## 3 Results
### Yeasts' Molecular Characterization and Phylogenetic Analysis
The neighbor-joining phylogenetic tree was reported in Figure 3. The bootstrap values higher than 50%, used to define the accuracy of branch prediction, were reported. According to sequence clusterization, the phylogenetic tree resulted in three well-defined clades, distinguishing with high accuracy the detected species, as reported by bootstrap values. The total average of dissimilarities among all phylotypes considered was 7.04%. The two major clades included _Saccharomyces_ and _Kazachstania_ species, supported by a bootstrap value of 98%. A total of four isolates, coming from sample B (LFB1, LFB10, LIB7, and LMB9), were grouped in _S. cerevisiae_ clade, having a percentage of identity of 100% with _S. cerevisiae_ CBS 1171T. The remaining _Saccharomyces_ species were grouped in three different clades, of which one includes _S. uvarum_ CBS 395T and the hybrid species from _S. uvarum_, _S. bayanus_ CBS 380T. and _S. pastoriamus_ CBS 1538T. Another cluster of _Saccharomyces_ species includes _S. kudriavzevii_ CBS 8840T and _S. arboricolus_ CBS 10644T, which share 99.2% of sequence identittt. The remaining clade of _Saccharomyces_ includes _S. carioanus_ CBS 8841T, _S. paradoxus_ CBS 432T, _S. jurei_ NCYC D5088T, and _S. mikatae_ CBS 8839T. Species included in this clade were phylogenetically closest, except for _S. mikatae_, sharing a dissimilarity percentage of about 1% with the other clade members. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Sample** & **Strains * & **Size of Amplicon** & **Amplicons Digestion** \\  & & **(bp)** & **with** _Hae_**III (Size Fragments, bp)** \\ \hline \multirow{3}{*}{MA} & LMA2, **LMA3 (UMCC 2973)**, LMA1, LMA4, **LMA5** & & \\  & **(UMCC 2974)**, LMA6, LMA7, LMA8, **LMA9 (UMCC & 630 & 400–230 \\  & **2975)**, LMA10 & & \\ \hline IMPA & **LIA1 (UMCC 2976)**, LIA9, LIA10, LIA2, LIA3, LIA4, & & \\  & **LIA5 (UMCC 2977)**, LIA7, **LIA6 (UMCC 2978)**, LIA8 & & \\ \hline \multirow{3}{*}{FINA} & **LFA1 (UMCC 2979)**, LFA2, **LFA3 (UMCC 2980)**, LFA4, & & \\  & LFA5, **LFA6 (UMCC 2981)**, LFA7, LFA8, LFA9, LFA10 & & \\ \hline \multirow{3}{*}{MB} & **LMB1 (UMCC 2982)**, LMB5 & 630 & 400–230 \\ \cline{2-5}  & LMB2, LMB3, LMB7, **LMB9 (UMCC 2984)**, LMB10 & 880 & 320–230–180–150 \\ \hline \multirow{3}{*}{IMPB} & LIB1, LIB4, **LIB7 (UMCC 2986)**, LIB10 & 880 & 320–230–180–150 \\ \cline{2-5}  & LIB2, LIB3, **LIB5 (UMCC 2985)**, LIB6, LIB8, LIB9 & 880 & 400–230 \\ \hline \multirow{3}{*}{FINNB} & **LFB1 (UMCC 2987)**, LFB2, LFB3, & & \\  & LFB4, LFB6, **LFB10 (UMCC 2988)** & 880 & 320–230–180–150 \\ \cline{2-5}  & LFB5, LFB7, LFB8, LFB9 & 630 & 400–230 \\ \hline \hline \end{tabular}  
* The strains sequenced are shown in bold and in brackets are indicated the correspondent UMCC code.  
\end{table} 
Table 3: RFLP-PCR of ITS-5.85 rDNA. Figure 2: Dendrogram obtained from unweighted pair group method with arithmetic mean (UPGMA) analysis, using Pearson’s coefficient, of the (GTG)\({}_{5}\) rep-PCR patterns. The similarity threshold for biotypes discrimination was 89% (blue line). The cophenetic coefficient is represented by numbers and dots colored as red, orange, yellow, or green, based on the branch quality. Two major clades are distinct with a threshold of 40% (red line). The _Kazachstania_ clade is branched out into three subclades. A clade including isolates coming from sample A and sample B was represented by _K. humilis_. Three isolates, represented by LMB1, LIB5, and LFA1, share 100% identity with _K. humilis_ CBS 5658T and _K. humilis_ H38. The remaining strains in the _K. humilis_ clade, including _C. milleri_ CBS 6897 (currently a synonym of _K. humilis_), are phylogenetically quite distant from _K. humilis_ CBS 5658T, which shares the 99.8% similarity, indicating a low divergence as also  
Figure 3: Neighbor-joining phylogenetic tree representing the phylogenetic distances of isolates from sourdough MA and MB and the strain types of identified yeasts species.