Uneven substrates constrain walking speed in ants through modulation of stride frequency more than stride length.

Natural terrain is rarely flat. Substrate irregularities challenge walking animals to maintain stability, yet we lack quantitative assessments of walking performance and limb kinematics on naturally uneven ground. We measured how continually uneven 3D-printed substrates influence walking performance of Argentine ants by measuring walking speeds of workers from laboratory colonies and by testing colony-wide substrate preference in field experiments. Tracking limb motion in over 8000 videos, we used statistical models that associate walking speed with limb kinematic parameters to compare movement over flat versus uneven ground of controlled dimensions. We found that uneven substrates reduced preferred and peak walking speeds by up to 42% and that ants actively avoided uneven terrain in the field. Observed speed reductions were modulated primarily by shifts in stride frequency instead of stride length (flat R 2: 0.91 versus 0.50), a pattern consistent across flat and uneven substrates. Mixed effect modelling revealed that walking speeds on uneven substrates were accurately predicted based on flat walking data for over 89% of strides. Those strides that were not well modelled primarily involved limb perturbations, including missteps, active foot repositioning and slipping. Together these findings relate kinematic mechanisms underlying walking performance on uneven terrain to ecologically relevant measures under field conditions.