Domitille Jarrige

Sajeet Haridas

Claudine Bleykasten-Grosshans

Muriel Joly

Thierry Nadalig

Martine Sancelme

Stéphane Vuilleumier

Igor V Grigoriev

Pierre Amato

Françoise Bringel
email: francoise.bringel@unistra.fr

Affiliations

High quality genome of the basidiomycete yeast Dioszegia hungarica PDD-24b-2 isolated from cloud water

Keywords: Tremellaceae, mitochondrial genome, de novo sequencing, fungi, airborne microorganisms, transposable elements, Dioszegia hungarica strain PDD-24b-2, aeromicrobiology, fungal spore, cold environment

Introduction

There is increasing evidence that airborne microorganisms participate in chemical transformations and physical processes in the atmosphere [START_REF] Šantl-Temkiv | Microbial ecology of the atmosphere[END_REF]. In particular, microorganisms found in clouds play a central role in reactions of carbon-containing compounds at night, whereas during the day, photochemistry is dominant [START_REF] Vaïtilingom | Long-term features of cloud microbiology at the puy de Dôme (France)[END_REF][START_REF] Vaïtilingom | Potential impact of microbial activity on the oxidant capacity and organic carbon budget in clouds[END_REF]. Both prokaryotic and eukaryotic microorganisms can be found in clouds [START_REF] Delort | Clouds: a transient and stressing habitat for microorganisms[END_REF]. Regarding eukaryotes, 1-3% of sequenced 18S rDNA amplicons belong to the class Tremellomycetes of basidiomycete yeasts [START_REF] Amato | Active microorganisms thrive among extremely diverse communities in cloud water (G. Moreno-Hagelsieb[END_REF], which include the genus Dioszegia (Order:

Tremellales; Family: Tremellaceae/ Bulleribasidiaceae) [START_REF] Liu | Towards an integrated phylogenetic classification of the Tremellomycetes[END_REF]. Dioszegia hungarica strain PDD-24b-2 was isolated from cloud water collected at the summit of the puy de Dôme, France [START_REF] Vaïtilingom | Long-term features of cloud microbiology at the puy de Dôme (France)[END_REF] (Figure S1). Strains identified as Dioszegia sp. are frequently isolated from cloud water sampled at this site (in 70% of studied samples; [START_REF] Vaïtilingom | Long-term features of cloud microbiology at the puy de Dôme (France)[END_REF]. This fungal taxon was repeatedly identified in various cold environments, such as snow and glacial meltwater rivers [START_REF] De García | Biodiversity of cold-adapted yeasts from glacial meltwater rivers in Patagonia, Argentina: yeasts from Patagonian glacial waters[END_REF], and is also associated with plants in Antarctica [START_REF] Ferreira | Taxonomy and richness of yeasts associated with angiosperms, bryophytes, and meltwater biofilms collected in the Antarctic peninsula[END_REF]). The D. hungarica type strain CBS 4214 T was isolated from soil in Külsó-tó, Hungary as described in [START_REF] Takashima | Emendation of Dioszegia with redescription of Dioszegia hungarica and two new combinations, Dioszegia aurantiaca and Dioszegia crocea[END_REF]. Also found in warmer environments, it is part of the core fungal community of the wheat phyllosphere (the aerial parts of plants) [START_REF] Karlsson | Organic farming increases richness of fungal taxa in the wheat phyllosphere[END_REF][START_REF] Sapkota | Spatiotemporal variation and networks in the mycobiome of the wheat canopy[END_REF]. D. hungarica was identified as one of the few 'microbial hub taxa' that, when influenced by plant host and abiotic factors, act on the plant microbiome. For example, it directly inhibits the growth of specific bacterial taxa on Arabidopsis thaliana seedlings, thus decreasing the phyllosphere bacterial community diversity [START_REF] Agler | Microbial hub taxa link host and abiotic factors to plant microbiome variation[END_REF]). The atmospheric environment in which airborne microbes are found represents both a source (immigration) and a sink (emigration) for the phyllosphere microbiome [START_REF] Kinkel | Microbial population dynamics on leaves[END_REF]. Examining the genome of D. hungarica may provide valuable information to better understand the dynamics of fungal diversity, especially at the plant/atmosphere interface, and its role in climate change-relevant ecosystems (e.g., clouds, cold environments, phyllosphere).

Dioszegia hungarica, formerly classified as Cryptococcus hungaricus and Bullera armeniaca [START_REF] Takashima | Emendation of Dioszegia with redescription of Dioszegia hungarica and two new combinations, Dioszegia aurantiaca and Dioszegia crocea[END_REF], is one of the 23 species of Dioszegia identified so far [START_REF] Li | Diversity and phylogeny of basidiomycetous yeasts from plant leaves and soil: proposal of two new orders, three new families, eight new genera and one hundred and seven new species[END_REF]. To date, genomes of three other Dioszegia species have been sequenced: D. aurantiaca strain JCM 2956 and D. crocea strain JCM 2961, isolated from overwintered nettle stems of Urtica sp. and strawberry phyllosphere, respectively [START_REF] Takashima | Draft genome sequences of basidiomycetous epiphytic phylloplane yeast type strains Dioszegia crocea JCM 2961 and Dioszegia aurantiaca JCM[END_REF], and D. cryoxerica strain ANT03-071 (https://mycocosm.jgi.doe.gov/Diocr1), isolated from moss in Antarctica [START_REF] Connell | Dioszegia antarctica sp. nov. and Dioszegia cryoxerica sp. nov., psychrophilic basidiomycetous yeasts from polar desert soils in Antarctica[END_REF]. Previous analyses of the internal transcribed spacer and D1/D2 regions of the large subunit rRNA gene showed that D. hungarica is phylogenetically distant from these genome-sequenced representatives of the genus [START_REF] Trochine | Description of Dioszegia patagonica sp. nov., a novel carotenogenic yeast isolated from cold environments[END_REF][START_REF] Li | Diversity and phylogeny of basidiomycetous yeasts from plant leaves and soil: proposal of two new orders, three new families, eight new genera and one hundred and seven new species[END_REF]. This makes the species D. hungarica a good candidate to further investigate fungal genetic diversity. In this study, we describe the highquality assembled genome sequence of D. hungarica strain PDD-24b-2 obtained by a hybrid PacBio and Illumina sequencing strategy. The assembled genome features 17 contigs, 16 large-scale linear contigs and a smaller contig representing the complete circular mitochondrial genome.

