The impact of anthropogenic disturbance on mycorrhizal fungi and their associations with rodents: insights from a temperate forest in Mexico

Ecosystem functioning is influenced by biological diversity, ecological interactions, abiotic conditions, and anthropogenic disturbance. This study examines how anthropogenic disturbance (i.e., land use change) affects arbuscular mycorrhizal fungi (AMF) and ectomycorrhizal fungi (EMF) in soil and rodent scat samples. We collected soil samples and rodent scats at five pairs of sites (i.e., anthropogenically disturbed vs. undisturbed temperate forests), in Michoacan, Mexico. Using DNA barcoding of partial internal transcribed region 1 (ITS) sequences, we identified 112 mycorrhizal fungi species. We found that there was a higher richness of EMF in undisturbed soil samples compared to disturbed soil samples and a higher AMF diversity in rodent scats for disturbed sites. Additionally, scat samples showed a high incidence of both AMF (75%) and EMF (100%) in rodent scats. However, we found significant differences in the diversity of both AMF and EMF depending on the rodent species acting as vectors. We also found a higher diversity of EMF in the wet season in the scat samples. Our study highlights the role of rodents as important vectors of mycorrhizal fungi, particularly for EMF, which could be essential to build up mycorrhizal fungi spore banks in disturbed forests.

Methods
This study was conducted in the municipality of Nuevo San Juan Parangaricutiro, Michoacan, Mexico. We selected ten sites in the area to assess the effects of anthropogenic disturbance (i.e., land use change) on mycorrhizal fungal communities and their associations with their putative rodent dispersers. We followed a paired design, where five of the sites were located under continuous forest cover (undisturbed) and the other five were in small patches that had undergone significant anthropogenic change (disturbed).

Soil samples were collected during the wet season (August 2022). Each sample was composed of ten soil cores collected with a sterilised PVC tube of 5 cm in diameter at a depth of 5 cm. For pine and shrub soil samples we selected five individual plants, spaced at least 20 m from each other, within the rodent trapping grid (see below). We then collected two soil cores at 30 cm to the base of each individual plant. For the bare soil samples, we collected a pair of soil cores along the closest access path, every 20 m until completing ten cores. Each collected sample was mixed in an ethanol-sterilised 2 L container for 2 minutes to homogenise the soil. Afterwards, a subsample of the homogenised soil was taken in a 1.5 mL Eppendorf tube.

Sampling for rodent scats was conducted in the dry (May 2022) and wet (August 2022) seasons. Pairs of disturbed and undisturbed sites were simultaneously sampled for two nights. We set up 50 Sherman traps at each sampling site in a 5 × 10 grid arrangement (spaced at 10 m). We completed a total sampling effort of 1,997 trap nights. We extracted DNA from all samples using the DNeasy PowerSoil Pro Kit (Qiagen®), following the manufacturer's protocol. DNA was dehydrated in a void vacuum to avoid degradation during shipping. The segment of the internal transcribed region, ITS1, was amplified using the primer set ITS1F and ITS1/2. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on an Illumina MiSeq following the manufacturer’s guidelines. The resulting DNA sequences were processed using the MR DNA analysis pipeline (MR DNA, Shallowater, TX, USA). All zOTUs were taxonomically classified using the Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI) Genbank online repository (www.ncbi.nlm.nih.gov). Only the species with a matching score >97% were included in further analyses. All the resulting species were classified by functional guild using the FUNGuild database (Nguyen et al. 2016) and the FUNGuildR R package (Furneaux and Song 2021; R Core Team 2024). We only included EMF and AMF taxa with a confidence ranking of “probable” or “highly probable”.