Seahorses, pipefishes, and seadragons are fishes from the family Syngnathidae that have evolved extraordinary traits including male pregnancy, elongated snouts, loss of teeth, and dermal bony armor. The developmental genetic and cellular changes that led to the evolution of these traits are largely unknown. Recent syngnathid genomes revealed suggestive gene content differences and provide the opportunity for detailed genetic analyses. We created a single cell RNA sequencing atlas of Gulf pipefish embryos to understand the developmental basis of four traits: derived head shape, toothlessness, dermal armor, and male pregnancy. We completed marker gene analyses, built genetic networks, and examined spatial expression of select genes. We identified osteochondrogenic mesenchymal cells in the elongating face that express regulatory genes bmp4, sfrp1a, and prdm16. We found no evidence for tooth primordia cells, and we observed re-deployment of osteoblast genetic networks in developing dermal armor. Finally, we found that epidermal cells expressed nutrient processing and environmental sensing genes, potentially relevant for the brooding environment. The examined pipefish evolutionary innovations are composed of recognizable cell types, suggesting derived features originate from changes within existing gene networks. Future work addressing syngnathid gene networks across multiple stages and species is essential for understanding how their novelties evolved.

The fasta file and annotation file shared here were used in the single cell RNA sequencing analysis described in the publication. The fasta nuclear genome file was downloaded from NCBI (GCF_024217435.2). The mitochondrial genome fasta file was downloaded from NCBI (NC_065499.1). The two fasta files were appended together to create the fasta file used in the analysis. To lengthen the 3' UTRs of the genome annotations, we produced two scISOrSeq libraries (one from a late embryonic stage and one from pregnant and non-pregnant brood pouch tissues) that were sequenced on seperate lanes on PacBio Sequel II - SMRT cells 8M. Reads were processed according to Healey et al. 2022 and aligned with the Gulf pipefish genome (GCF_024217435.2). Gene models from the scISOrSeq datasets were merged with the Gulf pipefish NCBI models (GCF_024217435.2) using TAMA merge.