Promoter architecture and developmental-expression divergence in the Daphnia pulex complex
Changes in gene regulation are central to developmental evolution, yet how transcription-initiation architecture contributes to developmental expression divergence remains poorly understood. Here, we integrated chromosome-level genome assemblies, developmental RNA-seq, and STRIPE-seq-based transcription start site (TSS) profiling to examine regulatory divergence between two lineages of the Daphnia pulex complex, KAP4 and CON21, across embryonic, juvenile, and adult stages. The two lineages shared a broadly conserved developmental transcriptional program, but KAP4 consistently expressed more protein-coding genes and long non-coding RNAs than CON21. Genes with stage-specific expression were weakly expressed and showed elevated nucleotide diversity and sequence divergence relative to persistently expressed genes, consistent with relaxed purifying selection. Comparative transcriptomic analyses revealed strong stage-dependent expression divergence between the two lineages: the greatest similarity occurred during late embryonic to early juvenile development rather than mid-embryogenesis, providing limited support for a canonical developmental hourglass pattern. STRIPE-seq uncovered a conserved two-peak TSS organization upstream of annotated translation-start sites, but the relative use of distal and proximal initiation regions differed between lineages. Motif analyses further indicated that these initiation regions are associated with distinct cis-regulatory sequence environments. Promoter shape was closely linked to transcriptional output, with broad promoters showing the highest expression, peaked promoters the lowest, and intermediate promoters occupying an intermediate state. Together, these findings suggest that developmental regulatory divergence in the D. pulex complex is shaped by fine-scale remodeling of TSS usage and promoter architecture.