# Yeast Diversity during Spontaneous Fermentations and
## 3 Results and Discussion  
### General Yeast Population Profile  
To evaluate yeast population profiles in spontaneous fermentations from grapes harvested within an individual vineyard, 48 duplicate samples covering eight grape varieties within the same vineyard, from the beginning, middle and end of fermentation were collected to analyse yeast population. Enumeration of yeast was performed using the traditional culture-dependent technique. Initially, yeast population was approximately \(8\times 10^{4}\) cfu/mL when fermentation started. Yeast viability gradually increased as fermentation processed, with the maximum population being observed at the middle fermentation stage (\(5\times 10^{7}\) cfu/mL, Table S1). Following that, viability decreased, finally reaching approximately \(7\times 10^{6}\) cfu/mL by the time fermentation terminated.  
A total of 712 isolates were collected from three stages during spontaneous fermentation (Table S2). Based on colony morphology described by [PERSON] et al. [34] and [PERSON] et al. [35], eight morphotypes were seen and six species were primarily identified (Figure 1A). Specifically, _Metschnikowia pulcherrima_ (three colonies, category I), _S. cerevisiae_ (290 colonies, category II, category VII), _Hansenispa vuarum_ (323 colonies, category III, category IV), _Pichia kluyveri_ (45 colonies, category V), _Saccharomyces pastorianus_ (47 colonies, category VI), and _Candida zemplinina_ (four colonies, category VIII) were identified among the 712 isolates (Table S3). The occurrence of these yeasts in spontaneous fermentations has been widely reported [8; 37; 45]. Notably, yeast isolates belonging to categories II and III were the most abundant, accounting for 39.33% (280 colonies) and 33.15% (236 colonies), respectively (Figure 1B). In contrast, isolates classified in categories I and VIII appeared sporadical, representing only 0.42% (three colonies) and 0.56% (four colonies) yeast presented spontaneous fermentation, respectively (Figure 1B).  
A total of 22 representative strains selected from across all colony morphotypes were subjected to species identification by sequencing analysis of the taxonomically distinctive 26S rRNA D1/D2 domain (Table S3). To ensure accuracy, PCR amplification and  
Figure 1: Species identification of 712 isolates obtained from spontaneous fermentations. (**A**): Colony morphotypes of eight yeast species on WLN agar medium. (**B**): Abundance of eight categories of morphotypes. (**C**): The neighbor-joining phylogenetic tree of selected isolates.  
sequencing were repeated multiple times. Molecular identification of 21 isolates at species level was identical with the outcomes gained from the WLN agar approach, highlighting the reliability of the traditional culture-dependent technique for preliminary wine yeast differentiation. Nonetheless, in only one case, we found that a _Metschnikova aff. fructicola_ strain F-b-10 verified by molecular species identification shared the same colony morphology as _M. pulcherrina_ isolates (Figure 1C). Empirically, colony morphology can be similar among different species. Therefore, apart from using the conventional WLN agar approach, sequencing of the 26S rRNA gene [16] or the ITS regions of the ribosomal DNA [46] is still warranted for more accurate species identification. Further analysis on the phylogenetic relationship between the tested isolates was consistent with the morphotype clustering result (Figure 1C).