Materials and Methods

Strain and growth conditions

Dioszegia hungarica strain PDD-24b-2 was isolated from cloud water collected at the summit of puy de Dôme, France on 17 January 2008 [START_REF] Vaïtilingom | Long-term features of cloud microbiology at the puy de Dôme (France)[END_REF]. R2A liquid medium was prepared as described previously [START_REF] Reasoner | A new medium for the enumeration and subculture of bacteria from potable water[END_REF]. Commercial dehydrated R2A agar (Oxoid, Hampshire, U. K.) was used as solid medium. Yeast mold (YM) medium (pH 6.2) contained per liter 3 g yeast extract, 3 g malt extract, 5 g peptone (pancreatic digest gelatin), 10 g D-glucose, and was supplemented with 20 g agar for solid medium. Liquid cultures were grown at 17°C with agitation (Sanyo MIR 254 refrigerated incubator, MA, USA). The ability to produce ballistospores was assessed on R2A solid medium, placing an inoculated Petri dish above a sterile one as described previously [START_REF] Ianiri | Phs1 and the synthesis of very long chain fatty acids are required for ballistospore formation[END_REF].

DNA extraction and PCR amplification

Total DNA was extracted from a 4-day aerobic culture (OD at 600 nm of 0.97) in 200 mL R2A medium incubated at 17°C, using the MasterPure TM complete DNA and RNA purification kit as described by the manufacturer (Lucigen, WI, USA). The 18S rRNA gene was PCR-amplified from total DNA (25 ng) using primers

Dios20F

(5'-GTGCGTCTGATTCTTGACTCC-3') and Dios11R

(5'-CCCGACCGTCCCTATTAATCA-3') and DreamTaq DNA polymerase, as recommended by the manufacturer (Thermo Fisher Scientific Baltics, Vilnius, Lithuania). The PCR program (Biometra TOne thermocycler, Analytik Jena, Jena, Germany) involved DNA denaturation at 95°C for 5 min, 30 cycles of 45 s at 93°C, 20 s at 56°C and 1 min at 72°C, and a final 10 min extension at 72°C. The amplified 1,080 bp PCR fragment was sequenced by the Sanger method (Microsynth France, Vaulx-en-Velin, France). [START_REF] Edman | Isolation of telomerelike sequences from Cryptococcus neoformans and their use in highefficiency transformation[END_REF], were searched and visualized in this assembly using IGV v 2.12.0 [START_REF] Robinson | Integrative genomics viewer[END_REF]. Completeness of the D. hungarica PDD-24b-2 genome assembly was assessed with BUSCO v5.2.2 [START_REF] Manni | BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes[END_REF]) against tremellomycetes_odb10, and compared to the 3 previously released genomes for the Dioszegia genus.

Genome sequencing, assembly and automatic annotation

The nuclear genome was deposited in the MycoCosm platform [START_REF] Grigoriev | MycoCosm portal: gearing up for 1000 fungal genomes[END_REF]) and automatically annotated, as previously described [START_REF] Kuo | Advances in Botanical Research[END_REF]) using the JGI Annotation Pipeline. The mitochondrial genome annotation pipeline combined ab initio predictions, homology-based predictions with a curated mitochondrial protein set, and HMM-based predictions, as described in [START_REF] Haridas | Fungal Genome Annotation[END_REF]). The KOG classification scheme was used to evaluate the number of genes associated with predicted processes with detailed gene ID available at https://mycocosm.jgi.doe.gov/cgi-bin/kogBrowser?type=KOG&db=Diohu1). The KEGG pathway database was used to identify metabolic pathway genes (https://mycocosm.jgi.doe.gov/cgibin/metapathways?db=Diohu1).

Identification of transposable elements

Putative transposable elements (TEs) were searched in the D. hungarica PDD-24b-2 genome sequence using two de novo approaches: the RepeatModeler v2.0.3 pipeline [START_REF] Flynn | RepeatModeler2 for automated genomic discovery of transposable element families[END_REF] with its LTR pipeline extensions, and the Extensive de novo TE Annotator (EDTA) pipeline [START_REF] Bell | Transposable element annotation in non model species: The benefits of species specific repeat libraries using semi automated EDTA and DeepTE de novo pipelines[END_REF]. TEs were also identified by protein homology with transposon sequences from other fungi (Saitozyma podzolica, Cryptococcus neoformans, Cryptococcus gattii, Rhodotorula toruloides, Candida glabrata) using BLAST+tblastx v2.11.0 [START_REF] Camacho | BLAST+: architecture and applications[END_REF]. Detected sequences were manually curated using CD Search (Marchler-Bauer and Bryant 2004;Marchler-Bauer et al. 2017) to predict conserved protein domains. Target Site Duplications (TSD) were identified by manually checking for direct repeats in sequences adjacent to identified TEs, and confirmed by surveying several copies. Detected putative TEs were classified into Orders and Superfamilies, as previously described [START_REF] Wicker | A unified classification system for eukaryotic transposable elements[END_REF]. Unique candidate TEs for each family, with typical TE domains (e.g.

