Abstract
Mounting discoveries of avian neo-sex chromosomes are providing opportunities to understand the extent of variation in fundamental aspects of avian neo-sex chromosome evolution. We integrated cytogenetic data, long-read assemblies, and whole-genome resequencing to test phylogenetic hypotheses of recombination suppression and to elucidate the phylogenetic distribution of neo-sex chromosomes in honeyeaters (Aves: Meliphagidae). We find that neo-sex chromosomes in honeyeaters evolved through a fusion of the long arm of chromosome 5 and the pseudoautosomal region (PAR) of both ancestral Z and W sex chromosomes. Resequencing data from 11 species of honeyeaters and outgroups supports our cytogenetic evidence that these neo-sex chromosomes are derived within honeyeaters. Phylogenetic analyzes confirm that all tested honeyeaters share the same breakpoint for a new 17.4 Mb PAR at the end of the neo-sex chromosomes and suggest a single, large expansion of recombination suppression, encompassing 44.6 Mb, is most supported in the newly fused region of the neo-W. We also discovered phylogenetic discordance between the mapping of neo-sex chromosomes on the established nuclear and mitochondrial (mtDNA) phylogenies. We conclude that neo-sex chromosomes arose once in honeyeaters because they form a monophyletic clade on the mtDNA tree, which shares the phylogenetic history of the neo-W through matrilineal coinheritance. Overall, our findings provide new insights on recombination suppression dynamics of avian neo-sex chromosomes and demonstrate the value of comparing nuclear and mtDNA trees to determine the phylogenetic distribution of neo-sex chromosomes, especially in the presence of mitonuclear discordance, which is common across the avian tree of life.