Metagenomics Approach for Identification of Arbuscular Mycorrhizal Fungal Sequences in Historical Herbarium Specimens.

Metagenomics offers a powerful alternative to amplicon-based approaches for investigating arbuscular mycorrhizal fungi (AMF) in samples characterized by highly degraded DNA, such as historical herbarium specimens. Here, we present a dedicated bioinformatic pipeline for the retrieval and authentication of AMF sequences from shotgun metagenomic datasets generated from herbarium-associated soils. The workflow integrates quality control and reads preprocessing, taxonomic classification using Kraken2, targeted extrafction of AMF reads, and mapping to reference genomes using short-read-optimized alignment strategies. Authentication of historical DNA is achieved through the analysis of post-mortem damage patterns, including fragment length distributions and cytosine deamination profiles, using MapDamage2. This pipeline enables the detection of ancient AMF DNA, and it could provide a framework for studying long-term dynamics of plant-mycorrhizal associations from historical soil archives.