transposase, reverse transcriptase, RNase H, integrase, aspartic protease, gag domains) were then used to build a putative TE library (File S1) to screen the genome sequence of D. hungarica PDD-24b-2 using RepeatMasker v4.1.2-p1 and estimate putative TE copy number, including full-length and truncated copies. The RepeatMasker.out file was parsed with the tool "One code to find them all" (Bailly-Bechet et al. 2014) to assemble detected TE fragments. More information on the TE-mining process can be found in the following GitHub repository: https://github.com/JarrigeD/Dioszegia_hungarica_sequencing

Phylogenetic analysis

The Internal Transcribed Spacer (ITS) region (ITS1, 5.8S ribosomal RNA gene, ITS2 and large subunit ribosomal RNA gene partial sequence) phylogenetic tree of the Dioszegia genus was constructed entirely with MEGA11 [START_REF] Tamura | MEGA11: Molecular Evolutionary Genetics Analysis version 11[END_REF]). An alignment of the ITS region was generated using MUSCLE [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]) on a total of 462 positions for D. hungarica PDD-24b-2, reference strains of the 23 Dioszegia species described to date, and 2 Hannaella type strains used as outgroup (Table S2). The MEGA11 "find best fit substitution model" tool was used to choose the substitution model for tree building. The General Time Reversible model with rate heterogeneity across sites (GTR+G) [START_REF] Tavaré | Some probabilistic and statistical problems in the analysis of DNA sequences[END_REF][START_REF] Yang | Among-site rate variation and its impact on phylogenetic analyses[END_REF]) had the lowest Bayesian information criterion score and corrected Akaike information criterion score and was used to calculate the matrix of pairwise distances. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1890)). A total of 500 replicate trees were built with the Maximum Likelihood method to calculate bootstrap support values, and the best tree topology with the highest log likelihood (-2379.73) was selected.

Geographical distribution

A total of 2,068 whole genome shotgun (WGS) raw read metagenomic datasets were hand-picked to maximize geographic and environmental variety, and retrieved from the Sequence Read Archive (SRA) using sra-tools.

The presence of D. hungarica sequences was tested using sra-tools blastn_vdb megablast, with strain PDD-24-filtered to target members of the genus Dioszegia (≥ 45 nt with ≥ 99% identity to the PDD-24b-2 5.8S rRNA gene sequence) and of the species D. hungarica (≥ 15 nt to ITS1 or ITS2 sequences, E-value ≤ 10e -10 ). These thresholds were defined using alignments of ITS regions of D. hungarica PDD-24b-2 to those of fungal type strains in the NCBI ITS_RefSeq_Fungi database.

Results and discussion

Cell morphology and growth characteristics

Single ovoid cells of approximately 4 µm in length, dividing by polar budding, were observed by optical microscopy (Figure 1A). D. hungarica strain PDD-24b-2 grows in R2A and YM media with the characteristic deep orange color typical of this genus [START_REF] Inacio | Phylloplane yeasts from Portugal: seven novel anamorphic species in the Tremellales lineage of the Hymenomycetes (Basidiomycota) producing orange-coloured colonies[END_REF], which becomes more pronounced at higher cell density (Figure 1B,1C). Growth in YM was faster than in R2A (Figure 1D), with incubation temperature strongly affecting growth (Figure 1D). The shortest doubling times were observed at 17°C in both YM (297 ± 5 min) and R2A (381 ± 33 min) media. Incubation at 4°C and 25°C resulted in 3-fold and 2-fold longer doubling times, respectively. No growth was observed at 30°C and 37°C after 2 weeks under all tested conditions (data not shown). This is in line with the temperatures of low-altitude clouds at the French site from which the strain was isolated, i.e. 5°C mean and 17°C maximal temperature, respectively [START_REF] Vaïtilingom | Contribution of microbial activity to carbon chemistry in clouds[END_REF]). In addition, strain PDD-24b-2 was able to produce ballistospores, a launched spore type specific to basidiomycetes, at 17°C on R2A solid medium after 6 days of culture. Ballistospores are able to launch from an inoculated plate to a neighbouring sterile one, on which colonies will grow following incubation, forming a "mirror" image of the inoculated plate (Figure 1E). Ballistosporic basidiospores have been proposed to act as giant cloud condensation nuclei that could increase precipitation by coalescing smaller droplets [START_REF] Hassett | Mushrooms as rainmakers: how spores act as nuclei for raindrops[END_REF]. Unlike the strain studied in this work, however, one of the D. hungarica strains isolated from terrestrial habitats was unable to produce ballistospores [START_REF] Takashima | Emendation of Dioszegia with redescription of Dioszegia hungarica and two new combinations, Dioszegia aurantiaca and Dioszegia crocea[END_REF], suggesting that this trait is not conserved within D. hungarica.

