1 US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
2 Centre for Structural and Functional Genomics, Concordia University, Montreal, QC, Canada.
3 Departamento de Genética, University of Córdoba, 14071, Córdoba, Spain.
4 Los Alamos National Laboratory, Los Alamos, NM, USA.
5 HudsonAlpha Institute for Biotechnology, Huntsville, AL, USA.
6 National Microbiome Data Collaborative, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
7 Department of Human Genetics, McGill University, Montreal, QC, Canada.
8 Bioinformatics Research Center, North Carolina State University, Raleigh, NC, USA.
9 Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS, Aix Marseille Université, Marseille, France.
10 DTU Bioengineering, Technical University of Denmark, 2800 Kgs, Lyngby, Denmark.
11 Woods Hole Oceanographic Institution, Falmouth, MA, USA.
12 Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
13 Department of Biology, The University of New Mexico, Albuquerque, NM, USA.
14 Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Utrecht, the Netherlands.
15 Systems Design and Architecture, Sandia National Laboratories, Albuquerque, NM, 87123, USA.
16 US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. ivgrigoriev@lbl.gov.
17 Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA, USA. ivgrigoriev@lbl.gov.

Thermophily is a trait scattered across the fungal tree of life, with its highest prevalence within three fungal families (Chaetomiaceae, Thermoascaceae, and Trichocomaceae), as well as some members of the phylum Mucoromycota. We examined 37 thermophilic and thermotolerant species and 42 mesophilic species for this study and identified thermophily as the ancestral state of all three prominent families of thermophilic fungi. Thermophilic fungal genomes were found to encode various thermostable enzymes, including carbohydrate-active enzymes such as endoxylanases, which are useful for many industrial applications. At the same time, the overall gene counts, especially in gene families responsible for microbial defense such as secondary metabolism, are reduced in thermophiles compared to mesophiles. We also found a reduction in the core genome size of thermophiles in both the Chaetomiaceae family and the Eurotiomycetes class. The Gene Ontology terms lost in thermophilic fungi include primary metabolism, transporters, UV response, and O-methyltransferases. Comparative genomics analysis also revealed higher GC content in the third base of codons (GC3) and a lower effective number of codons in fungal thermophiles than in both thermotolerant and mesophilic fungi. Furthermore, using the Support Vector Machine classifier, we identified several Pfam domains capable of discriminating between genomes of thermophiles and mesophiles with 94% accuracy. Using AlphaFold2 to predict protein structures of endoxylanases (GH10), we built a similarity network based on the structures. We found that the number of disulfide bonds appears important for protein structure, and the network clusters based on protein structures correlate with the optimal activity temperature. Thus, comparative genomics offers new insights into the biology, adaptation, and evolutionary history of thermophilic fungi while providing a parts list for bioengineering applications.