Mycelial decision to migrate

We have shown that when mycelia of P. velutina grew from inoculum wood blocks and colonised new larger bait wood resources, if the interconnection was subsequently severed, mycelium was often no longer able to grow out of the original inoculum. We did not attempt to reisolate the fungus from the original inoculum, so we cannot be certain whether the fungus had completely lost its viability within the original inoculum. However, the observations certainly suggest more or less complete migration of active mycelial resources from the inoculum to the bait.

As predicted, larger baits induced complete migration more frequently than small baits. Interestingly, the threshold volume of the bait that induced dramatic change in frequency of regrowth from original inoculum was somewhere between 4 and 16 cm3 regardless of the inoculum volume (which ranged from 0.5 to 4 cm3). This suggests that the primary factor affecting a mycelial decision to migrate completely to a new resource is actual volume of the new resource rather than the relative size of new resource to original inoculum, at least within the range of wood volume tested in the present study. This may seem counter-intuitive, as the mycelial outgrowth pattern was determined only by the nutritional status of the wood resources, because a larger inoculum contains a larger amount of carbon available to mycelium compared with a smaller inoculum [19, 24]. However, since a mycelium is an integrated system, coordinated resource allocation within a mycelium may explain this behavior. P. velutina mycelium tends to allocate more phosphorus to large wood resources than to smaller ones [19, 25], suggesting that there is a relatively larger nutritional cost for early colonisation of a larger resource than of a smaller one. This may also explain why mycelial migration from large inocula to the baits is determined by a relatively strict bait volume, whereas this was not the case with migration from small inocula. Given a larger cost to maintain a mycelial presence in large inocula than in smaller inocula, the decision to keep or discard a large inoculum after finding new large resources may be strongly determined by nutritional economy of the mycelium, whereas with small inocula the decision to keep or discard the original inoculum may be more stochastic.

Although decay rates of wood blocks were not measured in the present study, size-dependent wood decay rate may also affect the fungal decision of whether or not to migrate. Since decay rate of smaller wood particles is generally faster than larger ones [26], the more rapidly decreasing energy content of smaller wood blocks may cause the mycelium to completely migrate to new resources sooner than from larger ones. Thus, incubation periods longer than 48 days may alter the relationships between migration and wood size.

Microbial competitors in soil may also affect the decision of whether or not to migrate. Since the soil used in the microcosm was unsterilised, the focal fungi have to defend their wood blocks from a variety of microbial competitors in soil, which has an energetic cost. Smaller wood territory is more difficult to defend against mycelia occupying larger territory [27, 28], supporting our results showing that P. velutina mycelium left the smallest inoculum more often than large baits.

It is not clear why the mycelial decision to migrate depended on a certain range of bait size, but not on relative size of bait to inoculum. A possible reason is the limitation in maximum possible size of mycelium in the microcosms regardless of the volume of wood resources within [19, 29]. Since wood is relatively poor in mineral nutrients, e.g. nitrogen and phosphorus [30], most of the nutrients necessary for initial mycelial establishment within new woody resources will be translocated in the foraging mycelium, originating from soil, stored or recycled within resources [4, 19, 20, 25]. To maintain the carbon to nutrient ratio of mycelium, the amount of carbon source (wood block) available for a mycelium is determined by nutrient acquisition, which largely depends on the size of mycelium [4]. In this context, the threshold volume of a bait that would make a mycelium decide to migrate completely would change according to the size of microcosm, and must be larger in the field where P. velutina mycelium colonises larger coarse woody debris [6]. The small size of microcosms may also be the reason why the distance between inoculum and bait wood blocks did not affect the results in the present study. P. velutina is known as a ‘long-range forager’ [10], often forming cord networks extending over many meters [6, 12]. Cords of P. velutina can translocate phosphorus at least 75 cm within 5 days in the field [31] and probably very much further and faster, given carbon transfer to 18 cm distance from inoculum within 20 min in laboratory microcosms [29]. These effects of microcosm size and incubation time on fungal decisions should be tested in the future. Furthermore, relationships between fungal decision and sizes of inoculum and bait wood blocks should also be tested in more detail using wood blocks with a wider size range and narrower size intervals, because the size range of wood blocks were not evenly distributed in this study. Although the use of unsterilised soil provided a realistic scenario, the systems were much simplified with various stresses (such as fluctuating temperature and moisture) and disturbance agents (such as soil arthropods) prevented. These may also affect the nutritional economy of the mycelium and thus alter the migration threshold in natural ecosystems.