Genome sequencing, assembly and completeness

The genome of D. hungarica PDD-24b-2 was sequenced by a hybrid strategy using a combination of PacBio (average coverage of 97x, median subread size of 3,584 bp and 474,621 subreads in total) and Illumina (average coverage of 101x; 9,901,968 read pairs of 151 bp) sequencing, yielding a high-quality assembly. The 28 contigs assembled from PacBio subreads were corrected with the Illumina pair-end reads. BLASTn alignment analysis identified two identical contigs which were merged, and 9 small contigs nearly identical to larger ones (between 99.96% and 100% identity) which could represent alternative haplotypes and were thus discarded from the final assembly. This yielded a final genome assembly of 18 contigs, with 17 linear contigs corresponding to the nuclear genome. One contig was circular, as evidenced by more than 500 Illumina reads bridging its ends (data not shown), and corresponded to the mitochondrial genome. Its size of 27,226 bp was in close agreement with that estimated for the D. hungarica strain CBS 4214 T from average contour-length on electron micrographs twenty years ago (27.3 kbp, [START_REF] Gácser | Organization of mitochondrial DNA in the basidiomycetous Dioszegia hungarica (Cryptococcus hungaricus) species[END_REF].

After assembly, the beginning 5' third and the remaining 3' parts of the 18S rRNA gene were located at the termini of two nuclear contigs, i.e. the smallest contig of 2 kbp (contig20) and the contig of 1.11 Mbp (contig11, which also contained the remainder of the rRNA-encoding region). To confirm the linkage between contig20 and contig11, PCR amplification of the 18S rRNA gene was performed using primers targeting contig20 and contig11. The full 18S rRNA gene sequence including the 19 nt gap initially left out of the assembly was sequenced. Accordingly, contig11 was merged with the smaller contig20 to restore a complete 18S rRNA gene within the reunited rRNA region composed of 5S rRNA, 18S rRNA, 5.8S rRNA and 25S rRNA genes. Genome regions with rRNA genes are notoriously difficult to resolve in eukaryotic genomic assemblies, as rRNA genes can be found in tens to thousands tandem copies [START_REF] Nelson | Mechanisms of rDNA copy number maintenance[END_REF]. For instance, Cryptococcus neoformans, another basidiomycetous yeast, contains around 55 tandem repeats of a single rRNA gene region [START_REF] Loftus | The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans[END_REF][START_REF] Ganley | Highly efficient concerted evolution in the ribosomal DNA repeats: total rDNA repeat variation revealed by whole-genome shotgun sequence data[END_REF]. The rRNA gene copy number is usually estimated by relative read coverage [START_REF] Lofgren | Genome based estimates of fungal rDNA copy number variation across phylogenetic scales and ecological lifestyles[END_REF]. For strain PDD-24b-2, Illumina read depth coverage of contig20 was 35 times higher than for the rest of the genome (Figure S2). As expected, a similar increased coverage was also observed for the terminal part of contig11 in which the rRNA gene cluster is located. This suggests that ribosomal RNA genes are present in about 35 copies in D. hungarica PDD-24b-2, although the precise number of tandem repeats remains unknown. To estimate the length of the whole region containing copies of the rRNA gene cluster, we multiplied the rRNA unit length (10.29 kbp) by its relative coverage of 35. The estimated length of the complete contig11 would thus be approximately 1.46 Mbp (Figure 2).

For 7 contigs, T 2 AG 3-5 telomeric repeats were detected at one of the ends only (Figure 2), suggesting incomplete resolution of the nuclear genome. Nevertheless, the statistics and characteristics of the obtained final genome assembly of D. hungarica strain PDD-24b-2, with 16 large-scale contigs and a complete mitochondrial genome contig, compare favorably with previously reported genomes for the genus Dioszegia (Table 1). Specifically, and with an L50 value of 4 and a N50 length of 2.17 Mbp, the genome assembly of D. hungarica PDD-24b-2 contains no gaps, unlike the three previously sequenced genomes of Dioszegia strains (Table 1). In particular, the assembly of the D. cryoxerica ANT 3-071 T genome was strongly fragmented, with 111 scaffolds under 2 kbp, a L50 of 96 and a N50 of 0.12 Mbp. In the case of D. aurantiaca JCM 2956 T and D. crocea JCM 2961 T , some large and a few small (<2 kbp) scaffolds were reported.

The GC content of D. hungarica is about 57%, similar to that reported for D. cryoxerica, and higher than about 53% for D. aurantiaca and D. crocea. A detailed comparative assembly assessment of the 4 genomes using BUSCO v5.2.2 [START_REF] Manni | BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes[END_REF]) was performed using the tremellomycetes_odb10 database. The estimated occurrence of complete genes was similar for the 4 compared Dioszegia genomes, yet at about 89.5% instead of the expected 100%. This suggests a lineage specific bias for the Dioszegia genus of the reference tremellomycetes_odb10 database. A notable difference between Dioszegia genomes is the high percentage of duplicated genes reported for D. cryoxerica (53.4%), possibly reflecting unresolved haplotypes in the diploid assembly of its genome (https://mycocosm.jgi.doe.gov/Diocr1). This would also be consistent with the twice larger length of its assembly (39.5 Mpb) compared to the 3 other reported genomes including D. hungarica.

Genome annotation for protein-coding genes and predicted metabolic pathways

The obtained number of 8,219 predicted protein-coding genes is close to that reported for D. aurantiaca JCM 2956 T and D. crocea JCM 2961 T (Table 1), and also to the total number of unique protein-coding genes of D. cryoxerica ANT 3-071 T . The KOG classification scheme was used to evaluate the number of genes involved in cellular processes and signaling (1,301), information storage and processing (1,067), metabolism (1,460), and genes with unknown functions (1,056) (detailed gene ID available at https://mycocosm.jgi.doe.gov/cgibin/kogBrowser?type=KOG&db=Diohu1). The largest gene families include transporters from the Major Facilitator Superfamily (138) and sugar transporters (66), protein kinases (102), and clusters of genes with WD domain (100) and helicase-domain (75). Secondary metabolism is represented by three NRPS-like gene clusters and a single PKS-like gene cluster.

