Retrotransposons are ubiquitously distributed in fungal species, albeit their structure is not conserved. The pol gene encodes a polyprotein composed of an aspartic protease, a reverse transcriptase, a ribonuclease H, and an integrase; the order and number of domains may differ between retrotransposon families, and most of the elements have incomplete pol genes. In addition, retrotransposons are generally characterized by the presence of LTR . As previously described , C. gattii retrotransposon sequences can be grouped into (i) LTRs with associated, but incomplete, internal regions; and (ii) partial internal regions with no identified LTRs.
The comparative analysis of C. gattii retrotransposons showed here revealed a large diversity of retrotransposons among diverse genotypes. It is noteworthy that the number of retrotransposons families number changes in a genotype-associated fashion. The VGI genotype strains possess the higher detectable retrotransposons and consequently the higher density compared to strains from VGII, VGIII and VGIV genotypes. This diversification of the number of retrotransposon families among different fungal strains is in agreement with previous reports comparing the prevalence of such mobile elements among closely related species and strains of Coccidioides immitis and Coccidioides posadasii , as well for Paracoccidioides spp. .
The genomic sequence of 16 C. gattii strains are available and their analysis described. Comparative genomic analysis revealed that chromosomal structure was very conserved among the four varieties (VGI, VGII, VGIII, and VGIV). Furthermore, this conservation is even more pronounced among VGII strains . As expected, our retrotransposon conservation analysis revealed the majority of these mobile elements present in R265 strain have orthologs in other C. gattii strains, most of them displaying also synteny.
Strains of the VGII genotype do not have a functional RNAi pathway, a highly conserved mechanism to control and inhibit retrotransposon expression through a homology-dependent gene-silencing complex . It is therefore feasible to assume that VGII strains do not have an active RNAi pathway and that increased retrotransposons numbers are a consequence of increased retrotransposon expression and mobility, as seen in RNAi-deficient mutant of C. neoformans . Unexpectedly, VGII strains displayed a smaller number of retrotransposon sequences compared to RNAi-proficient cryptococcal genotypes. Although R265 hypervirulence seems related to the absence of this regulatory mechanism, this observation suggests the existence of alternative control pathways or non-canonical Argonaute proteins, or the possibility that C. gattii (and not only VGII) has a more efficient (RNAi independent) system to control mobile element propagation. A predominance of LTR and DNA elements, LINEs, and low-complexity repeats in certain VGI strains was observed in C. gattii genotype analysis . Furthermore, AGO gene absence in R265 and other VGII strains was not associated with gaps in the R265 genome . However, it is unclear how far the incomplete assembly of genomes may alter the retrotransposon count. WM276 and NT10 have more retrotransposons and a well-assembled genome (WM276). Nevertheless, EJB2, E566, RU294, as both VGIII and VGIV strains, have RNAi pathways and exhibit similar sequence counts. The same explanation was suggested by Muszewska and colleagues  when analyzing differences in retrotransposon amounts between distinct strains of two Coccidioides species. Their findings further support that identified element amounts correlate with genomic sequences assembly lengths.
The retrotransposon expression analysis showed here revealed that all retrotransposons have detectable transcripts in distinct stress conditions. An uneven distribution of reads was detected among the C. gattii R265 transposons, with the large majority of reads matching one of the LTRs. This is in accordance to RNA-Seq assays using mRNA isolated from fission yeast .
To further evaluate retrotransposons expression, we performed a comparative transcriptome analysis using RNA-Seq data from C. gattii WT and zap1 mutant strains cultured under zinc restriction . Iron, copper, and zinc are essential metals for many processes in fungal pathogens. Zinc, specifically, is involved in transcriptional control, ROS detoxification, carbohydrate oxidation, and alcoholic fermentation [41, 42]. Thereby, in Cryptococcus, the ZIP family of plasma membrane transporters mediates zinc acquisition, especially in zinc-limiting conditions. The expression regulation of these transporter-encoding genes is performed by the transcriptional factor ZAP1. Our group previously demonstrated a severe impairment in ROS detoxification in cryptococcal cells lacking ZAP1 gene . This can be explained by the absence of indirect activation of many biological processes affected by zinc depletion in the zap1 mutant, since the complete removal of this metal harms all cellular functions of zinc-dependent metalloproteins. In this way, the comparison of WT and zap1 mutant cells allowed us to explore two distinct stress related conditions. However, our comparative analysis allowed the identification of only 2 stress-regulated retrotransposons.
Control of retrotransposon expression in fungal species is poorly characterized. Heterochromatin formation by H3mK9 histone methyltransferase recruitment in Schizosaccharomyces pombe is mediated by specific siRNAs acting in an Argonaute complex [43, 44]. Saccharomyces cerevisiae (which lacks the RNAi machinery for silencing mobile elements) produces antisense non-coding RNAs to regulate gene expression at the transcriptional level  and post-transcriptionally, as an auxiliary in copy number control mechanism. This maintains lower levels of mature integrase and reverse transcriptase, coupled with a truncated gag . At the transcriptional level, Ty1 antisense RNAs regulate Ty1 retrotransposon expression by alterations in chromatin function .
In C. neoformans, retrotransposon activity is controlled by the RNAi pathway . Stalled spliceosomes associate with a complex named SCANR, leading to the formation of siRNAs responsible for retrotransposon silencing via AGO dependent RNA degradation . As C. gattii VGII strains lack components of RNAi pathway, it is reasonable to assume that this strategy is not used by VGII strains to control retrotransposons activity. However, the detection of sRNAs that map to retrotransposon sequences with low expression claims that additional mechanisms for retrotransposon activity control may exist in C. gattii VGII strains. It is noteworthy that C. gattii R265 possess a functional DCR2 gene, which codes the enzyme responsible for the processing of siRNA precursors. In C. neoformans, Dcr2 was shown to be important for RNAi-mediated gene silencing . In addition, the C. gattii VGII genomes accumulate a large amount of lost genes  and a hypervirulent phenotype. The absence of a canonical RNAi pathway can be a major influence on this profile. Nevertheless, the production of sRNAs means that C. gattii R265 has a secondary, alternative retrotransposon silencing mechanism, which guarantees genetic variation without the deleterious invasion of parasitic elements.