Divergence in Reproductive Behaviors Is Associated with the Evolutionary Loss of Parental Care.

AbstractThe mechanisms underlying the divergence of reproductive strategies between closely related species are still poorly understood. Additionally, it is unclear which selective factors drive the evolution of reproductive behavioral variation and how these traits coevolve, particularly during early divergence. To address these questions, we quantified behavioral differences in a recently diverged pair of Nova Scotian three-spined stickleback (Gasterosteus aculeatus) populations, which vary in parental care, with one population displaying paternal care and the other lacking this. We compared both populations, and a full reciprocal F1 hybrid cross, across four major reproductive stages: territoriality, nesting, courtship, and parenting. We identified significant divergence in a suite of heritable behaviors. Importantly, F1 hybrids exhibited a mix of behavioral patterns, some of which suggest sex linkage. This system offers fresh insights into the coevolutionary dynamics of reproductive behaviors during early divergence and offers support for the hypothesis that coevolutionary feedback between sexual selection and parental care can drive rapid evolution of reproductive strategies.

Parallel evolution despite low genetic diversity in three-spined sticklebacks.

When populations repeatedly adapt to similar environments they can evolve similar phenotypes based on shared genetic mechanisms (parallel evolution). The likelihood of parallel evolution is affected by demographic history, as it depends on the standing genetic variation of the source population. The three-spined stickleback (Gasterosteus aculeatus) repeatedly colonized and adapted to brackish and freshwater. Most parallel evolution studies in G. aculeatus were conducted at high latitudes, where freshwater populations maintain connectivity to the source marine populations. Here, we analysed southern and northern European marine and freshwater populations to test two hypotheses. First, that southern European freshwater populations (which currently lack connection to marine populations) lost genetic diversity due to bottlenecks and inbreeding compared to their northern counterparts. Second, that the degree of genetic parallelism is higher among northern than southern European freshwater populations, as the latter have been subjected to strong drift due to isolation. The results show that southern populations exhibit lower genetic diversity but a higher degree of genetic parallelism than northern populations. Hence, they confirm the hypothesis that southern populations have lost genetic diversity, but this loss probably happened after they had already adapted to freshwater conditions, explaining the high degree of genetic parallelism in the south.

Field application of de novo transcriptomic analysis to evaluate the effects of sublethal freshwater salinization on Gasterosteus aculeatus in urban streams.

Freshwater salinization poses global challenges for aquatic organisms inhabiting urban streams, impacting their physiology and ecology. However, current salinization research predominantly focuses on mortality endpoints in limited model species, overlooking the sublethal effects on a broader spectrum of organisms and the exploration of adaptive mechanisms and pathways under natural field conditions. To address these gaps, we conducted high-throughput sequencing transcriptomic analysis on the gill tissue of the euryhaline fish Gasterosteus aculeatus, investigating its molecular response to salinity stress in the highly urbanized river Boye, Germany. We found that in stream sections with sublethal concentrations of chloride costly osmoregulatory systems were activated, evidenced by the differential expression of genes related to osmoregulation. Our enrichment analysis revealed differentially expressed genes (DEGs) related to transmembrane transport and regulation of transport and other osmoregulation pathways, which aligns with the crucial role of these pathways in maintaining biological homeostasis. Notably, we identified candidate genes involved in increased osmoregulatory activity under salinity stress, including those responsible for moving ions across membranes: ion channels, ion pumps, and ion transporters. Particularly, genes from the solute carrier family SLC, aquaporin AQP1, chloride channel CLC7, ATP-binding cassette transporter ABCE1, and ATPases member ATAD2 exhibited prominent differential expression. These findings provide insights into the potential molecular mechanisms underlying the adaptive response of euryhaline fish to salinity stress and have implications for their conservation and management in the face of freshwater salinization.

Positive Selection Drives cis-regulatory Evolution Across the Threespine Stickleback Y Chromosome.