Gene predictions were analyzed in the light of experimentally characterized metabolic traits in D. hungarica [START_REF] Takashima | Emendation of Dioszegia with redescription of Dioszegia hungarica and two new combinations, Dioszegia aurantiaca and Dioszegia crocea[END_REF]. Genes for glycolysis/gluconeogenesis (39 genes), the TCA cycle (19 genes), starch utilization and production (61 genes), and nitrite utilization (1 nitrite reductase-encoding gene) (KOG classification within the MycoCosm plateform) reflect the previously reported utilization by D. hungarica of glucose, succinic and citric acid, starch, and nitrite, respectively. Conversely, no genes were predicted for methanol or nitrate utilization, or for thiamine biosynthesis, confirming the reported inability of D. hungarica to use methanol or nitrate, and its thiamine auxotrophy. Identified genes for carotenoid biosynthesis (36 putative genes, KEGG annotations, JGI Annotation Pipeline) are in line with previous reports of carotenoids in Dioszegia strains [START_REF] Madhour | Biosynthesis of the xanthophyll plectaniaxanthin as a stress response in the red yeast Dioszegia (Tremellales, Heterobasidiomycetes, Fungi)[END_REF][START_REF] Amaretti | Isolation of carotenoid-producing yeasts from an alpine glacier[END_REF][START_REF] Villarreal | Tolerance to ultraviolet radiation of psychrotolerant yeasts and analysis of their carotenoid, mycosporine, and ergosterol content[END_REF], and also with the bright orange color culture observed for D. hungarica PDD-24b-2 (Figure 1). Carotenoids prevent oxidative stress [START_REF] Madhour | Biosynthesis of the xanthophyll plectaniaxanthin as a stress response in the red yeast Dioszegia (Tremellales, Heterobasidiomycetes, Fungi)[END_REF]) and act as photo-protectants [START_REF] Moliné | Photoprotective role of carotenoids in yeasts: response to UV-B of pigmented and naturally-occurring albino strains[END_REF]) and cryo-protectants [START_REF] Dieser | Carotenoid pigmentation in Antarctic heterotrophic bacteria as a strategy to withstand environmental stresses[END_REF], and may thus favor survival under the harsh conditions of clouds [START_REF] Šantl-Temkiv | Microbial ecology of the atmosphere[END_REF]. In this context, strain PDD-24b-2 also encodes a putative antifreeze protein (protein ID: 32937, with a predicted ice-binding protein domain (InterPro entry: IPR021884) and a predicted secretion signal. Secreted antifreeze proteins impair ice crystal formation and protect cell integrity under cold conditions [START_REF] Hashim | Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12[END_REF], suggesting a role of this protein in cold protection of D. hungarica in the cloud environment that remains to be experimentally validated .

Transposable elements

A total of 311 putative sequences related to transposable elements (TEs) were detected, and classified in sixteen TE families (Table S1; [START_REF] Babu | The natural history of the WRKY-GCM1 zinc fingers and the relationship between transcription factors and transposons[END_REF]. The 10 bp long TSD supports an assignation to the Mutator Superfamily. Four families of non-autonomous Miniature Inverted-Repeat Transposable Elements (MITE) were also detected. One of them is related to the aforementioned Mutator element (same TIR and 10 bp long TSD). The others may be related to the hAT superfamily, according to their TSD length of 8 bp. However, we could not detect the corresponding autonomous copies encoding the transposases to confirm their annotation. In total, putative transposon-related sequences (around 130 kbp) represent 0.63% of the D. hungarica PDD-24b-2 genome, among the lowest so far for basidiomycete fungi [START_REF] Castanera | Biology, dynamics, and applications of transposable elements in basidiomycete fungi[END_REF]. However, reported TE contents are highly variable (ranging between 0.1 and 42 %), possibly also reflecting in part differences in sequence assembly and TE annotation protocols [START_REF] Castanera | Biology, dynamics, and applications of transposable elements in basidiomycete fungi[END_REF].

Circular mitochondrial genome

This study provides the first complete and circular mitochondrial genome for D. hungarica. Organization of the mitochondrial genome of strain PDD-24b-2 differs from that of other D. hungarica strains basing on previously reported physical maps [START_REF] Gácser | Organization of mitochondrial DNA in the basidiomycetous Dioszegia hungarica (Cryptococcus hungaricus) species[END_REF]. This is not unexpected as mitochondrial genome maps differed between D. hungarica strains.

The mitochondrial genome of strain PDD-24b-2 is smaller (27 kbp) than those of D. changbaiensis (35 kbp;

Tan and Wang, 2021) and D. cryoxerica ANT 03-071 T (36 kbp; L. B. Connell, personal communication) but of similar GC content (40-42%). The PDD-24b-2 mitochondrial genome contains all 15 known core protein-coding genes of mitochondria in Basidiomycetes, 23 tRNAs and 2 rRNAs (Table 2).

