Hybridization breaks species barriers in long-term coevolution of a cyanobacterial population

Reviewer #1 (Public Review):

Summary:

What are the overarching principles by which prokaryotic genomes evolve? This fundamental question motivates the investigations in this excellent piece of work. While it is still very common in this field to simply assume that prokaryotic genome evolution can be described by a standard model from mathematical population genetics, and fit the genomic data to such a model, a smaller group of researchers rightly insists that we should not have such preconceived ideas and instead try to carefully look at what the genomic data tell us about how prokaryotic genomes evolve. This is the approach taken by the authors of this work. Lacking a tight theoretical framework, the challenge of such approaches is to devise analysis methods that are robust to all our uncertainties about what the underlying evolutionary dynamics might be.

The authors here focus on a collection of ~300 single-cell genomes from a relatively well-isolated habitat with relatively simple species composition, i.e. cyanobacteria living in hotsprings in Yellowstone National Park, and convincingly demonstrate that the relative simplicity of this habitat increases our ability to interpret what the genomic data tells us about the evolutionary dynamics.

Using a very thorough and multi-faceted analysis of these data, the authors convincingly show that there are three main species of Synechococcus cyanobacteria living in this habitat, and that apart from very frequent recombination within each species (which is in line with insights from other recent studies) there is also a remarkably frequent occurrence of hybridization events between the different species, and with as of yet unidentified other genomes. Moreover, these hybridization events drive much of the diversity within each species. The authors also show convincing evidence that these hybridization events are not neutral but are driven by selected by natural selection.

Strengths:

The great strength of this paper is that, by not making any preconceived assumptions about what the evolutionary dynamics is expected to look like, but instead devising careful analysis methods to tease apart what the data tells us about what has happened in the evolution in these genomes, highly novel and unexpected results are obtained, i.e. the major role of hybridization across the 3 main species living in this habitat.

The analysis is very thorough and reading the detailed supplementary material it is clear that these authors took a lot of care in devising these methods and avoiding the pitfalls that unfortunately affect many other studies in this research area.

The picture of the evolutionary dynamics of these three Synechococcus species that emerge from this analysis is highly novel and surprising. I think this study is a major stepping stone toward the development of more realistic quantitative theories of genome evolution in prokaryotes.

The analysis methods that the authors employ are also partially novel and will no doubt be very valuable for analysis of many other datasets.

Weaknesses:

I feel the main weakness of this paper is that the presentation is structured such that it is extremely difficult to read. I feel readers have essentially no chance to understand the main text without first fully reading the 50-page supplement with methods and 31 supplementary materials. I think this will unfortunately strongly narrow the audience for this paper and below in the recommendations for the authors I make some suggestions as to how this might be improved.

A very interesting observation is that a lot of hybridization events (i.e. about half) originate from species other than the alpha, beta, and gamma Synechococcus species from which the genomes that are analyzed here derive. For this to occur, these other species must presumably also be living in the same habitat and must be relatively abundant. But if they are, why are they not being captured by the sampling? I did not see a clear explanation for this very common occurrence of hybridization events from outside of these Synechococcus species. The authors raise the possibility that these other species used to live in these hot springs but are now extinct. I'm not sure how plausible this is and wonder if there would be some way to find support for this in the data (e.g that one does not observe recent events of import from one of these unknown other species). This was one major finding that I believe went without a clear interpretation.

The core entities in the paper are groups of orthologous genes that show clear evidence of hybridization. It is thus very frustating that exactly the methods for identifying and classifying these hybridization events were really difficult to understand (sections I and V of the supplement). Even after several readings, I was unsure of exactly how orthogroups were classified, i.e. what the difference between M and X clusters is, what a `simple hybrid' corresponds to (as opposed to complex hybrids?), what precisely the definitions of singlet and non-singlet hybrids are, etcetera. It also seems that some numbers reported in the main text do not match what is shown in the supplement. For example, the main text talks about "around 80 genes with more than three clusters (SM, Sec. V; fig. S17).", but there is no group with around 80 genes shown in Fig S17! And similarly, it says "We found several dozen (100 in α and 84 in β) simple hybrid loci" and I also cannot match those numbers to what is shown in the supplement. I am convinced that what the authors did probably made sense. But as a reader, it is frustrating that when one tries to understand the results in detail, it is very difficult to understand what exactly is going on. I mention this example in detail because the hybrid classification is the core of this paper, but I had similar problems in other sections.