Mycelial memory of direction of growth

Reallocation of mycelial biomass and mycelial growth in the direction of the bait, as seen in period I, is in line with previous findings (reviewed in [10]). The completely novel finding is the dominant regrowth, in period II, from the inoculum side that had originally been joined to the newly colonised resource in period I. This is a kind of memory of mycelial systems for spatial navigation and is likely to be advantageous for quickly repairing damaged network connections, by regrowing towards self and growing in a direction where resources have been found to be plentiful. In other circumstances, exploring a new area and avoiding effort in a previously recently explored region, might increase the chance of finding new resources. With regard to mechanisms, larger and newer wood baits have a greater flux of nutrients towards them [25, 32, 33], probably attributable to the large metabolic demand of an invasive mycelium in newly-colonized wood [24]. Previous studies found that destructive disturbance of cord networks of P. velutina, removing several baits [34] or severing cords [35], caused polarised growth in the undisturbed direction. Such polarisation may be attributable to undisturbed hyphae forming a ‘dominant-sink’ for translocation within the mycelial system.

In the present study, it is not appropriate to say that the mycelium remembered the direction of the bait since the effects of bait itself and difference in soil area between bait-side and opposite-side could not be evaluated separately. Further, absence of a second control comprising systems without an added bait wood block did not allow us to completely evaluate the effects of bait wood blocks on the hyphal growth in period II. However, the design allowed us to confirm that mycelia had no preference in growth direction before addition of bait, and thus we can say that there was memory of the predominant direction in which the mycelium developed. Previous studies have categorised the biotic mechanisms of memory in organisms (or swarms) without (central) nervous systems into two types [36]: (1) external memory, which detects signals deposited in the environment; (2) somatic memory, achieved by storage of both epigenetic and/or non-genetic changes of cell physiology. An example of (1) is foraging plasmodia of slime moulds which avoid areas that have previously been explored by detecting deposited extracellular slime [17]. Foraging ants, on the other hand, use trace pheromones to attract (rather than repel) conspecific individuals to trails which allows sharing information about food, nest or mate location [37]. However, such external memory is not likely to be the case in the present study because the inocula were moved to completely fresh soil trays without any deposits from previous activity. Furthermore, previous disturbance studies without changing the soil tray showed no evidence of positive or negative effects of the area previously covered with mycelium [35].

Evidence for the possibility of (2), somatic memory, in fungi was provided in a recent study on Saccharomyces cerevisiae, which showed that epigenetic transcriptomic change in mother cells that had experienced environmental change could be transferred to daughter cells, which had not experienced the environmental change [38]. Further, non-genetic changes induced by the environment, such as chemical concentrations and bioelectricity within a single cell [39,40,41,42], or in networks across multiple cells in multicellular organisms [43], can also act to maintain memories of polarised growth or habituation if cells were stored after disturbance, dormancy, or regenerations. The third possibility for explaining preferential bait-side regrowth in the present study is a carry-over effect of differential distribution of mycelium within the inoculum wood block, without any physiological change in the mycelium with more mycelium in the inoculum on the side nearest the bait. It is also valid to consider this to be a part of a memory mechanism because the mechanisms of memory in the human brain includes this kind of non-physiological, non-epigenetic phenotypic level change in neuronal network structure [44]. However, although we appreciate that there may be semantic conflicts in the concept of non-neuronal memory among scientists as it is a novel and developing study field [36], we believe that recognizing such a carry-over effect as a kind of memory is a first step in the study of non-neuronal intelligence (in the words of Solé et al. [36] ‘liquid brain’) in a broader sense. Determining which of the above-mentioned mechanisms are involved in a mycelial memory in our system is the next experimental challenge.

It is interesting that larger inocula tended to remember their direction of growth better than smaller inocula in the present study. Previous studies on P. velutina also reported that mycelium growing from large (8 cm3) wood blocks showed stronger polarity in nutrient transfer [24] and growth [19] compared with mycelium growing from smaller wood blocks. However, the mechanisms of polarity and memory in fungal mycelium have been poorly explored and are a challenging topic for the future. Whatever the mechanisms involved in the memory of mycelium, the results presented here show that mycelium of P. velutina remembered its growth direction after the complete removal of outgrowing hyphae from wood inocula. Recognizing that fungal mycelium has a primitive intelligence with decision-making ability and memory is an important step towards understanding mycelial foraging behaviour, with consequences for carbon and nutrient dynamics on the forest floor.