One major difference between the mitochondrial genome of D. hungarica and that of D. changbaiensis, the only other Dioszegia annotated mitochondrial genome to date, is the presence in D. hungarica of an additional tRNA gene, trnR(ucu) for arginine (Table 2). Although similar arginine codon usages are found in both strains, this is not the case for the aga and agg codons that are exclusively found in one of the mitochondrial genome. It is possible that this additional tRNA-Arg(ucu) in D. hungarica is used to translate the agg codon [START_REF] Agris | tRNA's wobble decoding of the genome: 40 years of modification[END_REF]). On the other hand, in the absence of tRNA-Arg(ucu) the translation of the aga codon in D. changbaiensis remains unexplained.

A noticeable similarity between the mitochondrial genome of D. hungarica and that of D. changbaiensis is a high cgu codon usage for arginine (16.2% of arginine codons for D. hungarica and 20.8% for D. changbaiensis) (Table 2). Thus, as this cgu codon cannot be canonically translated by either tRNA-Arg(ucg) or tRNA-Arg(ucu) without post-transcriptional modifications [START_REF] Phizicky | tRNA biology charges to the front[END_REF], experiments are needed to identify yet unknown modification processes and their roles in translation in D. hungarica and D. changbaiensis mitochondria.

Phylogenetic analysis and environmental distribution

A phylogenetic tree based on the analysis of the Internal Transcribed Spacer (ITS) region was constructed for 24 strains of the genus Dioszegia, including strain PDD-24b-2, with 2 strains of the genus Hannaella, as outgroups, using the Maximum Likelihood method (Table S2). In this tree, D. hungarica PDD-24b-2 and the D. hungarica type strain are clustered together and distinct from genome-sequenced strains of other Dioszegia species (Figure 3), in accordance with previous taxonomical studies [START_REF] Trochine | Description of Dioszegia patagonica sp. nov., a novel carotenogenic yeast isolated from cold environments[END_REF][START_REF] Li | Diversity and phylogeny of basidiomycetous yeasts from plant leaves and soil: proposal of two new orders, three new families, eight new genera and one hundred and seven new species[END_REF]).

Geographical distribution and potential habitat specificity of D. hungarica were investigated with a large set of public metagenomes selected to represent a wide diversity of environments, using the ITS region of strain PDD-24b-2 as a query (Figure 4A). D. hungarica was detected at diverse latitudes around the world (Figure 4B), and mostly in aerial biomes. In contrast, representatives of the Dioszegia genus were found to be more diversely distributed (Figure 4C). Strikingly, ITS sequences specific of D. hungarica were not detected in marine samples in our dataset of selected metagenomes, nor in the Mar and TARA Ocean Gene Atlas databases (no fungal hits with over 97% identity were found). This suggests that D. hungarica is scarce in open sea environments. On the other hand, D. hungarica sequences were not detected in soil metagenomes either (Figure 4C). This was surprising since the Dioszegia hungarica type strain was isolated from soil [START_REF] Takashima | Emendation of Dioszegia with redescription of Dioszegia hungarica and two new combinations, Dioszegia aurantiaca and Dioszegia crocea[END_REF]. However, when using the GlobalFungi database, which is a terrestrial soil-focused database, ITS sequences of D hungarica were detected in soil samples. Considering the significant differences in types of sequences between metagenomes (WGS, short raw reads) and the GlobalFungi database (targeted amplification of longer ITS sequences), the stringency of search parameters used in our analysis may contribute to explain this discrepancy, especially in environments with low abundance of D. hungarica communities. Nevertheless, the low occurrence of D. hungarica in oceans is somewhat paradoxical considering that strain PDD-24b-2 was isolated from a cloud of oceanic origin (Figure S1). We thus hypothesize that D. hungarica was picked up during air mass travel across France through the puy de Dôme sampling site. As such, the detection of D. hungarica in cloud water could serve as an indicator of air mass contact with terrestrial surfaces in future studies where detailed characterization of investigated cloud microbiomes is of interest.

In conclusion, the obtained high-quality assembled and annotated genome of the orange-pigmented psychrotrophic yeast D. hungarica PDD-24b-2, a major representative of the cloud microbiome, now provides a blueprint for future functional genomics analyses of this environmentally relevant fungus. This will help characterize its mechanisms of resistance to UV radiation [START_REF] Inacio | Phylloplane yeasts from Portugal: seven novel anamorphic species in the Tremellales lineage of the Hymenomycetes (Basidiomycota) producing orange-coloured colonies[END_REF] and of survival in cold environments [START_REF] Dalluge | Toward a rapid method for the study of biodiversity in cold environments: the characterization of psychrophilic yeasts by MALDI-TOF mass spectrometry[END_REF], contribute to develop yeast enzymatic processes at low temperatures [START_REF] Vaz | The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica[END_REF], and help to identify and characterize the biotic factors that play a role in cloud chemistry.

Tables Table 1: , atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, rps3 atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6 S3 for detailed information).

(C) Environmental distribution of D hungarica and Dioszegia sp. compared to that of the complete metagenomic dataset.

  File S1). TEs are dominated by Class I elements representing twelve families. Of those, seven families of Copia and one family of Gypsy Long Terminal Repeat (LTR) TE were found. Class I non-LTR elements putative families were distributed in three LINE families and one DIRS family. Four families of Class II Terminal Inverted Repeat (TIR) elements were also detected. Only one family encodes a transposase gene carrying a cl24015 domain attributed to MULE TE DDE transposases

Fig. 1 .