Allele-specific gene expression evolves rapidly on heteromorphic sex chromosomes. Over time, the accumulation of mutations on the Y chromosome leads to widespread loss of gametolog expression, relative to the X chromosome. It remains unclear if expression evolution on degrading Y chromosomes is primarily driven by mutations that accumulate through processes of selective interference, or if positive selection can also favor the down-regulation of coding regions on the Y chromosome that contain deleterious mutations. Identifying the relative rates of cis-regulatory sequence evolution across Y chromosomes has been challenging due to the limited number of reference assemblies. The threespine stickleback (Gasterosteus aculeatus) Y chromosome is an excellent model to identify how regulatory mutations accumulate on Y chromosomes due to its intermediate state of divergence from the X chromosome. A large number of Y-linked gametologs still exist across 3 differently aged evolutionary strata to test these hypotheses. We found that putative enhancer regions on the Y chromosome exhibited elevated substitution rates and decreased polymorphism when compared to nonfunctional sites, like intergenic regions and synonymous sites. This suggests that many cis-regulatory regions are under positive selection on the Y chromosome. This divergence was correlated with X-biased gametolog expression, indicating the loss of expression from the Y chromosome may be favored by selection. Our findings provide evidence that Y-linked cis-regulatory regions exhibit signs of positive selection quickly after the suppression of recombination and allow comparisons with recent theoretical models that suggest the rapid divergence of regulatory regions may be favored to mask deleterious mutations on the Y chromosome.

Gut Microbiota Uniqueness Is Associated with Lake Size, a Proxy for Diet Diversity, in Stickleback Fish.

AbstractOrganismal divergence can be driven by differential resource use and adaptation to different trophic niches. Variation in diet is a major factor shaping the gut microbiota, which is crucial for many aspects of their hosts' biology. However, it remains largely unknown how host diet diversity affects the gut microbiota, and it could be hypothesized that trophic niche width is positively associated with gut microbiota diversity. To test this idea, we sequenced the 16S ribosomal RNA gene from intestinal tissue of 14 threespine stickleback populations from lakes of varying size on Vancouver Island, Canada, that have been shown to differ in trophic niche width. Using lake size as a proxy for trophic ecology, we found evidence for higher gut microbiota uniqueness among individuals from populations with broader trophic niches. While these results suggest that diet diversity might promote gut microbiota diversity, additional work investigating diet and gut microbiota variation of the same host organisms will be necessary. Yet our results motivate the question of how host population diversity (e.g., ecological, morphological, genetic) might interact with the gut microbiota during the adaptation to ecological niches.

Late Pleistocene stickleback environmental genomes reveal the chronology of freshwater adaptation.

Directly observing the chronology and tempo of adaptation in response to ecological change is rarely possible in natural ecosystems. Sedimentary ancient DNA (sedaDNA) has been shown to be a tractable source of genome-scale data of long-dead organisms1,2,3 and to thereby potentially provide an understanding of the evolutionary histories of past populations.4,5 To date, time series of ecosystem biodiversity have been reconstructed from sedaDNA, typically using DNA metabarcoding or shotgun sequence data generated from less than 1 g of sediment.6,7 Here, we maximize sequence coverage by extracting DNA from ∼50× more sediment per sample than the majority of previous studies1,2,3 to achieve genotype resolution. From a time series of Late Pleistocene sediments spanning from a marine to freshwater ecosystem, we compare adaptive genotypes reconstructed from the environmental genomes of three-spined stickleback at key time points of this transition. We find a staggered temporal dynamic in which freshwater alleles at known loci of large effect in marine-freshwater divergence of three-spined stickleback (e.g., EDA)8 were already established during the brackish phase of the formation of the isolation basin. However, marine alleles were still detected across the majority of marine-freshwater divergence-associated loci, even after the complete isolation of the lake from marine ingression. Our retrospective approach to studying adaptation from environmental genomes of three-spined sticklebacks at the end of the last glacial period complements contemporary experimental approaches9,10,11 and highlights the untapped potential for retrospective "evolve and resequence" natural experiments using sedaDNA.

The source of microbial transmission influences niche colonization and microbiome development.

Early life microbial colonizers shape and support the immature vertebrate immune system. Microbial colonization relies on the vertical route via parental provisioning and the horizontal route via environmental contribution. Vertical transmission is mostly a maternal trait making it hard to determine the source of microbial colonization in order to gain insight into the establishment of the microbial community during crucial development stages. The evolution of unique male pregnancy in pipefishes and seahorses enables the disentanglement of both horizontal and vertical transmission, but also facilitates the differentiation of maternal versus paternal provisioning ranging from egg development, to male pregnancy and early juvenile development. Using 16S rRNA amplicon sequencing and source-tracker analyses, we revealed how the distinct origins of transmission (maternal, paternal and horizontal) shaped the juvenile internal and external microbiome establishment in the broad-nosed pipefish Syngnathus typhle. Our data suggest that transovarial maternal microbial contribution influences the establishment of the juvenile gut microbiome whereas paternal provisioning mainly shapes the juvenile external microbiome. The identification of juvenile key microbes reveals crucial temporal shifts in microbial development and enhances our understanding of microbial transmission routes, colonization dynamics and their impact on lifestyle evolution.

