## 3 Discussion

Table 3: Permutational multivariate analysis of variance applied to variables to compare the variance in the volatile profiles of _Malassezia_ spp. similar volatile profiles than _M. globosa_, according to the grouping given by multivariate analysis; however, the phylogenetic relationships show that _M. restricta_ is close to _M. globosa_ according to their genomes [6]. Since the growth times of _M. restricta_ and _M. sympodialis_ were the most similar, they likely influence the volatile profile, making these species more similar, sharing compounds such as 2-methylpropanal and butane-2,3-dione, which have also been identified in staphylococcal species [45]. Additionally, supervised and unsupervised multivariate analyses confirmed that the species and growth phase produced differences in the volatile profiles released by _M. globosa_, _M. restricta_, and _M. sympodialis_. Comparison of the volatile profiles of the _Malassezia_ species evaluated in this study with that previously reported for _M. furfur_[12] demonstrated similarities, and compounds such as carbon dioxide, butan-1-ol, pentan-2-one, 3-methylbutan-1-ol, 2-methylbutan-1-ol, hexan-1-ol, and heptyl acetate produced by _M. furfur_ were identified in all the species in this study. Additionally, the influence of the growth phase on the volatile profile was determined; for instance, compounds such as 2,5-dimethylhexane are produced by _M. furfur_ in the stationary phase when the medium is supplemented with palmitic acid, and this compound is identified in lower proportions in _M. globosa_ and _M. sympodialis_, which present difficulty in the assimilation of this fatty acid [6], suggesting that lipid metabolism affects VOC production. A similar observation was made for the volatile 2-methylpropan-1-ol produced by _M. globosa_, _M. restricta_, and _M. sympodialis_ in the stationary phase, with the highest peak corresponding to _M. furfur_, which produced the same compound in the same phase but in a lower proportion [12]. Other compounds that share all species, including _M. furfur_ only in the stationary phase, are 3-methylpentan-2-one and non-3-en-1-ol. These results confirm the statement that the stage of development is strongly implicated in VOC production. Volatiles are probably produced due to different pathways involved in processes related to a later stage of development. A previous study on volatiles in _Malassezia_ also identified 2-phenylethanol and hexan-1-ol despite the differences in conditions [33]. These results also indicate differences between volatile profiles; for instance, gamma lactones identified in a study in 1979 [33] were not identified in our study, probably because the experiments used different culture media, such as Littman agar and Sabouraud-destrose agar, and, as demonstrated by [PERSON] et al. 2019 [12], the type of culture medium used affects volatile production. Other differential conditions in that first study [33] were an incubation temperature of 37 \({}^{\circ}\)C in contrast to an incubation temperature of 33 \({}^{\circ}\)C in our study. Additionally, they supplemented the media with lecithin, oleic acid, and triolein, which probably affected the volatile profile considering that a recent study proved that volatile profiles are influenced by media supplementation [12]. Finally, the differences between the results of the study performed in 1979 with those of the study presented here performed in 2019 could be explained by the methodology employed since the sampling and identification through GC-MS are different between both studies [12; 33]. These findings suggest that volatile profiles depend on the natural variability of the species, the stage of development, and the experimental conditions.