 1 Fig. 1. Morphological and growth characteristics of D. hungarica strain PDD-24b-2. (A) Cellular morphology observed with a Leica DM4000 B microscope at 1000x magnification after growth in YM broth at 17°C. (B) Colonies on R2A solid medium at 17°C. (C) Liquid cultures after growth at different temperatures for 4 days. Highest cell density was observed at 17°C. No growth was observed above 25°C or in sterile YM medium. (D) Effect of culture incubation temperatures on growth rate (µ). For each medium (YM or R2A), the mean growth rate was the mean of two biological replicates. (E) Ballistospore production on solid R2A medium. The inoculated plate (left) was placed on top of the uninoculated one (right). After 6 days of incubation, colonies also appeared on the bottom plate as a partial mirror image of the top inoculated plate.

Fig. 2 .

 2 Fig. 2. Telomeric sequences distribution in assembled contigs of D. hungarica PDD-24b-2. Detected telomeric T 2 AG 3-5 repeats are highlighted in orange (not drawn to scale). The complete ribosomal RNA gene region (with an estimated 35 copies of the ribosomal RNA gene cluster) is highlighted in blue. Identification labels for assembled contigs are given as in MycoCosm (https://mycocosm.jgi.doe.gov/Diohu1).

Fig. 3 .

 3 Fig. 3. Phylogenetic analysis of the Dioszegia genus based on the internal transcribed spacer (ITS) region. The tree was obtained from a sequence alignment of 462 nt of the ITS region with the Maximum Likelihood method and General Time Reversible model. Branch lengths (number of substitutions per site) are indicated in black, bootstrap support values (percentage of replicate trees in which the associated taxa clustered together in

Fig. 4 .

 4 Fig. 4. Geographical and environmental distribution of the Dioszegia genus and D. hungarica species. (A) Environmental metagenome exploration pipeline. The megablast hits filtering process is indicated with unfiltered hits (in black), filtered hits representing D. hungarica (in orange) or Dioszegia sp. sequences (in purple) mapped on D. hungarica PDD-24b-2 ITS region. (B) Geographical distribution of sequences assigned to D hungarica or Dioszegia sp. in environmental metagenomic datasets (see TableS3for detailed information).

  Libraries were sequenced using the MiSeq Illumina platform and the PacBio platform (SMRT cell Pacbio Sequel). Illumina and PacBio reads were quality checked with FastQC v0.11.9(Andrews, Braham

	Bioinformatics).
	Illumina adapter sequences were removed with CutAdapt v2.10 (Martin 2011) and paired-end reads cleaned with
	Prinseq v0.20.4 (Schmieder and Edwards 2011): the first 15 nucleotides of each read were cut, nucleotides with
	a Phred score under 30 were cut from the read 3' end, reads shorter than 60 nucleotides were discarded, reads
	with a mean Phred score under 30 were discarded, as well as those containing undetermined nucleotides. Only
	paired reads were conserved. After these processing steps 8,383,275 read pairs were obtained.
	PacBio subreads were assembled with Flye v2.8.2 (Kolmogorov et al. 2019). The cleaned Illumina read pairs
	were used to correct the PacBio assembly using BOWTIE2 v2.4.1 (Langmead and Salzberg 2012; Langmead et
	al. 2019) and Pilon v1.23 (Walker et al. 2014). Contigs were aligned to each other using BLASTn v2.10.1
	(Camacho et al. 2009) to resolve alternative haplotypes. Telomeric repeats of the sequence T 2 AG 3-5 , akin to
	those of Cryptococcus neoformans

Illumina library preparation (Nextera XT kit), PacBio library preparation (SMRTbell express template prep kit 2.0) and high throughput sequencing of D. hungarica PDD-24b-2 were performed by GenoScreen (Lille, France).

  Maps of WGS datasets with D. hungarica or Dioszegia sp. hits

	were plotted in Python v3.10.2 using Matplotlib v3.5.1 and GeoPandas v0.10.2. Details of metadata and dataset
	accessions, homolog search scripts, filtering parameters and mapping processes are available at
	https://github.com/JarrigeD/Dioszegia_hungarica_sequencing.
	The GlobalFungi database (accessible at https://globalfungi.com/) (17,000 ITS amplicons environmental
	samples) (Větrovský et al. 2020), the MarDB database (accessible at https://mmp.sfb.uit.no/blast/) (14,600

marine microbial genomes)

[START_REF] Klemetsen | The MAR databases: development and implementation of databases specific for marine metagenomics[END_REF][START_REF] Priyam | Sequenceserver: A modern graphical user interface for custom BLAST databases[END_REF]

and TARA Ocean Gene Atlas databases

[START_REF] Villar | The Ocean Gene Atlas: exploring the biogeography of plankton genes online[END_REF]

) (accessible at https://tara-oceans.mio.osupytheas.fr/ocean-gene-atlas) EUK_SMAGs (

713

eukaryotic plankton Metagenome Assembled Genomes: MAGs)

[START_REF] Delmont | Functional repertoire convergence of distantly related eukaryotic plankton lineages abundant in the sunlit ocean[END_REF]

), MATOUv1_metaG (116.8 million eukaryotic expressed genes + 530 Arctic Ocean MAGs)

[START_REF] Carradec | A global ocean atlas of eukaryotic genes[END_REF]

) and OM-RGC_v2_metaG (370 marine metagenomes + 530 MAGs)

[START_REF] Salazar | Gene expression changes and community turnover differentially shape the global ocean metatranscriptome[END_REF]

were also searched for close homologs of the ITS region of Dioszegia hungarica PDD-24b-2.

Genome assembly and predicted annotation data of D. hungarica PDD-24b-2 compared to the three other genome-sequenced Dioszegia sp.

