Summary: High‐throughput molecular studies of museum specimens (museomics) have great potential in biodiversity research, but fungal historical collections have scarcely been examined, leading to no comprehensive methodological assessments. Here we present a whole genome sequencing (WGS) project conducted at the Fungarium of the Royal Botanic Gardens, Kew. DNA was extracted from 2104 specimens collected between 1770 and 2023, and we found that the specimen age had the smallest effect, while DNA purification and taxonomic identity had the greatest effect on DNA yield. We barcoded 771 specimens, and WGS was conducted on a subset of 394 specimens that were empirically selected for in‐depth analysis. We developed an automated assembly pipeline, integrating 16 different approaches. Starting from 220 libraries (excluding lichenised specimens), we produced 3143 assemblies using these approaches and found that there is no universal assembly method that can provide good‐quality genomes for all; rather, different approaches should be used depending on the library size and the specimen's age. Producing high‐quality genomes from specimens over 100 yr old is possible by using customised DNA extraction protocols and applying a multimethod bioinformatic approach. Whole genomes from historical collections will enrich genomics resources, accelerating biodiversity and evolutionary research, amongst others.

This study revises the genus Cyanoboletus (Boletaceae) in the Mediterranean Basin, integrating single-locus and multi-locus phylogenetic analyses (ITS, LSU, tef1-α, and rpb2), morphological characterisation, ecology, and arsenic accumulation in basidiomes. Morphological descriptions (including a new form, Cyanoboletus mediterraneensis f. pallidus), comprehensive sampling, type studies, biogeography, macro- and microphotographs, an identification key, and a historical overview of the nomenclatural issues surrounding C. pulverulentus, C. poikilochromus, and C. mediterraneensis are given. An epitype collection is designated for C. pulverulentus. A new method to measure spore suprahilar depression has been proposed, which allowed more clear morphological separation between C. mediterraneensis and C. pulverulentus. This method may prove useful for species delimitation in other fungal groups that have asymmetric basidiospores. Additionally, we generated a new ITS sequence of the C. sinopulverulentus holotype and inferred its conspecificity with the later described C. flavocontextus. Furthermore, notes on the taxonomy of Boletus gabretae are presented, and its placement in the genus Neoboletus is suggested. Cyanoboletus is confirmed as a strongly supported generic clade encompassing 21 monophyletic species-level clades, 14 of which represent known species, and seven are undescribed taxa. The synonymy of Cupreoboletus with Cyanoboletus is also verified. This publication provides the tools to delimit Cyanoboletus species that have important conservation value, which can be used by conservationists, ecologists, and citizen scientists. It also highlights species-specific arsenic hyperaccumulation in C. pulverulentus, contributing to a better understanding of fungal metal uptake. Our study indicates that within Cyanoboletus, only C. pulverulentus demonstrates this characteristic and is the only known member of Boletales that possesses a high arsenic accumulation ability.