Large inversions in Lake Malawi cichlids are associated with habitat preference, lineage, and sex determination

Reviewer #1 (Public review):

Summary:

Using high-quality genomic data (long-reads, optical maps, short-reads) and advanced bioinformatic analysis, the authors aimed to document chromosomal rearrangements across a recent radiation (Lake Malawi Cichlids). Working on 11 species, they achieved a high-resolution inversion detection and then investigated how inversions are distributed within populations (using a complementary dataset of short-reads), associated with sex, and shared or fixed among lineages. The history and ancestry of the inversions is also explored.

On one hand, I am very enthusiastic about the global finding (many inversions well-characterized in a highly diverse group!) and impressed by the amount of work put into this study. On the other hand, I have struggled so much to read the manuscript that I am unsure about how much the data supports some claims. I'm afraid most readers may feel the same and really need a deep reorganisation of the text, figures, and tables. I reckon this is difficult given the complexity brought by different inversions/different species/different datasets but it is highly needed to make this study accessible.

The methods of comparing optical maps, and looking at inversions at macro-evolutionary scales can be useful for the community. For cichlids, it is a first assessment that will allow further tests about the role of inversions in speciation and ecological specialisation. However, the current version of the manuscript is hardly accessible to non-specialists and the methods are not fully reproducible.

Strengths:

(1) Evidence for the presence of inversion is well-supported by optical mapping (very nice analysis and figure!).

(2) The link between sex determination and inversion in chr 10 in one species is very clearly demonstrated by the proportion in each sex and additional crosses. This section is also the easiest to read in the manuscript and I recommend trying to rewrite other result sections in the same way.

(3) A new high-quality reference genome is provided for Metriaclima zebra (and possibly other assemblies? - unclear).

(4) The sample size is great (31 individuals with optical maps if I understand well?).

(5) Ancestry at those inversions is explored with outgroups.

(6) Polymorphism for all inversions is quantified using a complementary dataset.

Weaknesses:

(1) Lack of clarity in the paper: As it currently reads, it is very hard to follow the different species, ecotypes, samples, inversions, etc. It would be useful to provide a phylogeny explicitly positioning the samples used for assembly and the habitat preference. Then the text would benefit from being organised either by variant or by subgroups rather than by successive steps of analysis.

(2) Lack of information for reproducibility: I couldn't find clearly the filters and parameters used for the different genomic analyses for example. This is just one example and I think the methods need to be re-worked to be reproducible. Including the codes inside the methods makes it hard to follow, so why not put the scripts in an indexed repository?

(3) Further confirmation of inversions and their breakpoints would be valuable. I don't understand why the long-reads (that were available and used for genome assembly) were not also used for SV detection and breakpoint refinement.

(4) Lack of statistical testing for the hypothesis of introgression: Although cichlids are known for high levels of hybridization, inversions can also remain balanced for a long time. what could allow us to differentiate introgression from incomplete lineage sorting?

(5) The sample size is unclear: possibly 31 for Bionano, 297 for short-reads, how many for long-reads or assemblies? How is this sample size split across species? This would deserve a table.

(6) Short read combines several datasets but batch effect is not tested.

(7) It is unclear how ancestry is determined because the synteny with outgroups is not shown.

(8) The level of polymorphism for the different inversions is difficult to interpret because it is unclear whether replicated are different species within an eco-group or different individuals from the same species. How could it be that homozygous references are so spread across the PCA? I guess the species-specific polymorphism is stronger than the ancestral order but in such a case, wouldn't it be worth re-doing the PCa on a subset?

Reviewer #2 (Public review):

Summary:

Chromosomal inversions have been predicted to play a role in adaptive evolution and speciation because of their ability to "lock" together adaptive alleles in genomic regions of low recombination. In this study, the authors use a combination of cutting-edge genomic methods, including BioNano and PacBio HiFi sequencing, to identify six large chromosomal inversions segregating in over 100 species of Lake Malawi cichlids, a classic example of adaptive radiation and rapid speciation. By examining the frequencies of these inversions present in species from six different linages, the authors show that there is an association between the presence of specific inversions with specific lineages/habitats. Using a combination of phylogenetic analyses and sequencing data, they demonstrate that three of the inversions have been introduced to one lineage via hybridization. Finally, genotyping of wild individuals as well as laboratory crosses suggests that three inversions are associated with XY sex determination systems in a subset of species. The data add to a growing number of systems in which inversions have been associated with adaptation to divergent environments. However, like most of the other recent studies in the field, this study does not go beyond describing the presence of the inversions to demonstrate that the inversions are under sexual or natural selection or that they contribute to adaptation or speciation in this system.