Secondary Contact, Introgressive Hybridization, and Genome Stabilization in Sticklebacks.

Advances in genomic studies have revealed that hybridization in nature is pervasive and raised questions about the dynamics of different genetic and evolutionary factors following the initial hybridization event. While recent research has proposed that the genomic outcomes of hybridization might be predictable to some extent, many uncertainties remain. With comprehensive whole-genome sequence data, we investigated the genetic introgression between 2 divergent lineages of 9-spined sticklebacks (Pungitius pungitius) in the Baltic Sea. We found that the intensity and direction of selection on the introgressed variation has varied across different genomic elements: while functionally important regions displayed reduced rates of introgression, promoter regions showed enrichment. Despite the general trend of negative selection, we identified specific genomic regions that were enriched for introgressed variants, and within these regions, we detected footprints of selection, indicating adaptive introgression. Geographically, we found the selection against the functional changes to be strongest in the vicinity of the secondary contact zone and weaken as a function of distance from the initial contact. Altogether, the results suggest that the stabilization of introgressed variation in the genomes is a complex, multistage process involving both negative and positive selection. In spite of the predominance of negative selection against introgressed variants, we also found evidence for adaptive introgression variants likely associated with adaptation to Baltic Sea environmental conditions.

Paternal protein provisioning to embryos during male seahorse pregnancy.

In brief: Seahorses exhibit male pregnancy and are thus valuable comparative models for the study of the physiology and evolution of pregnancy. This study shows that protein is transported from fathers to developing embryos during gestation, and provides new knowledge about paternal contributions to embryonic development. Abstract: Syngnathid embryos (seahorses, pipefishes and seadragons) develop on or in the male in a specialised brooding structure (brood pouch). Seahorse brood pouches supply nutrients, including lipids, to developing embryos (patrotrophy). We tested the hypothesis that proteins, vital for gene regulation and tissue growth during embryogenesis, are also transported from father to embryos, using the Australian pot-bellied seahorse, Hippocampus abdominalis. We used dry masses and total nitrogen content to estimate the total protein content of newly fertilised egg and neonate H. abdominalis. Neonates contained significantly greater protein mass than newly fertilised eggs. This result indicates that paternal protein transport to developing embryos occurs during H. abdominalis pregnancy. This study is the first to show paternal protein transport during pregnancy in seahorses, and furthers our understanding of paternal influence on embryonic development in male pregnant vertebrates.

Effect of cryopreservation on motility, DNA integrity and gene expression in grey mullet, Mugil cephalus sperm.

This study documents the effect of cryopreservation on motility, DNA integrity, and gene expression in Mugil cephalus sperm. Fresh sperm were cryopreserved using V2 extender (V2E) or 0.3 M glucose, each in combination with one of three cryoprotective agents (CPAs), i.e., 10 % of dimethylsulfoxide, ethylene glycol, or glycerol, all at once. After two different storage (7- vs 60- day) periods in liquid nitrogen, sperm samples were thawed. Single-cell gel electrophoresis was used to detect the DNA integrity. Heat shock proteins (HSPs), HSP70, HSP90 and glutathione peroxidase (GPx2) genes mRNA expression levels was documented using qRT-PCR. The results demonstrated that among 0.3 M glucose + CPAs combinations, EG recorded higher frozen-thawed motility 69 % (7- day) and 59 % (60- day). Similarly, in V2E + CPAs combinations, EG recorded higher frozen-thawed motility 31 % (7- day) and 26 % (60- day). The DNA integrity of all thawed sperm (both periods) did not differ from that of fresh sperm. The qRT-PCR results revealed that in the combination of 0.3 M glucose + CPAs, the level of HSP90 and GPx2 gene expression was found to be upregulated in frozen-thawed sperm on both periods. Whereas, the expression level of the HSP70 gene was down-regulated. On the contrary, in the combination of V2E + CPAs, the expression levels of HSP70, HSP90 and GPx2 genes could not be detected on both periods. Overall, the findings of this study demonstrate that the cryomedium (extender + cryoprotectant) has a more influential role in the motility and levels of gene expression in the frozen-thawed sperm of M. cephalus.

Light-specific wavelengths differentially affect the exploration rate, opercular beat, skin color change, opsin transcripts, and the oxi-redox system of the longsnout seahorse Hippocampus reidi.

