**1. Introduction**

Spontaneous fermentation is the most traditional way and a low-intervention method for conducting alcoholic fermentation in wineries, giving rise to the most complex wine profiles. This complexity develops because of the large number of different yeast species involved (*Saccharomyces* spp. and non-*Saccharomyces*) [1]. However, the presence of unknown microbiota makes it a risky and unpredictable practice. For this reason, the inoculation with single culture inocula of *Saccharomyces cerevisiae* has become widespread in the modern wine industry to reduce the risk of wine spoilage. Nevertheless, some authors have pointed out that the use of the same

yeasts in all the winegrowing regions of the world can cause a loss of typicity and have a negative effect on the biodiversity of natural yeasts present in the wineries [2].

*S. cerevisiae* is the predominant yeast species in alcoholic fermentation and the main element responsible for the characteristics of the wines. Many surveys carried out with spontaneous fermentations in different wine-producing regions have demonstrated that there is high genetic diversity within this species in each vinification [3]. However, in most cases, only a small number of strains of *S. cerevisiae* are dominant, mainly in the tumultuous and final fermentation stages, representing a high percentage out of the total number of strains identified.

Earlier studies have shown that some strains of *S. cerevisiae* have been isolated in several consecutive years in the same winery, which is why some authors have suggested the term "winery effect" [4], and also some strains of these species were detected in different wineries of the same wine-producing area, suggesting that they were representative of a specific enological ecosystem [5, 6]. Knight et al. [7] even found specific genotypes from a particular region. These findings suggest that specific native *S. cerevisiae* strains could be associated with a *terroir* and have an influence on terroir-associated wine characteristics [8].These authors found a correlation between genotypic and phenotypic groups and the geographical origin of the strains, supporting the concept that there can be a microbial aspect to *terroir*.

Nowadays, many wineries and regions are carrying out strain selection programs with yeasts isolated from their vineyard/winery ecosystems, based on the idea that these yeasts are better adapted to their musts, which have characteristics determined by the grape varieties and the *terroir* [9, 10]. Thus, the use of these typical strains as starter yeasts could provide wines with distinctive characteristics of a particular winery or region. For this reason, studying the existence of strains which are specific to one winery or enological area is very interesting for the wine industry [11].

Rioja is a wine region in Spain with qualified designation of origin status. It is subdivided into three sub-zones: Rioja Alta, Rioja Oriental, and Rioja Alavesa. Rioja Alta is located on the western edge of the region and at higher elevations with an Atlantic climate. Rioja Oriental, the eastern section, is strongly influenced by a Mediterranean climate which makes this area the warmest and driest part of the region. Rioja Alavesa, with a similar climate to Rioja Alta, produces different wines due to the relatively poor condition of the soil. Each sub-zone has its own character, which results in different wines derived from the different compositions and origins of the soils and the climate conditions.

The study of the ecology and biodiversity of the yeast population during alcoholic fermentation is an interesting and important step in the research into, and understanding of, a winegrowing area and should be a step prior to the selection and subsequent employment of the yeasts isolated from that area as starters. The aim of this work was to study the ecology of spontaneous fermentations in 11 wineries from all over the Rioja Designation of Origin during 3–4 consecutive years in order to establish the existence of typical strains belonging to wineries, sub-zones, and regional ecosystems. The wineries under study were distributed throughout the three sub-zones of the Rioja designation of origin.
