Road disturbance shifts root fungal symbiont types and reduces the connectivity of plant-fungal co-occurrence networks in mountains

Roads are currently one of the most disruptive anthropogenic disturbances to mountain ecosystems worldwide. These disturbances can have a profound effect on roadside soil properties and vegetation, typically favouring fast-growing and ruderal species. However, their effect on plant-associated fungal communities and plant-fungal interactions remains largely unknown. In this study, we examined the changes in root-associated fungal communities as well as plant-fungal and fungal-fungal co-occurrence networks along mountain roads from four biogeographical regions. We found that roadsides consistently altered plant and fungal community composition, generally favouring arbuscular mycorrhizal fungi and putative plant pathogens at the expense of ectomycorrhizal fungi. Moreover, roadsides consistently reduced the complexity of plant-fungal and fungal-fungal co-occurrence networks (with 66% to 95% and 40% to 94% reduction in total edge density, respectively), even though the richness of fungal communities was not reduced and many of the naturally occurring highly-connected taxa were still present. Our findings suggest that altered and transient conditions in the roadsides may favour more generalist symbionts like AMF and pathogens with low fidelity for particular hosts as opposed to surrounding natural vegetation which is dominated by symbionts with higher specificity for the host (like ectomycorrhizal fungi). We conclude that road disturbance may have a consistent negative imprint on connectivity between plants and fungi; a consequence that deserves attention as it could render mountain roadside systems unstable and vulnerable to further pressures such as climate change and invasive species.

Methods
All sampling sites were located along mountain roads in four distinct and distant regions: Norway, Spain (Tenerife), Chile, and Argentina. The Norwegian sites were situated in a subarctic climate along three roads spanning elevations from 13 to 683 m a.s.l. (6-7 elevation points per road; 20 sites in total). Vegetation transitioned from birch forests with ericaceous understory at lower elevations to dwarf-shrub tundra at higher elevations. In Tenerife, sites were located along three paved roads from 24 to 2377 m a.s.l. (7-8 elevation points per road; 22 sites in total) on the southern slopes of the Teide volcano, where vegetation ranged from thermophilous scrubs at lower elevations to Canary pine forests and high mountain scrublands at higher elevations. Chilean sites were located along two paved roads from 378 to 1645 m a.s.l. (8 and 11 elevation points per road; 19 sites in total), with vegetation shifting from Mediterranean and temperate forests to resinous forests and scrublands at higher elevations. The Argentinean sites were distributed along three gravel roads spanning elevations from 1755 to 3782 m a.s.l. (6-7 elevation points per road; 20 sites in total), transitioning from shrublands and herbaceous steppes at lower elevations to sparse cushion shrub and herbaceous vegetation at higher elevations.

The experimental setup expanded on a global road survey initiative to monitor plant species composition along mountain roads. Each sampling site (81 in total) consisted of two large plots in a T-shape: one parallel to the road (2 m × 50 m) and the other perpendicular, extending from 2 to 102 m into the background vegetation. The parallel plots were designated as roadside (R) plots, and the perpendicular plots as background (B) plots. Within each plot, five subplots of 20 cm × 20 cm were selected, spaced approximately 10 m apart in the roadside plots and at increasing distances (5 m, 10 m, 20 m, 40 m, and 80 m) in the background plots. In Norway, due to logistical constraints, up to four subplots (10 m, 20 m, 40 m, and 80 m) were sampled instead of five. A total of 810 samples were expected (81 sites × 2 plots × 5 subplots), but due to occasional sampling challenges and failed sequencing, the final dataset included 673 samples with matching plant and fungal data.

Vegetation surveys were conducted during the peak growing season in 2017 for Norway, in 2019 for Tenerife, and in 2018-2019 for Argentina and Chile. Presence/absence and percentage cover of all plant species were recorded for each subplot. During surveys, root samples were collected from a random subset of plants in each subplot. Roots from all plants in a subplot were pooled, washed with ionized water, and stored in Ziplock bags at 4 °C before being shipped for analysis of root-associated fungi using Illumina amplicon sequencing (ITS1 region). Subplots were designed to ensure root sampling of most observed plant species, maximizing the likelihood of capturing interactions between plants and their associated fungi.

The dataset includes an OTU table of root-associated fungi per subplot together with taxonomy and lifestyles, plant community per subplot and metadata per subplot indicating region, latitude, longitude, mean annual temperature, elevation, distance from the road, etc.