Strengths:

All analyses are very well done, and the conclusions about the presence of the six inversions in Lake Malawi cichlids, the frequencies of the inversions in different species, and the presence of three inversions in the benthic lineages due to hybridization are well-supported. Genotyping of 48 individuals resulting from laboratory crosses provides strong support that the chromosome 10 inversion is associated with a sex-determination locus.

Weaknesses:

The evidence supporting a role for the chromosome 11 inversion and the chromosome 9 inversion in sex determination is based on relatively few individuals and therefore remains suggestive. The authors are mostly cautious in their interpretations of the data. However, there are a few places where they state that the inversions are favored by selection, but they provide no evidence that this is the case and there is no consideration of alternative hypotheses (i.e. that the inversions might have been fixed via drift).

Reviewer #3 (Public review):

This is a very interesting paper bringing truly fascinating insight into the genomic processes underlying the famous adaptive radiation seen in cichlid fishes from Lake Malawi. The authors use structural and sequence information from species belonging to distinct ecotypic categories, representing subclades of the radiation, to document structural variation across the evolutionary tree, infer introgression of inversions among branches of the clade, and even suggest that certain rearrangements constitute new sex-determining loci. The insight is intriguing and is likely to make a substantial contribution to the field and to seed new hypotheses about the ecological processes and adaptive traits involved in this radiation.

I think the paper could be clarified in its prose, and that the discussion could be more informative regarding the putative roles of the inversions in adaptation to each ecotypic niche. Identifying key, large inversions shared in various ways across the different taxa is really a great step forward. However, the population genomics analysis requires further work to describe and decipher in a more systematic way the evolutionary forces at play and their consequences on the various inversions identified.

The model of evolution involving multiple inversions putatively linking together co-adapted "cassettes" could be better spelled out since it is not entirely clear how the existing theory on the recruitment of inversions in local adaptation (e.g. Kirkpatrick and Barton) operates on multiple unlinked inversions. How such loci correspond to distinct suites of integrated traits, or not, is not very easy to envision in the current state of the manuscript.

The role of one inversion in sex determination is apparent and truly intriguing. However, the implication of such locus on ecological adaptation is somewhat puzzling. Also, whether sex determination loci can flow across species via introgression seems quite important as a route to chromosomal sex determination, so this could be discussed further.

Author response:

We thank the reviewers for the careful review of our manuscript. Overall, they were positive about our use of cutting-edge methods to identify six inversions segregating in Lake Malawi. Their distribution in ~100 species of Lake Malawi species demonstrated that they were differentially segregating in different ecogroups/habitats and could potentially play a role in local adaptation, speciation, and sex determination. Reviewers were positive about our finding that the chromosome 10 inversion was associated with sex-determination in a deep benthic species and its potential role in regulating traits under sexual selection. They agree that this work is an important starting point in understanding the role of these inversions in the amazing phenotypic diversity found in the Lake Malawi cichlid flock.

There were two main criticisms that were made which we summarize:

(1) Lack of clarity. It was noted that the writing could be improved to make many technical points clearer. Additionally, certain discussion topics were not included that should be.

We will rewrite the text and add additional figures and tables to address the issues that were brought up in a point-by-point response. We will improve/include (1) the nomenclature to understand the inversions in different lineages, (2) improved descriptions for various genomic approaches, (3) a figure to document the samples and technologies used for each ecogroup, and 4) integration of LR sequences to identify inversion breakpoints to the finest resolution possible.

(2) We overstate the role that selection plays in the spread of these inversions and neglect other evolutionary processes that could be responsible for their spread.

We agree with the overarching point. We did not show that selection is involved in the spread of these inversions and other forces can be at play. Additionally, there were concerns with our model that the inversions introgressed from a Diplotaxodon ancestor into benthic ancestors and incomplete lineage sorting or balancing selection (via sex determination) could be at play. Overall, we agree with the reviewers with the following caveats. 1. Our analysis of the genetic distance between Diplotaxodons and benthic species in the inverted regions is more consistent with their spread through introgression versus incomplete lineage sorting or balancing selection. 2. This question of selection is much more complicated in the context of the Lake Malawi cichlid radiation with ~800 different species. We believe the role of these inversions must be considered in a species- and time-specific way. In other words, the evolutionary forces acting on these inversions at the time of their formation are likely different than the role of the evolutionary forces acting now. Further the role of these inversions is likely different in different species. For example, the inversion of 10 and 11 play a role in sex determination in some species but not others and the potential pressures acting on the inverted and non-inverted haplotypes will be very different. These are very interesting and important questions booth for understanding the adaptive radiations in Lake Malawi and in general, and we are actively studying crosses to understand the role of these inversions in phenotypic variation between two species. We will modify the text to make all of these points clearer.