Light is a strong stimulus for the sensory and endocrine systems. The opsins constitute a large family of proteins that can respond to specific light wavelengths. Hippocampus reidi is a near-threatened seahorse that has a diverse color pattern and sexual dimorphism. Over the years, H. reidi's unique characteristics, coupled with its high demand and over-exploitation for the aquarium trade, have raised concerns about its conservation, primarily due to their significant impact on wild populations. Here, we characterized chromatophore types in juvenile and adult H. reidi in captivity, and the effects of specific light wavelengths with the same irradiance (1.20 mW/cm2) on color change, growth, and survival rate. The xanthophores and melanophores were the major components of H. reidi pigmentation with differences in density and distribution between life stages and sexes. In the eye and skin of juveniles, the yellow (585 nm) wavelength induced a substantial increase in melanin levels compared to the individuals kept under white light (WL), blue (442 nm), or red (650 nm) wavelengths. In addition, blue and yellow wavelengths led to a higher juvenile mortality rate in comparison to the other treatments. Adult seahorses showed a rhythmic color change over 24 h, the highest reflectance values were obtained in the light phase, representing a daytime skin lightening for individuals under WL, blue and yellow wavelength, with changes in the acrophase. The yellow wavelength was more effective on juvenile seahorse pigmentation, while the blue wavelength exerted a stronger effect on the regulation of adult physiological color change. Dramatic changes in the opsin mRNA levels were life stage-dependent, which may infer ontogenetic opsin functions throughout seahorses' development. Exposure to specific wavelengths differentially affected the opsins mRNA levels in the skin and eyes of juveniles. In the juveniles, skin transcripts of visual (rh1, rh2, and lws) and non-visual opsins (opn3 and opn4x) were higher in individuals under yellow light. While in the juvenile's eyes, only rh1 and rh2 had increased transcripts influenced by yellow light; the lws and opn3 mRNA levels were higher in juveniles' eyes under WL. Prolonged exposure to yellow wavelength stimulates a robust increase in the antioxidant enzymes sod1 and sod2 mRNA levels. Our findings indicate that changes in the visible light spectrum alter physiological processes at different stages of life in H. reidi and may serve as the basis for a broader discussion about the implications of artificial light for aquatic species in captivity.

Influence of EE2 exposure, age and sex on telomere length in European long-snouted seahorse (Hippocampus guttulatus).

After a Telomere Lengthening in juvenile stage, a progressive telomere shortening occurs with age despite higher telomerase level. Telomere Length (TL) may also reflect past physiological state such as a chronic chemical stress. Several studies have revealed a correlation between TL, ageing and/or sex in vertebrates, including teleosts; however, the patterns of telomere dynamics with telomerase mRNA expression, sex, lifespan or chemical stress in teleosts are unclear. The first aim of this study is to verify if telomere length is age and sex-dependent. The second aim is to consider if TL is a useful indicator of stress response in European long-snouted seahorse, Hippocampus guttulatus, an ectothermic and non-model system. We showed that after telomere lengthening during the juvenile stage, a telomeric attrition became significant in sexually mature individuals (p = 0.042). TL decreased in older seahorses despite the presence of somatic telomerase mRNA expression at all life stages studied. There was no difference in TL between males and females, but telomerase mRNA expression was consistently higher in females than males. Exposure to EE2 had no effect on TL in young seahorses, but was correlated with a significant increase in telomerase mRNA expression and various physiological disruptions. Here, a growth retardation of -10 % for body length (p = 0.016) and approximately -45 % for mass (p = 0.001) compared to healthy juvenile seahorses was observed. Our data suggest that telomere dynamics alone should not be used as a marker of EE2 exposure in juvenile seahorses.

Fish gut and skin microbiota dysbiosis induced by exposure to commercial sunscreen formulations.

UV filters (organic or mineral) present in sunscreen products are emerging contaminants of coastal aquatic environments. There is an urgent need to understand marine organisms responses to these compounds. In this study, we investigated the effect of exposure to dilutions of commercial sunscreen formulations on bacterial communities of mullet (Chelon sp.). The gut and skin mucus microbial communities were characterized using a metabarcoding approach targeting the 16S rRNA gene. Our results revealed that mullets had its own bacterial communities that differ from their surrounding habitats and specific to tissue. The dilutions of commercial sunscreens modified the relative abundance of Actinobacteroita, Bacteriodota and Proteobacteria for both gut and skin microbiota. They also allowed to bacteria affiliated to Mycobacterium, Nocardia and Tenacibaculum genera, known to house pathogenic species, to colonize the epithelium which may have implications for fish host health.

