## 2 Materials and Methods  
The _Neonectria lugdunensis_ strain was obtained from the internal collection of DSMZ (Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures) with the assigned identifier DSM 113088. Originally, this strain was derived from ascospores of the perithecal morph on a twig of an unknown plant species in Slovakia (GenBankak accession number of ITS sequence DQ24777) by [PERSON] and deposited in the Czech Collection of Microorganisms under CCM F-13783. The fungus was cultivated on agar plates containing 2% malt extract (Feelwell, Garrenburg, Germany) using Oxoid brand agar (Basingstoke, UK) at a temperature of 16 \({}^{\circ}\)C within a designated cooling facility for DNA extraction. To confirm the taxonomic assignation of the strain cultures, we employed Sanger sequencing of the beta tubulin marker gene. This was accomplished using the pair of primers T1 (5\({}^{\prime}\)-AAC-ATG-CGT-GAG-ATT-GTA-AGT-3\({}^{\prime}\)) and T2 (5\({}^{\prime}\)-TAG-TGAC-CCG-CCG-CAG-TTG-3\({}^{\prime}\)). The obtained sequences were manually curated using the Sequencer tool v5.4.6 ([[http://www.genecodes.com](http://www.genecodes.com)]([http://www.genecodes.com](http://www.genecodes.com)), accessed on 15 November 2023). Subsequently, a BLAST analysis was conducted against the National Center for Biotechnology Information (NCBI) Nucleotide Database ([[https://www.ncbi.nlm.nih.gov/](https://www.ncbi.nlm.nih.gov/)]([https://www.ncbi.nlm.nih.gov/](https://www.ncbi.nlm.nih.gov/)), accessed on 20 November 2023). After the taxonomic confirmation of the strain, the biomass was transferred to 1 litter flask each with potato-glucose liquid medium ([PERSON], Karlsruhe, Germany) and transferred to a 16 \({}^{\circ}\)C cooling room over a shaker (Bottmingen, Switzerland) at 120 RPM. The samples were kept in a shaker until sufficient biomass (14 g) for genome extraction was obtained (see Supplementary File). The genomic DNA, with fragment sizes greater than fifteen kilobases, verified with an Agilent Femto Pulse (Agilent, Santa Clara, CA, USA), was subjected to Hifi genome sequencing at Macrogen, the Netherlands. The raw sequence files were obtained from Macrogen using Flye v2.9 [57], and the quality was assessed with Busco v5.2.2 [58] using the ascomycota_odb10 database. For the comparative genomics of the _Nectriaceae_ family, we obtained 17 genomes of _Nectriaceae_ from the NCBI database (see Table 1). We aimed to select representative genomes of each genus at random but taking into consideration the lifestyle of each fungal species, and we added all genomes available for the _Neonectria_ genus. We also added 2 outgroup genomes for phylogenomics analysis, belonging to the _Ophicordycipitaceae_ family, which is phylogenetically closely related to _Nectriaceae_[36], and the newly assembled genome of _Neonectria lugdunensis_. The genomes were verified according to their genome completeness to ensure values higher than 95%, according to the results obtained with BUSCO and the ascomycota_odb10 database. An all-vs.-all genome-to-genome alignment and comparison analysis was performed using the DNADIFF program from MUMMER3 [59]. All genomes were aligned and compared against each other to obtain the average nucleotide identity (ANI), and genomes with similarity higher than 95% were considered to be of the same species [60]. The integrative method of RepeatModeler2 [61] was employed to generate a comprehensive _denovo_ species-specific repeat library for all genomes. The identification of new repeats by RepeatModeler v2.05 resulted in a library of consensus sequences for each species, combined with repeat annotation carried out using RepeatMasker v4.1.5 [62], via sequence comparison against the Dfam library, generating transposable element results, while soft-masking the genome. The soft-masked genome was used to predict the tRNAsequences determined by tRNAscan-SE 2.0.12 [63] with default parameters for eukaryotic organisms.