## 4 Discussion  
Here, the molecular characterization at the strain level of _K. humilis_, _S. cerevisiae_ and _L. santrancisensis_ isolates occurring in PDO Tuscan bread sourdough was performed. Rep-PCR and inter-delta region analyses revealed the highest intraspecific diversity in _K. humilis_ and _S. cerevisiae_, respectively, while the combination of three RAPD-PCR analyses was the best discriminating method for _L. santrancisensis_. The DNA fingerprinting methods rep-PCR with (GTG)5 primer and RAPD-PCR with two different sets of primers, used to characterize _L. santrancisensis_ isolates, showed a high discriminatory power, particularly the two multiplex RAPD-PCR techniques (D \(\geq\) 0.95). RAPD-PCR with P4, P7 and M13 primers clustered the isolates in accordance with the polymorphism observed within 16S rRNA gene and revealed a large degree of intraspecific variability among _L. santrancisensis_ strains isolated from PDO Tuscan bread sourdough. Actually, the occurrence of only one LAB species, _L. santrancisensis_, may have boosted a marked intraspecific differentiation. Similar findings were found by [PERSON] et al. (2005), who, studying the LAB communities of 5 different sourdoughs, found _L. santrancisensis_ intraspecific diversity only in the sourdough where such microorganism was the only LAB species [19]. Concerning the yeast community of PDO Tuscan bread sourdough, the intraspecific characterization has been carried out comparing 6 different molecular methods. Inter-delta regions amplification resulted in the best discrimination tool for the typization of _S. cerevisiae_, differentiating two biotypes among the three _S. cerevisiae_ isolates. Similar results were obtained by [PERSON] et al. (2009), who used this method to investigate the intraspecific diversity of the _S. cerevisiae_ isolates characterizing the sourdough from the Marche region [20]. Also, [PERSON] et al. (2004) found a high degree of polymorphism among _S. cerevisiae_ isolated from Sicilian sourdoughs, using inter-delta region analysis [21]. Conversely, [PERSON] et al. (2012), studying _S. cerevisiae_ strains isolated from Legaccio and Panettone mother sponges, revealed only one inter-delta profile for each sourdough [22]. The rep-PCR with (GTG)5 primer method showed the best discriminatory index (0.95) for _K. humilis_. Cluster analyses grouped the 65 _K. humilis_ in 9 biotypes, revealing a high degree of intraspecific polymorphism. Accordingly, a high genetic diversity at the strain level within _C. milleri_ and _Candida humilis_ (reclassified as _K. humilis_) isolated from traditional Italian sourdoughs, using (GTG)5 microsatellite analyses was reported [11]. By contrast, a very low polymorphism among _C. milleri_ isolates of traditional sourdoughs was detected using MV1-RAPD PCR [22]. In conclusion, our work showed a high degree of genetic diversity among LAB and yeast strains of PDO Tuscan bread sourdough. Such highly complex microbial communities could be further studied for their functional traits conferring peculiar organoleptic, rheological and nutritional properties to PDO Tuscan bread. Actually, preliminary tests carried out on these isolates revealed the ability to solubilize phytate in 18 out of the 96 _L. santrancisensis_ strains, in the 3 _S. cerevisiae_ and in 3 out of 65 _K. humilis_ isolates, 50% of which showed also protease activity [12]. The molecular and functional characterization of PDO Tuscan bread sourdough microbiota will allow the selection of autochthonous Tuscan sourdough yeasts and LAB to be used as starters in the years to come. Conceptualization, M.P., M.G. and M.A. ; methodology, C.C. ; software, M.P. and C.C. ; validation, M.P. and M.A. ; writing--original draft preparation, M.P. ; writing--review and editing, M.A. and M.G. ; supervision, M.A. and M.G. ; funding acquisition, M.G. All authors have read and agreed to the published version of the manuscript.