Signatures of transposon-mediated genome inflation, host specialization, and photoentrainment in Entomophthora muscae and allied entomophthoralean fungi

Reviewer #1 (Public Review):

Summary:
The authors present a detailed study of a nearly complete Entomophthora muscae genome assembly and annotation, along with comparative analyses among related and non-related entomopathogenic fungi. The genome is one of the largest fungal genomes sequenced, and the authors document the proliferation and evolution of transposons and the presence/absence of related genetic machinery to explore how this may have occurred. There has also been an expansion in gene number, which appears to contain many "novel" genes unique to E. muscae. Functionally, the authors were interested in CAZymes, proteases, circadian clock related genes (due to entomopathogenicity/ host manipulation), other insect pathogen-specific genes, and secondary metabolites. There are many interesting findings including expansions in trahalases, unique insulinase, and another peptidase, and some evidence for RIP in Entomophthoralean fungi. The authors performed a separate study examining E. muscae species complex and related strains. Specifically, morphological traits were measured for strains and then compared to the 28S+ITS-based phylogeny, showing little informativeness of these morpho characters with high levels of overlap.

This work represents a big leap forward in the genomics of non-Dikarya fungi and large fungal genomes. Most of the gene homologs have been studied in species that diverged hundreds of millions of years ago, and therefore using standard comparative genomic approaches is not trivial and still relatively little is known. This paper provides many new hypotheses and potential avenues of research about fungal genome size expansion, entomopathogenesis in zygomycetes, and cellular functions like RIP and circadian mechanisms.

Strengths:
There are many strengths to this study. It represents a massive amount of work and a very thorough functional analysis of the gene content in these fungi (which are largely unsequenced and definitely understudied). Too often comparative genomic work will focus on one aspect and leave the reader wondering about all the other ways genome(s) are unique or different from others. This study really dove in and explored the relevant aspects of the E. muscae genome.

The authors used both a priori and emergent properties to shape their analyses (by searching for specific genes of interest and by analyzing genes underrepresented, expanded, or unique to their chosen taxa), enabling a detailed review of the genomic architecture and content. Specifically, I'm impressed by the analysis of missing genes (pFAMs) in E. muscae, none of which are enriched in relatives, suggesting this fungus is really different not by gene loss, but by its gene expansions.

Analyzing species-level boundaries and the data underlying those (genetic or morphological) is not something frequently presented in comparative genomic studies, however, here it is a welcome addition as the target species of the study is part of a species complex where morphology can be misleading and genetic data is infrequently collected in conjunction with the morphological data.

Weaknesses:
The conclusions of this paper are mostly well supported by data, but a few points should be clarified.

In the analysis of Orthogroups (OGs), the claim in the text is that E. muscae "has genes in multi-species OGs no more frequently than Enotomophaga maimaiga. (Fig. 3F)" I don't see that in 3F. But maybe I'm really missing something.

Also related, based on what is written in the text of the OG section, I think portions of Figure 3G are incorrect/ duplicated. First, a general question, related to the first two portions of the graph. How do "Genes assigned to an OG" and "Genes not assigned to an OG" not equal 100% for each species? The graph as currently visualized does not show that. Then I think the bars in portion 3 "Genes in species-specific OG" are wrong (because in the text it says "N. thromboides had just 16.3%" species-specific OGs, but the graph clearly shows that bar at around 50%. I think portion 3 is just a duplicate of the bars in portion 4 - they look exactly the same - and in addition, as stated in the text portion 4 "Potentially species-specific genes" should be the simple addition of the bars in portion 2 and portion 3 for each species.

In the introduction, there is a name for the phenomenon of "clinging to or biting the tops of plants," it's called summit disease. And just for some context for the readers, summit disease is well-documented in many of these taxa in the older literature, but it is often ignored in modern studies - even though it is a fascinating effect seen in many insect hosts, caused by many, many fungi, nematodes (!), etc. This phenomenon has evolved many times. Nice discussions of this in Evans 1989 and Roy et al. 2006 (both of whom cite much of the older literature).

Reviewer #2 (Public Review):

In their study, Stajich and co-authors present a new 1.03 Gb genome assembly for an isolate of the fungal insect parasite Entomophthora muscae (Entomophthoromycota phylum, isolated from Drosophila hydei). Many species of the Entomophthoromycota phylum are specialised insect pathogens with relatively large genomes for fungi, with interesting yet largely unexplored biology. The authors compare their new E. muscae assembly to those of other species in the Entomophthorales order and also more generally to other fungi. For that, they first focus on repetitive DNA (transposons) and show that Ty3 LTRs are highly abundant in the E. muscae genome and contribute to ~40% of the species' genome, a feature that is shared by closely related species in the Entomophthorales. Next, the authors describe the major differences in protein content between species in the genus, focusing on functional domains, namely protein families (pfam), carbohydrate-active enzymes, and peptidases. They highlight several protein families that are overrepresented/underrepresented in the E. muscae genome and other Entomophthorales genomes. The authors also highlight differences in components of the circadian rhythm, which might be relevant to the biology of these insect-infecting fungi. To gain further insights into E. muscae specificities, the authors identify orthologous proteins among four Entomophthorales species. Consistently with a larger genome and protein set in E. muscae, they find that 21% of the 17,111 orthogroups are specific to the species. To finish, the authors examine the consistency between methods for species delineation in the genus using molecular (ITS + 28S) or morphological data (# of nuclei per conidia + conidia size) and highlight major incongruences between the two.

Although most of the methods applied in the frame of this study are appropriate with the scripts made available, I believe there are some major discrepancies in the datasets that are compared which could undermine most of the results/conclusions. More precisely, most of the results are based on the comparison of protein family content between four Entomophthorales species. As the authors mention on page 5, genome (transcriptome) assembly and further annotation procedures can strongly influence gene discovery. Here, the authors re-annotated two assemblies using their own methods and recovered between 30 and 60% more genes than in the original dataset, but if I understand it correctly, they perform all downstream comparative analyses using the original annotations. Given the focus on E. muscae and the small sample size (four genomes compared), I believe performing the comparisons on the newly annotated assemblies would be more rigorous for making any claim on gene family variation.

The authors also investigate the putative impact of repeat-induced point mutation on the architecture of the large Entomophthorales genomes (for three of the eight species in Figure 1) and report low RIP-like dinucleotide signatures despite the presence of RID1 (a gene involved in the RIP process in Neurospora crassa) and RNAi machinery. They base their analysis on the presence of specific PFAM domains across the proteome of the three Entomophthorales species. In the case of RID1, the authors searched for a DNA methyltransferase domain (PF00145), however other proteins than RID1 bear such functional domain (DNMT family) so that in the current analysis it is impossible to say if the authors are actually looking at RID1 homologs (probably not, RID1 is monophyletic to the Ascomycota I believe). Similar comments apply to the analysis of components of the RNAi machinery. A more reliable alternative to the PFAM analysis would be to work with full protein sequences in addition to the functional domains.