*3.3. Phylogenetic Analysis of CYPs*

Phylogenetic analysis of CYPs was carried out following the procedure described elsewhere [36,53,56,57]. Briefly, first, the protein sequences were aligned by MAFFT v6.864 [58], and embedded on the T-REX web server [59]. Then, the alignments were automatically subjected to tree inferring and optimization by the T-REX web server. Finally, the best-inferred trees were visualized and colored by iTOL (http://itol.embl.de/about.cgi) [60].

#### *3.4. Generation of CYP Profile Heat-Maps*

The presence or absence of CYPs in species of *Tremellomycetes* was shown with heat-maps generated using CYP family data following the method described elsewhere [54,61]. The data were represented as −3 for gene absence (green) and 3 for gene presence (red). A tab-delimited file was imported into multi-experiment viewer (MeV) [62]. Hierarchical clustering using a Euclidean distance metric was used to cluster the data. Twenty-three species of *Tremellomycetes* form the horizontal axis and 38 CYP families form the vertical axis.

#### *3.5. CYP Diversity Percentage Analysis*

CYP diversity percentage analysis was carried out as described elsewhere [53,57,63,64]. Briefly, the CYP diversity percentage in species of *Tremellomycetes* was measured as a percentage contribution of the number of CYP families in the total number of CYPs.

#### *3.6. Functional Prediction of CYPs*

Literature was searched for characterized CYPs from species of *Tremellomycetes*, if any. Furthermore, functional prediction of CYPs was carried out based on the characterized homolog CYPs from different fungal organisms. CYP family level functional prediction was presented in the article.

#### **4. Conclusions**

Infections caused by human pathogenic species of *Tremellomycetes* are regarded as neglected diseases. Research on unraveling the infectious fungal pathogens' physiology and development of novel drugs against these pathogens is seldom done because of the lack of a lucrative market. However, cryptococcal meningitis remains a huge killer among people living with HIV in Sub-Saharan Africa and some of the species in the genus *Trichosporon* are now emerging human pathogens. This study's results provide insight into the CYP enzymes in the species of *Tremellomycetes*. This study revealed that cryptococcal species have almost 50% fewer CYP genes than their non-pathogenic counterparts and furthermore have the highest CYP diversity. Four CYP families were found to be conserved in pathogenic *Cryptococcus* species, indicating their important role in these pathogens. Interestingly, the CYP5139 family was bloomed with 17 CYP subfamilies in species of *Tremellomycetes*, indicating its possible key role in the physiology of these organisms. This study serves as a reference for

future annotation of CYPs and has opened new vistas for the characterization of CYPs in the species of *Tremellomycetes*.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/12/ 2889/s1.

**Author Contributions:** Conceptualization, K.S.; methodology, O.O.A., T.P., W.C., A.P.K., J.-H.Y., D.R.N. and K.S.; validation, O.O.A., T.P., W.C., A.P.K., J.-H.Y., D.R.N. and K.S.; formal analysis, O.O.A., T.P., W.C., A.P.K., J.-H.Y., D.R.N. and K.S.; investigation, O.O.A., T.P., W.C., A.P.K., J.-H.Y., D.R.N. and K.S.; resources, K.S. and A.P.K.; data curation, O.O.A., T.P., W.C., A.P.K., J.-H.Y., D.R.N. and K.S.; writing—original draft preparation, O.O.A., W.C., J.-H.Y., D.R.N. and K.S.; writing—K.S.; visualization, O.O.A., T.P., W.C., D.R.N. and K.S.; supervision, K.S.; project administration, K.S.; funding acquisition, A.P.K. and K.S.

**Funding:** Khajamohiddin Syed expresses sincere gratitude to the University of Zululand Research Committee for funding (Grant No. C686) and to the National Research Foundation (NRF), South Africa for a research grant (Grant No. 114159). Tiara Padayachee is grateful to the NRF, South Africa for an honors bursary. Abidemi Paul Kappo is grateful to the South African Medical Research Council (SAMRC) for a research grant (Grant No. PC57009).

**Acknowledgments:** The authors want to thank Laurie Connell, School of Marine Sciences, The University of Maine, Maine, USA and Joey Spatafora, Dept. Botany & Plant Pathology, Oregon State University, Oregon, USA for permission to use *Naganishia vishniacii* (formerly *Cryptococcus vishniacii*) and *Cryptococcus wieringae* genome data for CYP analysis. The authors also want to thank Barbara Bradley, Pretoria, South Africa for English language editing.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.
