Interview with the authors: Genomic basis of Y‐linked dwarfism in cichlids pursuing alternative reproductive tactics

In a recent paper in Molecular Ecology, Singh et al. used genome sequencing, bioinformatics and population genetic analyses to gain insight into the genetics and evolution of a fascinating mating system. The species in question, Lamprologous callipterus, exhibits a mating system with two males morphs. Large “bourgeois” males carry empty snail shells that are inhabited and used as nests by the females. An alternate male morph, much smaller than the “bourgeois” males, also exists and inhabits shells along with the females. Previous genetic work had established that this mating system was Y-linked and that the male body size was a Mendelian trait, but the sex-determining locus had not been identified until this study.

We sent some questions to Pooja Singh, the author who led this work, to get more detail on this study.

Photo credit: Drawing by Pooja Singh, based on Barbara Taborsky’s original image.

What is the biggest or most surprising innovation highlighted in this study? 

The most novel aspect of this study is that we found an example of a young sex chromosome that may have evolved due to sexual antagonism over body size. While the sexual antagonism theory is considered the classical model of sex chromosome evolution, few empirical examples exist to support it. The other exciting finding was that the candidate body size/dwarfism gene that we propose for L. callipterus, GHRHR, is a well-known dwarfism gene in mammals. Fish and mammals shared a common ancestor over 440 million years ago, so the body size development pathway is genetically constrained through deep evolutionary time.

What difficulties did you run into along the way? 

The major challenge for me was that I knew little about sex chromosome evolution when I started this project, so I really had to do a lot of groundwork reading relevant literature and researching methods to be able to get things going. I had to start thinking beyond the classical XY and WZ old sex systems and familiarize myself with the workings of early stages of sex chromosome evolution.

When I read your paper, I had never heard of the fascinating mating system of these cichlids. They reminded me of the ruff, and the multiple inversions that seem to be involved in the different reproductive strategies in that system. You mention in the paper that you were not able to identify inversions based on the bioinformatic approaches you used. Is there a sense for how much chromosome evolution during the radiation? Could the use of the divergent reference genome have anything to do with the lack of a signal of inversions? 

To my knowledge the broad scale chromosomal structure of African cichlid species is similar. However, small scale structural variations (inversions, indels, translocations etc.) have not been investigated systematically. So yes, it is totally possible that our short read data and the divergent (and fragmented) reference genome assembly may have hindered our ability to detect inversions. The system really needs a long-read de novo genome assembly to resolve the inversion question.

In the Discussion, you talk about the possibility of different male Y-haplotypes. Is your data sufficiently high resolution that you could examine insertion/deletion polymorphisms in your dataset? 

Yes, we could technically identify small insertions/deletions in our data. Might certainly be something to investigate in the future, in combination with long-read Y assembly.

A recently proposed model of sex-chromosome evolution indicates that gene expression differences may predominate at the early stages of sex chromosome evolution (Lenormand and Roze 2022 Science – This is intriguing given that you didn’t find any smoking gun loci with signals of sexual antagonism. Do you have plans to look at patterns of gene expression across the different morphs?

While Lenormand and Roze’s theory is certainly exciting for the field of sex chromosome evolution, I think it is less plausible for the L. callipterus mating system because antagonistic body sizes in females and males are crucial shell-brooding success and fitness. And because we found the candidate sex-determining gene and body-size gene to be physically linked. I am certainly not a proponent of the ‘one classical theory explains all’ narrative and I really look forward to seeing what RNA-seq data reveal about sex chromosome evolution in this species. It would be especially interesting to see the landscape of cis/trans eQTLs of genes in our proposed L. callipterus sex chromosome and how much it reflects the expectations from Lenormand and Roze’s model. Beyond just this species, cichlid fishes are an excellent system to test the sexual antagonism vs gene regulation models of sex chromosome evolution.

Regarding the coverage analysis you used to identify the putative sex-linked locus. Given the hypothesis that the divergence of the sex chromosomes is recent, reads sampled from Y-linked regions may still map well to the orthologous region on the putative X-chromosome.  Did you tweak mapping quality filters at all?

I did run a less stringent mapping analysis, which overall had slightly higher mapping statistics, but the reduced coverage pattern on the L. callipterus sex chromosome was still significant.

Snail shell nest of L. callipterus with the nest owner in the left centre.
Photo credit: Koblmueller et al. 2007

Could you describe the significance of this research for the general scientific community in one sentence?

Sexual antagonism over body-size may have driven sex chromosome evolution in a shell-brooding cichlid fish where giant and dwarf male reproductive types have evolved.

Moving forward, what are the next steps in this area of research (unless otherwise covered)?

Our main priority right now is to keep the L. callipterus dwarf males alive and breeding. Our fish were recently moved from the University of Bern in Switzerland to the University of Graz in Austria, and it has proved difficult to get the dwarf males happy and breeding in the new facility. This is (probably) the only collection in the world of L. callipterus dwarf males outside Lake Tanganyika so they are very precious. Our next step is to write a convincing grant to get funding to build a long-read improved genome assembly, conduct RNA-seq, and sample and sequence more individuals from natural populations. I would like to use the RNA-seq data to map expression QTLs and investigate the regulatory interactions of candidate genes related to sex, morphology, physiology, and behaviour that we found in or around the L. callipterus sex region. It would also be interesting to study sex chromosomes in related lamprologine species, as our pre-liminary analysis in this manuscript suggests that the sex region may be shared across the lamprologine tribe