Although I generally was quite convinced by the methods and it was clear that the authors were doing a very thorough job, there were some instances where I did not understand the analysis. For example, the way orthogroups were built is very much along the lines used by many in the field (i.e. orthoMCL on the graph of pairwise matchings, building phylogenies of connected components of the graph, splitting the phylogenies along long branches). But then to subdivide orthogroups into clusters of different species, the authors did not use the phylogenetic tree already built but instead used an ad hoc pairwise hierarchical average linkage clustering algorithm.

Reviewer #2 (Public Review):

Summary:
Birzu et al. describe two sympatric hotspring cyanobacterial species ("alpha" and "beta") and infer recombination across the genome, including inter-species recombination events (hybridization) based on single-cell genome sequencing. The evidence for hybridization is strong and the authors took care to control for artefacts such as contamination during sequencing library preparation. Despite hybridization, the species remain genetically distinct from each other. The authors also present evidence for selective sweeps of genes across both species - a phenomenon which is widely observed for antibiotic resistance genes in pathogens, but rarely documented in environmental bacteria.

Strengths:
This manuscript describes some of the most thorough and convincing evidence to date of recombination happening within and between cohabitating bacteria in nature. Their single-cell sequencing approach allows them to sample the genetic diversity from two dominant species. Although single-cell genome sequences are incomplete, they contain much more information about genetic linkage than typical short-read shotgun metagenomes, enabling a reliable analysis of recombination. The authors also go to great lengths to quality-filter the single-cell sequencing data and to exclude contamination and read mismapping as major drivers of the signal of recombination.

Weaknesses:
Despite the very thorough and extensive analyses, many of the methods are bespoke and rely on reasonable but often arbitrary cutoffs (e.g. for defining gene sequence clusters etc.). Much of this is warranted, given the unique challenges of working with single-cell genome sequences, which are often quite fragmented and incomplete (30-70% of the genome covered). I think the challenges of working with this single-cell data should be addressed up-front in the main text, which would help justify the choices made for the analysis. The conclusions could also be strengthened by an analysis restricted to only a subset of the highest quality (>70% complete) genomes. Even if this results in a much smaller sample size, it could enable more standard phylogenetic methods to be applied, which could give meaningful support to the conclusions even if applied to just ~10 genomes or so from each species. By building phylogenetic trees, recombination events could be supported using bootstraps, which would add confidence to the gene sequence clustering-based analyses which rely on arbitrary cutoffs without explicit measures of support.

The manuscript closes without a cartoon (Figure 4) which outlines the broad evolutionary scenario supported by the data and analysis. I agree with the overall picture, but I do think that some of the temporal ordering of events, especially the timing of recombination events could be better supported by data. In particular, is there evidence that inter-species recombination events are increasing or decreasing over time? Are they currently at steady-state? This would help clarify whether a newly arrived species into the caldera experiences an initial burst of accepting DNA from already-present species (perhaps involving locally adaptive alleles), or whether recombination events are relatively constant over time. These questions could be answered by counting recombination events that occur deeper or more recently in a phylogenetic tree. The cartoon also shows a 'purple' species that is initially present, then donates some DNA to the 'blue' species before going extinct. In this model, 'purple' DNA should also be donated to the more recently arrived 'orange' species, in proportion to its frequency in the 'blue' genome. This is a relatively subtle detail, but it could be tested in the real data, and this may actually help discern the order of the inferred recombination events.

The abstract also makes a bold claim that is not well-supported by the data: "This widespread mixing is contrary to the prevailing view that ecological barriers can maintain cohesive bacterial species..." In fact, the two species are cohesive in the sense that they are identifiable based on clustering of genome-wide genetic diversity (as shown in Fig 1A). I agree that the mixing is 'widespread' in the sense that it occurs across the genome (as shown in Figure 2A) but it is clearly not sufficient to erode species boundaries. So I believe the data is consistent with a Biological Species Concept (sensu Bobay & Ochman, Genome Biology & Evolution 2017) that remains 'fuzzy' - such that there are still inter-species recombination events, just not sufficient to erode the cohesion of genomic clusters. Therefore, I think the data supports the emerging picture of most bacteria abiding by some version of a BSC, and is not particularly 'contrary' to the prevailing view.

The final Results paragraph begins by posing a question about epistatic interactions, but fails to provide a definitive answer to the extent of epistasis in these genomes. Quantifying epistatic effects in bacterial genomes is certainly of interest, but might be beyond the scope of this paper. This could be a Discussion point rather than an underdeveloped section of the Results.