A Haplotype-Resolved Genome Assembly of the Long-Spined Sea Urchin Diadema antillarum, a Keystone Caribbean Reef Herbivore.

Background/Objectives: The long-spined sea urchin Diadema antillarum is a keystone herbivore whose grazing maintains Caribbean coral reefs; basin-wide mass mortalities in 1983-1984 and 2022 have made genomic resources a conservation priority, yet no nuclear genome existed for the species. We aimed to generate the first nuclear reference and to resolve the high heterozygosity that complicates genome assembly in broadcast-spawning marine invertebrates. Methods: For the assembly, we combined PacBio HiFi, Oxford Nanopore, and Illumina sequencing. Genome size and heterozygosity were estimated by k-mer profiling. We compared standard and haplotype-aware assembly strategies (hifiasm), evaluated completeness with BUSCO, and annotated repeats using a species-specific RepeatModeler library. Results: k-mer profiling estimated a haploid genome of ~703 Mb with 2.52% heterozygosity. Standard assembly then produced an inflated 1.75 Gb assembly (98.4% BUSCO-complete but 84.4% duplicated), indicating retention of both haplotypes. Haplotype-aware reassembly separated this into a collapsed primary assembly (1.03 Gb) and two phased haplotypes (0.95 and 0.89 Gb), each comparable in size to the chromosome-level congener D. antillarum (886 Mb). BUSCO completeness reached 99.0%, with single-copy orthologs rising to 85-90%, and reference-free consensus quality reached QV 44.5 (Merqury; initial assembly). This genome is repeat-rich (42.84% repetitive; 29.96% unclassified). Conclusions: We provide the collapsed primary assembly together with both phased haplotypes as a haplotype-resolved reference for D. antillarum, establishing a foundation for immunogenomic, comparative, and population-genetic studies and for monitoring and restoration of this ecologically critical species. More broadly, the study shows that haplotype-aware assembly is essential for resolving such highly heterozygous genomes and delivers the genomic foundation needed to guide the conservation of this keystone Caribbean reef species.