Modulation of tooth regeneration through opposing responses to Wnt and BMP signals in teleosts.

Most vertebrate species undergo tooth replacement throughout adult life. This process is marked by the shedding of existing teeth and the regeneration of tooth organs. However, little is known about the genetic circuitry regulating tooth replacement. Here, we tested whether fish orthologs of genes known to regulate mammalian hair regeneration have effects on tooth replacement. Using two fish species that demonstrate distinct modes of tooth regeneration, threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio), we found that transgenic overexpression of four different genes changed tooth replacement rates in the direction predicted by a hair regeneration model: Wnt10a and Grem2a increased tooth replacement rate, whereas Bmp6 and Dkk2 strongly inhibited tooth formation. Thus, similar to known roles in hair regeneration, Wnt and BMP signals promote and inhibit regeneration, respectively. Regulation of total tooth number was separable from regulation of replacement rates. RNA sequencing of stickleback dental tissue showed that Bmp6 overexpression resulted in an upregulation of Wnt inhibitors. Together, these data support a model in which different epithelial organs, such as teeth and hair, share genetic circuitry driving organ regeneration.

Ultrastructural analysis of throat dermal tissue and chromatophore components in the threespine stickleback (Gasterosteus aculeatus).

The threespine stickleback (Gasterosteus aculeatus) is an important model for studying the evolution of nuptial coloration, but histological analyses of color are largely lacking. Previous analyses of one nuptial coloration trait, orange-red coloration along the body, have indicated carotenoids are the main pigment producing this color. In addition, recent gene expression studies found variation in the correlates of throat coloration between the sexes and between populations, raising the possibility of variation in the mechanisms underlying superficially similar coloration. We used transmission electron microscopy (TEM) to investigate the histological correlates of color in the throat dermal tissue of threespine stickleback from Western North America, within and between sexes, populations, and ecotypes. Ultrastructural analysis revealed carotenoid-containing erythrophores to be the main chromatophore component associated with orange-red coloration in both males and females across populations. In individuals where some darkening of the throat tissue was present, with no obvious orange-red coloration, erythrophores were not detected. Melanophore presence was more population-specific in expression, including being the only chromatophore component detected in a population of darker fish. We found no dermal chromatophore units within colorless throat tissue. This work confirms the importance of carotenoids and the erythrophore in producing orange-red coloration across sexes, as well as melanin within the melanophore in producing darkened coloration, but does not reveal broad histological differences among populations with similar coloration.

Microbiome study of a coupled aquaponic system: unveiling the independency of bacterial communities and their beneficial influences among different compartments.

To understand the microbiome composition and interplay among bacterial communities in different compartments of a coupled freshwater aquaponics system growing flathead grey mullet (Mugil cephalus) and lettuces (Lactuca sativa), 16S rRNA gene amplicon sequencing of the V3-V4 region was analysed from each compartment (fish intestine, water from the sedimentation tank, bioballs from the biological filter, water and biofilm from the hydroponic unit, and lettuce roots). The bacterial communities of each sample group showed a stable diversity during all the trial, except for the fish gut microbiota, which displayed lower alpha diversity values. Regarding beta diversity, the structure of bacterial communities belonging to the biofilm adhering to the hydroponic tank walls, bioballs, and lettuce roots resembled each other (weighted and unweighted UniFrac distances), while bacteria from water samples also clustered together. However, both of the above-mentioned bacterial communities did not resemble those of fish gut. We found a low or almost null number of shared Amplicon Sequence Variants (ASVs) among sampled groups which indicated that each compartment worked as an independent microbiome. Regarding fish health and food safety, the microbiome profile did not reveal neither fish pathogens nor bacterial species potentially pathogenic for food health, highlighting the safety of this sustainable food production system.

A distinct neurogenomic response to a trade-off between social challenge and opportunity in male sticklebacks (Gasterosteus aculeatus).

Animals frequently make adaptive decisions about what to prioritize when faced with multiple, competing demands simultaneously. However, the proximate mechanisms of decision-making in the face of competing demands are not well understood. We explored this question using brain transcriptomics in a classic model system: threespined sticklebacks, where males face conflict between courtship and territorial defence. We characterized the behaviour and brain gene expression profiles of males confronted by a trade-off between courtship and territorial defence by comparing them to males not confronted by this trade-off. When faced with the trade-off, males behaviourally prioritized defence over courtship, and this decision was reflected in their brain gene expression profiles. A distinct set of genes and biological processes was recruited in the brain when males faced a trade-off and these responses were largely non-overlapping across two brain regions. Combined, these results raise new questions about the interplay between the neural and molecular mechanisms involved in decision-making.

