No abstract

Supplementary table S1. Coordinates of centromeres and their GC content in M. sympodialis.Coordinates and length of Mtw1-enriched regions in comparison with that of the core centromeres in M. sympodialis. Chromosome numberCore centromere

15Centromeres are rapidly evolving across eukaryotes, despite performing a conserved 16 function to ensure high fidelity chromosome segregation. CENP-A chromatin is a hallmark of 17 a functional centromere in most organisms. Due to its critical role in kinetochore architecture, 18 the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric 19 chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal 20 kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with 21 the evolutionarily conserved CENP-C, but assembles a monocentric chromosome with a 22 localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved 23 kinetochore proteins were found to bind nine short overlapping regions, each comprising an 24 2 ~200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the 25 structure of budding yeast point centromeres. Resembling fungal regional centromeres, these 26 core centromere regions are embedded in large genomic expanses devoid of genes yet marked 27 by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi 28 pathway. Our results suggest that these hybrid features of point and regional centromeres arose 29 from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging 30 fungus. 31 Introduction 32Accurate chromosome segregation is crucial to maintain genome integrity during cell 33 division. The timely attachment of microtubules to centromere DNA is essential to achieve 34proper chromosome segregation. This is accomplished by a specialized multilayered protein 35 complex, the kinetochore which links microtubules to centromere DNA. This protein bridge is 36 divided into two layersthe inner and outer kinetochore. The fundamental inner kinetochore 37 protein is the histone H3 variant CENP-A. It binds directly to centromere DNA and lays the 38 foundation to recruit other essential proteins of the kinetochore complex, playing a fundamental 39 role in centromere structure and function, and hence, precise chromosome segregation 1,2 . 40 CENP-A is also found at all identified neocentromeres 3 and at the active centromeres of 41 dicentric chromosomes 4 , acting as an epigenetic determinant of centromeric identity. 42Despite its conserved function, the centromere is one of the most rapidly evolving 43 regions of the genome 5 . This so-called "centromere paradox" has led to centromeres of diverse 44 sizes and content. The first centromeres identified in Saccharomyces cerevisiae were found to 45 be point centromeres -small regions of ~120 bp defined by specific DNA sequences 6,7 . In 46 contrast to point centromeres described in only a few budding yeasts of the phylum 47 Ascomycota, most other fungi and metazoans have regional centromeres that are larger, 48 ranging from a few kilobases to several megabases 8 . Regional centromeres are often 49 3 interspersed with repetitive sequences and are mostly defined by epigenetic fac...

The thermotolerant multidrug-resistant ascomycete Candida auris rapidly emerged since 2009 causing systemic infections worldwide and simultaneously evolved in different geographical zones. The molecular events that orchestrated this sudden emergence of the killer fungus remain mostly elusive. Here, we identify centromeres in C. auris and related species, using a combined approach of chromatin immunoprecipitation and comparative genomic analyses. We find that C. auris and multiple other species in the Clavispora/Candida clade shared a conserved small regional GC-poor centromere landscape lacking pericentromeres or repeats. Further, a centromere inactivation event led to karyotypic alterations in this species complex. Interspecies genome analysis identified several structural chromosomal changes around centromeres. In addition, centromeres are found to be rapidly evolving loci among the different geographical clades of the same species of C. auris. Finally, we reveal an evolutionary trajectory of the unique karyotype associated with clade 2 that consists of the drug-susceptible isolates of C. auris. IMPORTANCE Candida auris, the killer fungus, emerged as different geographical clades, exhibiting multidrug resistance and high karyotype plasticity. Chromosomal rearrangements are known to play key roles in the emergence of new species, virulence, and drug resistance in pathogenic fungi. Centromeres, the genomic loci where microtubules attach to separate the sister chromatids during cell division, are known to be hot spots of breaks and downstream rearrangements. We identified the centromeres in C. auris and related species to study their involvement in the evolution and karyotype diversity reported in C. auris. We report conserved centromere features in 10 related species and trace the events that occurred at the centromeres during evolution. We reveal a centromere inactivation-mediated chromosome number change in these closely related species. We also observe that one of the geographical clades, the East Asian clade, evolved along a unique trajectory, compared to the other clades and related species.

34Genomic rearrangements associated with speciation often result in chromosome number 35 variation among closely related species. Malassezia species show variable karyotypes ranging 36 between 6 and 9 chromosomes. Here, we experimentally identified all 8 centromeres in M. 37 sympodialis as 3 to 5 kb long kinetochore-bound regions spanning an AT-rich core and depleted 38 of the canonical histone H3. Centromeres of similar sequence features were identified as CENP-39 A-rich regions in Malassezia furfur with 7 chromosomes, and histone H3 depleted regions in 40 Malassezia slooffiae and Malassezia globosa with 9 chromosomes each. Analysis of synteny 41 conservation across centromeres with newly generated chromosome-level genome assemblies 42 suggests two distinct mechanisms of chromosome number reduction from an inferred 9-43 chromosome ancestral state: (a) chromosome breakage followed by loss of centromere DNA 44 and (b) centromere inactivation accompanied by changes in DNA sequence following 45 chromosome-chromosome fusion. We propose AT-rich centromeres drive karyotype diversity in 46 the Malassezia species complex through breakage and inactivation. 47 107 5 that exhibit chromosome number variation. Malassezia species are lipid-dependent 108 basidiomycetous fungi that are naturally found as part of the animal skin microbiome (Theelen et 109 al. , 2018). The Malassezia genus presently includes 18 species divided into three clades -A, B, 110 and C. These species also have unusually compact genomes of less than 9 Mb, organized into 6 111 to 9 chromosomes as revealed by electrophoretic karyotyping of some of these species 112 (Boekhout and Bosboom, 1994, Boekhout et al., 1998, Wu et al., 2015. Fungemia-associated 113 species like Malassezia furfur belong to Clade A. Clade B includes common inhabitants of 114 human skin that are phylogenetically clustered into two subgroups namely Clade B1 that 115 contains Malassezia globosa and Malassezia restricta and Clade B2 that contains Malassezia 116 sympodialis and related species. Clade C includes Malassezia slooffiae and Malassezia cuniculi 117 which diverged earlier from a Malassezia common ancestor (Wu et al., 2015, Lorch et al., 2018). 118 Besides humans, Malassezia species have been detected on the skin of animals. For 119 example, M. slooffiae was isolated from cows and goats, M. equina from horses, M. brasiliensis 120 and M. psittaci from parrots, and a cold-tolerant species M. vesperilionis isolated from bats 121 (Lorch et al., 2018. Additionally, culture-independent studies of fungi from 122 environmental samples showed that the Malassezia species closely realted to those found on 123 human skin were also detected in diverse niches such as deep-sea vents, soil invertebrates, 124 hydrothermal vents, corals, and Antarctic soils (Amend, 2014). More than ten Malassezia species 125 have been detected as a part of the human skin microbiome (Findley and Grice, 2014). The 126 human skin commensals such as M. globosa, M. restricta, and M. sympodialis have been 127 associ...