Peer review of: A mechanistic explanation of the transition to simple multicellularity in fungi (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This is a beautifully written manuscript with excellent diagrams that clarify the text. The hypothesis 
that fungal hyphae explore and exhaust resources while transporting newly acquired resources (e.g., 
C, N, and P) to older part of the cytoplasmic community is not new, but it is very nicely extended here 
to explain (or at least hypothesize) how multicellularity evolved. I would note that the same logic can 
be applied to coenocytic algae that employ a "search" strategy to "explore" substrate resources. 

Reviewer #2 (Remarks to the Author): 

My name is Laszlo G. Nagy, a proponent of open review. Using a computational analysis, Heaton et al 
explain why a hyphal thallus is a best-fit solution in habitats where nutrient sources are spatially 
heterogeneous. I think the ms provides a much needed explanation for why fungi live where they live 
and why they are dominant microbes in several key processes, such as wood-decay and mycorrhizal 
associations. I really enjoyed reading the ms and I only have some smaller, discretionary comments 
for the authors. 

Main comments: 
I feel that mixed concepts exist for exoenzyme synthesis and transport in the ms. At some places it is 
assumed that enzymes are secreted by the tip and remain in place and active (l146), whereas in other 
places long-range transport of the enzymes themselves are assumed (l250-252). This latter feels 
problematic to me (see below). 

A cost for synthesising exoenzymes - I think this would be an important parameter for such a model, 
yet it seems like this wasn’t incorporated. See comment below and suggestion for additional analyses 
incorporating this parameter. 

I feel a small disconnect between multicellularity and what the authors are testing. Strictly speaking, 
their model explains why tubular structures like hyphae thrive in habitats where nutrient sources are 
spatially heterogeneous. It is clear that a hyphal structure is adaptive in this case, but the model 
doesn’t explain why a multicellular hypha is so. I realize it is hard to separate the emergence of 
multicellularity from the emergence of hyphae in fungi, but I feel that the results pertain to the tubular 
nature of hyphae and not necessarily to multicellularity and so some effort is in order. Maybe this 
could be mentioned in the discussion (coenocytic vs septate hyphae) and the title/abstract slightly 
reconsidered. This does not reduce the coolness of the study in my eyes, however. 

I feel the abstract is a bit technical and does not highlight the far-reaching implications of this work. I 
suggest mentioning how hyphae predisposed fungi for being mycorrhizal partner of plants too. 

Specific comments: 
l48 - might be worth noting that hyphae follow a fractal-like branching pattern, which confers 
additional advantages in exploring the substrate and avoiding conflict 
l55 - or even earlier than hyphal MC, in unicellular chytrids? See Chang et al 2015 GBE on pectinolytic 
capabilities of early fungi. 
l63 - “a carbon use efficiencies” 
Fig1 legend: synthesis —> synthesise 
l90 - remove ‘specific’ 
l121-134 - was a cost for synthesising exoenzymes built into the model? This sounds like an important 
parameter to me, with a potential to take a toll on how advantageous hyphal growth is. This might be 
worth adding to the model and running some additional analyses. Alternatively, the assumption of 
enzyme synthesis having a negligible cost should be mentioned here. Also, I suggest you mention the 

parameter for the cost of transport here (its only in the supplement now). 
l154-155 - I think you don’t need to assume that enzymes are only synthesised in the hyphal tip. 
l229-230 - note here that its because of the ability to transport assimilates. 
l248 - is there evidence for the transport of hydrolases (better written as exoenzymes - btw, please 
standardise terminology in the ms) 
l250 - delete ‘eukaryotic’ 
l250-252 - isn’t vesicle-enabled macromolecule transport restricted to cell wall components and the 
corresponding enzymes? Refs 30-32 point in this direction, rather than a useful long-term transport of 
assimilates or hydrolytic enzymes. 
l255 - ‘h yphal morphology inevitably results’ this sounds like a case of evolutionary determinism, 
which is usually suggested for phenotypes that emerged repeatedly several times. Maybe just say 
‘advantageous/adaptive’ 

Fig4 suggests that there are specific substrates where fungi have an advantage irrespective of being 
terrestrial or aquatic. This has two implications: 
- did this advantage drive the emergence of filamentous fungi in addition to the availability of 
terrestrial habitats? 
- if this is indeed the case, why dont we see more aquatic fungi dominating submerged complex 
polymers (wood)? 