	Content	D. hungarica PDD-24b-2	D. aurantiaca JCM 2956 T	D. crocea JCM 2961 T	D. cryoxerica ANT 03-071 T
	BioProject accession n°	PRJNA809585	PRJDB3721	PRJDB3718	PRJNA196046
	Reference	this study	Takashima et al., 2019 Takashima et al., 2019	(Connell L.B. personal communication)
	Sequencing and assembly a statistics
	Sequencing read coverage depth	97x (PacBio Sequel) +	112x (Illumina HiSeq	176x (Illumina HiSeq	98.5x (Illumina HiSeq
	(technology)	101x (Illumina MiSeq)	2500)	2500)	2500)
	Assembly size (Mbp)	20.96	19.34	20.60	39.52
	Scaffolds/contigs	18/18	52/139	26/86	865/1,318
	Longest scaffold (Mbp)	5.56	4.12	3.59	0.43
	L50 scaffold value	4	5	10	96
	N50 scaffold length (Mbp)	2.17	1.28	1.95	0.12
	Scaffolds over 2 kbp	17 b	44	21	754
	GC content (%)	57.2	53.6	53.2	56.9
	Gaps (%)	0.00	0.82	0.59	1.3
	Linear contigs	16 b	139	86	1.318
	Mitochondrial genome (kbp)	27 (circular)	NA c	NA	36
	Annotation statistics
	Gene models	8,219	8,106	8,753	15,948
	Average transcript length (bp)	1,538	1,817	1,801	1,415
	Average exon/intron length (bp)	247/67	258/59	259/61	264/61
	Average exons per gene	6.23	5.90	5.80	5.36
	Average protein length (aa)	513	507	500	429
	Genes with GO annotations	3,925	NA	NA	6,951
	BUSCO v5.2.2 assembly completeness assessment (%) d
	Complete	89.3	89.0	89.3	90.0
	Single	88.8	88.9	88.7	36.6
	Duplicated	0.5	0.1	0.6	53.4
	Fragmented	3.3	3.9	3.9	3.2
	Missing	7.4	7.1	6.8	6.8

a Haploid assemblies except for D. cryoxerica ANT 03-071 T which is probably diploid. b Scaffolds and linear contigs left after merging contig20 and contig11. c NA. Not Available.

d

BUSCO reference dataset: tremellomycetes_odb10 (2021-06-28)

.

Table 2 : Comparison of the mitochondrial genomes of D. hungarica and

 2

	Content	D. hungarica PDD-24b-2	D. changbaiensis CGMCC AS 2.2309 T
	Accession n°	JAKWFO000000000	MT755637
	Reference	this study	Tan and Wang, 2021
	General statistics
	Size (bp)	27,226	34,853
	GC content (%)	40.6	41.9
	Mitochondrial genome contents
	Protein coding genes	atp6

D. changbaiensis. The additional tRNA gene in D hungarica is underlined.

Acknowledgements

We thank Léa Eck for her help with DNA extraction and Amandine Moreno for physiological tests and microscopy photographs, Dr. Laurie B Connell for giving access to the D. cryoxerica ANT 03-071 T genome prior to publication, Dr. Joseph Schacherer for discussion of sequencing strategy and Prof. Hubert Becker for his help with tRNA analysis. This article is dedicated to Dr. Anne-Marie Delort, pioneer of investigations on the role of microorganisms in atmospheric chemistry, on the occasion of her retirement.

FB initiated the study. FB and DJ wrote the manuscript. FB, DJ and SV revised the manuscript. MJ and MS cultivated the strain under supervision of PA. FB performed wet lab experiments. SH performed genome annotation on the MycoCosm platform under supervision of IG. DJ performed the phylogenetic analysis and geographic distribution study. DJ performed the transposable element search under the guidance of CBG. TN contributed to discussion. All authors have read, edited and approved the final manuscript.

Funding

This study and the postdoctoral grant to DJ were funded by the French National Agency, grant ANR-19-CE01-0004-02, project METACLOUD. Genome annotation was performed by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, supported by the Office of Science of the U.S. Department of Energy under contract no. DE-AC02-05CH11231.

Data availability

The Dioszegia hungarica PDD-24b-2 Whole Genome Shotgun project was deposited at DDBJ/ENA/GenBank under accession number JAKWFO000000000. The genome version used in this report is JAKWFO010000000. The raw Illumina and PacBio reads were deposited at the Sequence Read Archive under accessions numbers SRR18177991 and SRR18177990 respectively. Details on genome assembly and gene model properties are provided on the MycoCosm genome portal (https://mycocosm.jgi.doe.gov/Diohu1). Strain Dioszegia hungarica PDD-24b-2 is available upon request to Dr. Pierre Amato or Dr. Françoise Bringel. Representative sequences of putative D. hungarica PDD-24b-2 TE families are in File S1. Putative TEs detected in D. hungarica PDD-24b-2 are in Table S1. ITS sequences used to construct the phylogenetic tree of the Dioszegia genus are in Table S2.

Environmental samples in which D. hungarica and Dioszegia species were searched are provided in Table S3.

The air mass trajectory of the cloud from which D. hungarica strain PDD-24b-2 was isolated is shown in Figure S1. A close-up of the Illumina read depth coverage of D. hungarica PDD-24b-2 rRNA gene region is provided in Figure S2. The homolog search scripts, environmental metagenome dataset, as well as more information on biogeographic analyses and TE mining are available at https://github.com/JarrigeD/Dioszegia_hungarica_sequencing.

Conflicts of interest

None Declared.