Coprinellus comprises mushroom-forming saprotrophic fungi that colonize decomposing plant-derived substrates, yet chromosome-complete genomic resources for this genus remain limited. To resolve chromosome architecture and lineage-specific evolution in this fungal group, we generated chromosome-complete assemblies of Coprinellus xanthothrix and C. saccharinus by integrating Oxford Nanopore Technologies (ONT) long reads, DNA nanoball sequencing (DNBSEQ) short reads, Hi-C, and RNA-seq data. Each genome comprised 13 gapless chromosomes with all 26 telomeres recovered; assembly sizes were 46.92 and 54.81 Mb, with BUSCO completeness of 99.20% and 99.10%, respectively. The larger C. saccharinus genome was primarily associated with greater retroelement content, whereas functional annotation profiles and CAZyme repertoires were broadly comparable between species. Phylogenomic analyses grouped C. xanthothrix with C. radians and C. saccharinus with C. micaceus, with estimated divergence times of 54.4 and 32.8 Ma, respectively. Both divergence events occurred within the Paleogene. Gene family analyses identified lineage-specific expansions enriched in nucleotide metabolism, DNA replication and repair, glutathione metabolism, redox regulation, endocytosis, cytoskeletal organization, and cell-cycle processes. Synteny and Ks analyses further revealed chromosome-level rearrangements and lineage-specific small-scale duplication but no strong evidence of recent whole-genome duplication. Together, the temporal placement of these divergences and the associated genomic patterns raise a testable hypothesis that long-term climatic and vegetation reorganization, together with changes in plant-derived substrates and microhabitats, may have contributed to lineage establishment and ecological differentiation. However, because direct paleoecological evidence and functional validation are currently lacking, this proposed relationship should not be interpreted as causal. These chromosome-complete assemblies provide important resources for comparative genomics in Coprinellus and future experimental studies of saprotrophic adaptation.

Telomere-to-telomere (T2T) genomes provide a framework for resolving fungal chromosome architecture and lineage-specific evolution. We generated chromosome-complete assemblies of Coprinellus xanthothrix and C. saccharinus by integrating Oxford Nanopore, DNBSEQ, Hi-C, and RNA-seq data. Each genome comprised 13 gapless chromosomes with all 26 telomeres recovered; assembly sizes were 46.92 and 54.81 Mb, with BUSCO completeness of 99.20% and 99.10%, respectively. The larger C. saccharinus genome was primarily associated with greater retroelement content, whereas functional annotation profiles and CAZyme repertoires were broadly comparable between species. Phylogenomic analyses grouped C. xanthothrix with C. radians and C. saccharinus with C. micaceus, with estimated divergence times of 54.4 and 32.8 Ma, respectively. Both divergence events occurred within the Paleogene. Gene-family analyses identified lineage-specific expansions enriched in nucleotide metabolism, DNA replication and repair, glutathione metabolism, redox regulation, endocytosis, cytoskeletal organization, and cell-cycle processes. Synteny and Ks analyses further revealed chromosome-level rearrangements and lineage-specific small-scale duplication but no strong evidence of recent whole-genome duplication. Together, the temporal placement of these divergences and the associated genomic patterns raise a testable hypothesis that long-term climatic and vegetation reorganization, together with changes in plant-derived substrates and microhabitats, may have contributed to lineage establishment and ecological differentiation. However, because direct paleoecological evidence and functional validation are currently lacking, this proposed relationship should not be interpreted as causal. These chromosome-complete assemblies provide important resources for comparative genomics in Coprinellus and future experimental studies of saprotrophic adaptation.