Myxomycetes are multinucleate unicellular organisms. They form a plasmodium that moves by protoplasmic flow and prey on microorganisms. When encountering intraspecifics, the plasmodium has the capacity for ‘fusion’, actively approaching and fusing its cells, or 'avoidance', altering its direction to avoid the other individual. This is an allorecognition ability. However, it remains unclear whether the range of allorecognition extends to other species, and its ecological significance is also obscure. Here, we conduct a quantitative evaluation of contact responses from closely related species of plasmodium to clarify the recognition range of the allorecognition system in Myxomycetes. Behavioural assays demonstrate that the allorecognition system recognizes individuals within the same species while failing to recognize those of different species. The allorecognition is an extremely narrow and inward-focused mechanism, arguing for a highly specialized system of self-other recognition.

Behavioral tests to verify the allorecognition behavior of Myxomycetes plasmodia were conducted based on a previously established method (Masui et al., 2018). Two plasmodia were placed 3 mm apart on 2% agar medium and recorded by time-lapse photography every minute until the two individuals fused or two days had elapsed. The experimental environment was kept at 25°C, with no direct light exposure, and the samples were watered with sterile water to prevent the surface of the medium from drying out. Both Physarum rigidum and Physarum roseum were used in intraspecies and interspecies behavioral tests. In each behavioral test, an 'encounter' was defined as behavior in which plasmodia stopped moving within 3 mm of each other for at least 2 minutes or in which cell membranes made contact with each other, and was assessed using the following framework. Behavioral tests were qualitatively assessed using a tripartite framework comprising ‘fusion’, ‘avoidance’, and ‘no reaction (ignore)’. These indicators were meticulously documented and categorized up to the point of either ultimate fusion or total cellular separation. Encounter cases were classified using time-lapse photography. Fusion was defined as the merging of cells with each other. Avoidance was defined as ceasing movement or a change in direction of travel without fusion occurring during observation. If the cells did not exhibit either of these two behaviors and exhibited the behavior of riding over the other individual, it was classified as ‘no response (ignore)’.