Lievens et al. present an impressive body of work. The topic is interesting and the approach is sensible. Experimental evolutions studies can be challenging, and the effort here should be commended. I do however have some issues, listed below. Among those, I think the most important involve the interpretation of the results. First, if I understand correctly, the infectious exposure came from diverse stock, it then seems likely that any adaptation was selection on standing genetic variation rather than mutations. Second, I have trouble wrapping my head around the explanation that the absence of adaptation to experimental conditions represents strong trade-off, as opposed to simply a negative result.

Line-by-line comments

L66: on -> in L68: "in host use limit the evolution of specialization of two naturally occurring parasite" Previously talk about barriers to generalism. Here inverted. I get that these are two sides to the same coin, but good to be consitant to ensure readers follow. Similar around L78

L97: what is meant by suboptimal virulence? Too high or too low?

L102: Genetic diversity of parasite stocks unknown, but collected from various sites

parag around 115: italics for Latin names

L125: "we did not control the number of spores that were transmitted from one group of hosts to the next. Thus, the size of the inoculum and of the microsporidian population in all passages after P1 were dependent on the infection dynamics that developed within each replicate line." Is this a problem? different doses selection for what then?

L150 missing ref

L171: surviving and revived lines... what does that mean? passsage 10 vs 6? Figured it out later, but could do with clarifying first use of 'revived'

L181: only includes infected individuals in survival analysis. How many/what proportion were excluded because they were exposed but not infected?

Results

Pretty complicated results. Not unsurprisingly. Lots of stuff going on.

L252: A. rigaudi is most virulent when assayed on A. parthenogenetica if passaged on other host. Suggests adaptation to host sp. and perhaps suboptimal virulence on the other. This is an important result and a figure here would be useful. This is shown in Fig4 but figure not mentioned in text here.

Same for L274

L257: "but infected hosts did not die faster than controls" unclear here what is control. Presumably all were infected?

Discussion

L285-6: "infectivity readily evolved towards generalism" This doesn't appear true. E. aremiae did evolve greater infectivity on A.parthenogenetica but A. rigaudi did not. Similarly, "virulence played a minor role" doesn't seem quite true either as A. rigaudi evolved reduced virulence on A. parthenogenetica when tested on that host. Figure 4.

L286-288: "Our results are consistent with a strong trade-off acting on spore production and a weak trade-off on infectivity, and suggest that spore production is the key trait preventing the evolution of generalism in this system."

It is pushing it a bit to interpret no adaptation to the experimental evolution treatment as strong trade-off limiting the evolution of spore production.

Paragraph starting at L319: As genetic diversity of the initial infectious dose is unknown but taken from multiple source populations (L106) I would have thought that the most parsimonious explanation in the absence of genetic evidence is that there was selection upon standing genetic variation rather than accumulation of mutations of any flavour.

"It is worth noting that our conclusions would have been very different if we had not measured the parasites’ traits separately. Based on the overall fitness (Fig. 5), we would have concluded that A. rigaudi was unable to adapt to its mismatched host, while E. artemiae was able to evolve towards generalism after exposure to A. parthenogenetica. This would have suggested that the two parasites had asymmetrical fitness trade-offs between hosts: a strong trade-off for A. rigaudi, and a weaker trade-off for E. artemiae."

I certainly agree that measuring different components of parasite biology is informative. But I may have missed something here. This sounds precisely like the argument given by figure 5. That E. artemiae has weaker trade-offs, hence the blue arrow.

Fig4: gray is hard to make out. Also not obvious the star system. eg. stars over curly brackets, does this say that the values within this group are sig different from one another, or from teh other group in curly brackets. Looking at the figure I would have said within group but the values look too similar to be different from one another. I would recommend add square brackets to clarify that the stars refer to between host species.

Based on supplemental materials, there should be a strongly significant difference between virulence of E. artemiae in the different hosts (X=58.1, p<0.0001), but is not given in figure. Nor do the virulence values look very different in the different hosts.

Suppl

fig1: why so few evolved lines (dashed, hollow points, '+' signs?) - only three of the plots have one of those.

Lievens et al. provide here a study testing and discussing the evolution of niche specialization in a parasite, using an experimental evolution approach. After 10 generations of passaging separately two parasites on either their preferred host/occasional host/or both(alternatively between passages), they collect data on parasite spore production, infectivity of parasite (define as % of hosts infected during assay) and parasite virulence (defined as ratio of time until host death compared to control hosts) of the evolved lines on the two types of hosts.

The hosts species are A. franciscana and A. parthenogenetica - The parasites are microsporidia E. artemiae and A. rigaudi. The study shows that all 6 evolved lines maintained a higher fitness on their preferred host species. This results is mostly driven by a strong variation in spore production between parasite-host associations. Despite 10 generations of habituation to their occasional hosts A. franciscana , the A. rigaudi parasites evolved lines still produced as much spores as the A. rigaudi lines that have evolved on A. parthenogenetica, when both lines were put in contact with A. franciscana in a last essay. (And the other way around for E. artemiae evolved lines). The authors thus suggest that spore production is the barrier preventing the evolution of generalist strategy in these host/parasite systems. Additionally, E. artemiae evolved lines show variations in their fitness when put in contact to A. parthenogenetica in the last essay, while the three evolved lines show similar fitness. The authors here suggest that E. artemiae can evolve a more generalist strategy, while A. rigaudi can not.

In brief, the authors argue that a weak trade off between the ability of a parasite to successfully infect host1 and its ability to infect host2 could evolve a more generalist strategy. In contrast, if the trade offs are strong, the parasite is most likely to remain a specialist of host1. I believe such results might have important implications for the study of parasitism, highlighting potential constraints to their spread and evolution in natural environments.

I have little comments on this research article. I would simply ask the authors to more rapidly describe the type of trade-offs they are considering. I am an Ecologist and Evolutionary Biologist. My first idea of a trade-off is between two different traits pulling on opposite direction, here the trade-offs refer to one unique trait but pulled to it's opposite extremes in the two host environment. It might help if this is made clear from the abstract.

L146-150: This is not 100% informative. I would have prefer to have a clear explanation of when these tests were necessary and used; rather than have to guess when you actually used them.

Figure 2: I wonder if you could add information on which host species are each passage of the 'Alternating host' line.

Figure 4&5: to make it clear maybe, and stand alone figure; potentially add 'Evolved line of A.rigaudi' and 'evolved line of E. artemiae' at the top of figures. And to keep consistent with your text maybe 'Final assay host' at the bottom of your graph.

On L.248-249: You write: 'No infection was detected for the line---Replicate 1, so it was excluded from further analyses. This particular replicate is however included in Table 3, and is not counted as 'lost', Did I read this table wrongly?

Could you also discuss the fact that in the field, those parasites are not occurring on their own on those hosts, and competition between parasite is likely to influence each of the traits you have been studying here. Is there any data on whether one or the other parasite is more competitive? Or is there anything on other potential hosts or 'reservoirs' for these Microsporidians in their natural environments/regions where you collected them?

Thanks