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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. 

A major focus of host-microbe research is to understand how genetic differences, of various magnitudes, among hosts translate to differences in their microbiomes. This has been challenging for animal hosts, including humans, because it is difficult to control environmental variables tightly enough to isolate direct genetic effects on the microbiome. Our work in stickleback fish is a significant contribution because our experimental approach allowed strict control over environmental factors, including standardization of the microbiome from the earliest stage of development and unrestricted co-housing of fish in a truly common environment. Furthermore, we measured host genetic variation over 2,000 regions of the stickleback genome, comparing this information and microbiome composition data among fish from very similar and very different genetic backgrounds. Our findings highlight how differences in the host genome influence microbiome diversity and make a case for future manipulative microbiome experiments that use host systems with naturally occurring genetic variation. 

The Gulf pipefish Syngnathus scovelli has emerged as an important species for studying sexual selection, development, and physiology. Comparative evolutionary genomics research involving fishes from Syngnathidae depends on having a high-quality genome assembly and annotation. However, the first S. scovelli genome assembled using short-read sequences and a smaller RNA-sequence dataset has limited contiguity and a relatively poor annotation. Here, using PacBio long-read high-fidelity sequences and a proximity ligation library, we generate an improved assembly to obtain 22 chromosome-level scaffolds. Compared to the first assembly, the gaps in the improved assembly are smaller, the N75 is larger, and our genome is ~95% BUSCO complete. Using a large body of RNA-Seq reads from different tissue types and NCBI's Eukaryotic Annotation Pipeline, we discovered 28,162 genes, of which 8,061 are non-coding genes. Our new genome assembly and annotation are tagged as a RefSeq genome by NCBI and provide enhanced resources for research work involving S. scovelli. 

Seadragons are a remarkable lineage of teleost fishes in the family Syngnathidae, renowned for having evolved male pregnancy. Comprising three known species, seadragons are widely recognized and admired for their fantastical body forms and coloration, and their specific habitat requirements have made them flagship representatives for marine conservation and natural history interests. Until recently, a gap has been the lack of significant genomic resources for seadragons. We have produced gene-annotated, chromosome-scale genome models for the leafy and weedy seadragon to advance investigations of evolutionary innovation and elaboration of morphological traits in seadragons as well as their pipefish and seahorse relatives. We identified several interesting features specific to seadragon genomes, including divergent noncoding regions near a developmental gene important for integumentary outgrowth, a high genome-wide density of repetitive DNA, and recent expansions of transposable elements and a vesicular trafficking gene family. Surprisingly, comparative analyses leveraging the seadragon genomes and additional syngnathid and outgroup genomes revealed striking, syngnathid-specific losses in the family of fibroblast growth factors (FGFs), which likely involve reorganization of highly conserved gene regulatory networks in ways that have not previously been documented in natural populations. The resources presented here serve as important tools for future evolutionary studies of developmental processes in syngnathids and hold value for conservation of the extravagant seadragons and their relatives. 

Abstract Single-cell RNA sequencing is a powerful technique that continues to expand across various biological applications. However, incomplete 3′-UTR annotations can impede single-cell analysis resulting in genes that are partially or completely uncounted. Performing single-cell RNA sequencing with incomplete 3′-UTR annotations can hinder the identification of cell identities and gene expression patterns and lead to erroneous biological inferences. We demonstrate that performing single-cell isoform sequencing in tandem with single-cell RNA sequencing can rapidly improve 3′-UTR annotations. Using threespine stickleback fish (Gasterosteus aculeatus), we show that gene models resulting from a minimal embryonic single-cell isoform sequencing dataset retained 26.1% greater single-cell RNA sequencing reads than gene models from Ensembl alone. Furthermore, pooling our single-cell sequencing isoforms with a previously published adult bulk Iso-Seq dataset from stickleback, and merging the annotation with the Ensembl gene models, resulted in a marginal improvement (+0.8%) over the single-cell isoform sequencing only dataset. In addition, isoforms identified by single-cell isoform sequencing included thousands of new splicing variants. The improved gene models obtained using single-cell isoform sequencing led to successful identification of cell types and increased the reads identified of many genes in our single-cell RNA sequencing stickleback dataset. Our work illuminates single-cell isoform sequencing as a cost-effective and efficient mechanism to rapidly annotate genomes for single-cell RNA sequencing.