Monika Hospodářská

Reproductive traits contain some of the most bizarre and unintuitive innovations seen across the tree of life. Chief among these is the sperm dimorphism of butterflies and moths (Lepidoptera). Males make two types of sperm: traditional fertilizing sperm (eupyrene) and a second, non-fertilizing type (apyrene) that lacks a nucleus entirely. Despite decades of study, the function and evolution of this second sperm type has remained unclear. Here we explore the sperm biology of the evergreen bagworm moth, Thyridopteryx ephemeraeformis (Lepidoptera: Tineoidea: Psychidae), and find no evidence for non-fertilizing sperm in this species. We generate genomic, transcriptomic, and proteomic resources to characterize the apparently monomorphic sperm of this species and compare it to that of other Lepidoptera. The fertilizing sperm of the evergreen bagworm shows key differences from other studied species, especially a lack of proteolytic enzymes used to break down sperm bundles in other species. Combining this and other evidence, we offer new hypotheses for the evolution and function of this enigmatic reproductive trait. Notably, early diverging moths appear to produce far less non-fertilizing sperm in general than later diverging Lepidoptera. We infer that non-fertilizing sperm are not necessary in these taxa, perhaps because less robust packaging of fertilizing sperm enables greater mobility than in later diverging moths.

Chromosomal rearrangements act as barriers to gene flow and can thus promote speciation. In moths and butterflies (Lepidoptera), which possess holocentric chromosomes facilitating karyotype changes, chromosome fusions are more common than fissions. Yet, limited evidence suggests that when speciation involves chromosomal rearrangements, it is most often linked to fissions. Notable karyotypic variation is observed in three clades of the subfamily Polyommatinae (Lycaenidae), with chromosome numbers ranging from n = 10 to 225. We investigated genome sizes and karyotypes in several species of the genera Polyommatus and Lysandra with modal and derived high chromosome numbers. Our findings showed no support for polyploidy, confirming previous conclusions about karyotypic diversification via chromosome fragmentation in this butterfly family. Species with high chromosome numbers have slightly larger genomes, which indicate a potential role of repetitive sequences but contradict the hypothesis of holocentric drive. Ends of fragmented chromosomes were healed with telomeres synthesized de novo, which were significantly larger than those of species with modal karyotype. No interstitial telomeric sequences were detected on autosomes. Internal telomeric signals on sex chromosomes, however, revealed multiple sex chromosome systems in Polyommatus (Plebicula) dorylas and Polyommatus icarus, with two karyotype races differing in sex chromosome constitution in the latter. Notably, the W chromosome resisted fragmentation, presumably due to its epigenetic silencing.

Sex chromosomes have evolved repeatedly across eukaryotes. The emergence of a sex‐determining (SD) locus is expected to progressively restrict recombination, driving convergent molecular differentiation. However, evidence from taxa like teleost fishes, representing over half of vertebrate species with unmatched diversity in SD systems, challenges this model. Teleost sex chromosomes are often difficult to detect as they experience frequent turnovers, resetting the differentiation process. Nothobranchius killifishes, which include the XY system shared by N. furzeri and N. kadleci and X₁X₂Y systems in six other species, offer a valuable model to study sex chromosome turnovers. We characterised X₁X₂Y systems in five killifish species and found that sex chromosomes evolved at least four times independently. Sex‐determining regions resided near centromeres or predicted chromosome rearrangement breakpoints in N. brieni and N. guentheri, suggesting recombination cold spots may facilitate sex chromosome evolution. Chromosomes representing the XY system in N. furzeri/N. kadleci were sex‐linked also in the outgroup Fundulosoma thierryi, with several genes, including gdf6, residing in the region of differentiation. Although the X₁X₂Y systems of N. guentheri, N. lourensi (both Coastal clade), and N. brieni (Kalahari clade) involved different chromosomes, they shared a potential SD region. We uncovered two sex‐linked evolutionary strata of distinct age in N. guentheri. However, its potential SD gene amhr2 was located in the younger stratum and is hence unlikely to be the ancestral SD gene in this lineage. Our findings suggest recombination landscapes shape sex chromosome turnover and that certain synteny blocks are repeatedly co‐opted as sex chromosomes in killifishes.

Sex chromosomes of teleost fishes often have low levels of differentiation and undergo frequent turnovers. Annual Nothobranchius killifishes comprise representatives with male-heterogametic XY or X1X2Y sex chromosome systems, scattered across their phylogeny, nested within species lacking cytologically detectable sex chromosomes. They thus provide a suitable system to study sex chromosome evolution and turnover. Here, we combined molecular cytogenetics and genomic analyses to examine several multiple sex chromosome systems in Nothobranchius spp. and their outgroup Fundulosoma thierryi. We used fluorescence in situ hybridization with three sex chromosome-specific painting probes and bacterial artificial chromosomes (BAC) bearing eight orthologues of genes found to be repeatedly co-opted as master sex determining (MSD) genes in fishes. Our results suggest at least four independent origins of sex chromosomes in the genus Nothobranchius. The synteny block carrying amhr2 gene was shared by X1X2Y systems of N. brieni, N. guentheri and N. lourensi, but the autosomal additions and the overall neo-Y chromosome structure differed among these species. On the other hand, gdf6 gene was localized to neo-Y of F. thierryi. None of the mapped MSD gene candidates seems to determine sex in N. ditte. We further sequenced genomes of F. thierryi female and N. guentheri male by long-read platforms and performed analyses of male and female Pool-seq data and coverage to delimit their non-recombining regions, determine degree of their differentiation, and thus complement the cytogenetic data in assessing potential MSD genes. We found low level of sex chromosomes differentiation in F. thierryi. In N. guentheri, however, we identified two distinct evolutionary strata on neo-Y. The amhr2 gene resides in the younger stratum and has low allelic variation, which questions its role in sex determination.