species was observed for key genes involved in glycolysis such as *HXK2,* which encodes hexokinase, *PFK1*/*PFK2* subunits of phosphofructokinase, and *CDC19* that encodes pyruvate kinase. This homology could explain the improved fermentative performance observed for *H. vineae* compared with other *Hanseniaspora* species. The elevated number of copies of *ADH* genes in *H. vineae* might be associated with increased ethanol tolerance in the species. The presence of active

genes typically related to wine fermentation capacities in *H. vineae* and *H. osmophila* such as sulfite tolerance (*SSU1*) and sucrose hydrolyzing invertase (*SUC2*) differentiate both species from the other sequenced species of the genus. Taken together, findings reported here support the characterization of the *Hanseniaspora* genus into two different groups that are adapted to two different niches, fruit and juice fermentation. These results have contributed to the improved characterization of the genus and furthermore might support the importance of it as a model for further studies related to the genetic and evolutionary phenomena of yeas<sup>t</sup> domestication processes.

**Author Contributions:** M.J.V., E.B., E.D., and F.C. conceived the study and its design; M.J.V. and F.C. wrote the manuscript; M.J.V. performed laboratory experiments and data analysis; E.B. carried out statistical analysis. All authors read and approved the manuscript.

**Funding:** This research was funded by Agencia Nacional de Investigación e Innovación (ANII), Application of *Hanseniaspora vineae* Project ALI\_2\_2019\_1\_155314 with Lage y Cia-Lallemand, Uruguay.

**Acknowledgments:** We wish to thank our Universidad de la Republica for basic support of this work: CSIC Group Project 802 and Facultad de Quimica, Uruguay.

**Conflicts of Interest:** The authors declare no conflict of interest.