I like that the presentation of the model precedes the results (even though, strictly speaking that 
would be the methods section), I hope the Editors will allow this sequence of chapters to be retained. 

An interesting experiment with this model would be to assess the extent of conflict between individual 
hyphae when their assimilation zones overlap. We speculated that the fractal-like filling of the space 
by the mycelium minimizes this conflict, but we did not test this experimentally. I realize this is out of 
scope for this paper, just dropping the idea here. 

Reviewer #3 (Remarks to the Author): 

Heaton et al report on computational modelling of nutrient uptake and growth for different types of 
cellular organization. The authors compare fungal hyphae with various modes of unicellular 
organization. The computational modelling supports the conclusion that hyphal organization is 
advantageous when C/N ratio is high and the substrate is recalcitrant. They argue that the hyphal 
advantage is based on a continuous cytoplasm which enables transport and efficient mining of 
resources over time. The idea that hyphal organization represents an optimal solution to breaking 
down recalcitrant substrates is a long held view in mycology and is not in itself conceptually original. 
However, the extent to which the results of modelling can be considered definitive make this a 
potentially important study. 

Major points. 
1. The forms of cellular organization analysed by modelling correspond to existing organisms. 
However, the origin of “autolytic cell” is unclear. Do such cell types exist, or is this form of 
organization included as a theoretical alternative? If this type of organization is absent in nature, it 
surely suggests that it is a poor strategy. Thus, it is difficult to understand how motile and autolytic 
strategies appear to perform similarly well under many conditions (Figure 4). The authors should 
address this and provide more context on these issues. 
2. The model of hyphae allows for vesicle transport throughout the entire mycelium. However, it is 
long known that multicellular fungal colonies possess a “peripheral growth zone” where hyphal 
compartments actively contribute to tip growth (Journal of General Microbiology (1971),67,325-344). 
Septa in the peripheral growth zone are unplugged, while older septa are plugged. Moreover, the 
proteins associated with the formation of these plugs have been identified 

(www.pnas.org/cgi/doi/10.1073/pnas.1207467109). Plugs preclude vesicle trafficking, and this leads 
to the question of how the results from modelling would be affected if the authors imposed a 
peripheral growth zone. The authors may wish to argue that this consideration is excluded for 
simplicity. In this event, they should still make reference to the peripheral growth zone and this form 
of pore plugging since they are wel-established aspects of hyphal colony organization. 
3. Page 10, lines 186-192: when “a unit volume captures less resource than is needed to grow a unit 
volume”, it would be reasonable to imagine that the connectivity of a hyphal network would give fungi 
an advantage. However, the model predicts that motile cells will dominate. Is this because the cost of 
vesicle trafficking is too high? Along similar lines, different costs of vesicle trafficking (β) are examined 
in Fig. 4 and Supplementary Fig. 2. However, κ is also changed (in Fig. 4: κ = 2, 4, 6 cell radii, and in 
Supplementary Fig. 2, κ = 3, 5 cell radii). If there is no reason for using different sets of values, 
please standardize the κ values to make it easier to compare these figures. 
4. Page 13, Fig. 5c: Why is digestion rate of motile cells > 0.3 g ml-1 hr-1 even when κ = 1? 
According to Fig. 3a, when κ = 1, the extracellular volume from where cell can obtain nutrients is 0. 
Given this, how is a digestion rate greater than 0 possible? Related to this point, it would be helpful if 
the authors explain why the digestion rate of motile cells is always greater than that of other cell 
types when κ=1. 
5. The discussion lacks specific references to the author’s figures, so it is difficult to follow and 
understand how the conclusions are drawn from their data. 
6. In the discussion, the distinction between “metabolic theory” and the “information-centric 
perspective” theories of evolutionary transitions are not likely to be familiar to a broad audience, so 
their relevance to the present study should be explained in greater depth. 

Minor points. 
1. Supplementary information, page 1, lines 12 to 17: the authors conclude that the overall C:N ratio 
required for growth is 6:1. However, if half of C is allocated to biosynthesis and half is lost through 
respiration and the cells C:N ratio is 12:1, then should not the overall C:N requirement for growth be 
24:1? 

2. Page 6, lines 112-113: apparent growth rate is defined as “volume colonised per unit time per unit 
volume colonised”. This is a bit confusing. By contrast the full definition from the supplements is 
better. I suggest using the definition from the supplement in the main text.