Abstract
Neo-sex chromosomes provide a powerful system for studying the early stages of sex chromosome evolution and the genomic mechanisms that may contribute to reproductive isolation. Using PacBio long-read HiFi sequencing, Hi-C scaffolding, and sex-specific transcriptomic data, we generated six chromosome-level assemblies (male and female from three populations) of the mountain pine beetle (Dendroctonus ponderosae), a species known to harbor three partially reproductively isolated neo-Y haplogroups. These assemblies reveal that the large neo-X and neo-Y chromosomes formed through sequential fusions of the ancestral X with three autosomes, with recombination cessation occurring at ~8.6, ~6.3, and ~4.3 MYA for each event. Comparative analyses show that while neo-X chromosomes remain largely collinear across populations, neo-Ys exhibit dramatic structural divergence, with 900-1,200 inverted segments per haplogroup and only ~65% of sequence able to be aligned to the neo-X. Repeat analyses demonstrate moderate TE accumulation on the neo-Y, particularly LTR elements, and gene mapping analyses reveal extensive degeneration: ~62% of neo-Y genes exhibit gene loss, fragmentation, or disruptive mutations. All populations retain a single pseudoautosomal region (PAR), though PAR size and gene content vary due to neo-Y specific rearrangements. Across neo-Ys, 27 genes are uniquely missing in the Western haplogroup, including previously identified candidates implicated in hybrid male sterility. Broader comparisons among neo-Ys show widespread structural variation, population specific patterns of degeneration, and limited gene family expansions. Together, these results provide the first full characterization of neo-sex chromosome evolution in D. ponderosae, revealing rapid, lineage specific neo-Y degeneration and highlighting the potential for sex chromosome divergence to contribute to emerging reproductive incompatibilities within a single species.