Galectin 9 restricts viral replication in teleost via autophagy-antiviral pathway and polarizes M2 macrophages for anti-inflammatory response: New insights into functional properties of fish Galectin-9 from Planiliza haematocheilus.

Galectin 9 (Gal9) is a tandem repeat type ß-galactoside-binding galectin that mediates various cellular biochemical and immunological functions. Many studies have investigated the functional properties of Gal9 in mammals; however, knowledge of fish Gal9 is limited to antibacterial studies. In this context, our aim was to clone Gal9 from Planiliza haematocheilus (PhGal9) and investigate its structural and functional characteristics. We discovered the PhGal9 open reading frame, which was 969 base pairs long and encoded a 322 amino acid protein. PhGal9 had a projected molecular weight of 35.385 kDa but no signal peptide sequence. PhGal9 mRNA was ubiquitously produced in all investigated tissues but was predominant in the intestine, spleen, and brain. Its mRNA expression was increased in response to stimulation by Poly(I:C), LPS, and L. garvieae. The rPhGal9 exhibited a dose-dependent agglutination potential toward gram-positive and gram-negative bacteria at a minimum concentration of 50 µg/mL. Overexpression of PhGal9 promoted M2-like phenotype changes in mouse macrophages, and RT-qPCR analysis of M1 and M2 marker genes confirmed M2 polarization with upregulation of M2 marker genes. In the antiviral assay, the expression levels of Viral Hemorrhagic Septicemia Virus (VHSV) glycoproteins, phosphoproteins, nucleoproteins, non-virion proteins, matrix proteins, and RNA polymerase were significantly reduced in PhGal9-overexpressed cells. Furthermore, the mRNA expression of autophagic genes (sqstm1, tax1bp1b, rnf13, lc3, and atg5) and antiviral genes (viperin) were upregulated in PhGal9 overexpressed cells. For the first time in teleosts, our study demonstrated that PhGal9 promotes M2 macrophage polarization by upregulating M2-associated genes (egr2 and cmyc) and suppressing M1-associated genes (iNOS and IL-6). Furthermore, our results show that exogenous and endogenous PhGal9 prevented VHSV attachment and replication by neutralizing virion and autophagy, respectively. Gal9 may be a potent modulator of the antimicrobial immune response in teleost fish.

Extensive gene loss parallels kidney aglomerulism in Syngnathidae.

The eccentric seahorses, seadragons, pipehorses and pipefishes (Syngnathidae) have an aglomerular kidney1. Here, we show that nephron genes2 conserved in Bilateria are secondarily eroded/deleted in Syngnathidae genomes. A transcriptome enrichment analysis suggests the predominance of excretion processes in the Syngnathidae kidney. In a lineage where crypsis and idleness are tightly associated, we propose that aglomerulism evolved as an energy-saving strategy.

The United States dried seahorse trade: A comparison of traditional Chinese medicine and ecommerce-curio markets using molecular identification.

Tens of millions of dried seahorses (genus Hippocampus) are traded annually, and the pressure from this trade along with their life history traits (involved parental care and small migration distances and home ranges) has led to near global population declines. This and other forms of overexploitation have led to all seahorse species being listed in Appendix II under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). The signatory nations of CITES recommended a 10-cm size limit of seahorses to ensure harvested individuals have reached reproductive maturity, and have thus had the chance to produce offspring, to maintain a more sustainable global seahorse fishery. We assessed adherence to CITES recommendations using DNA barcoding and size measurements to compare two prominent U.S. dried seahorse markets: (1) traditional Chinese medicine (TCM), and (2) non-medicinal ecommerce and coastal curio (ECC). We also estimated U.S. import abundance from CITES records. Of the nine species identified among all samples (n = 532), eight were found in the TCM trade (n = 168); composed mostly (75%) of the Indo-Pacific species Hippocampus trimaculatus, and Hippocampus spinosissimus, and the Latin American Hippocampus ingens. In contrast, ECC samples (n = 344) included 5 species, primarily juvenile Indo-Pacific Hippocampus kuda (51.5%) and the western Atlantic Hippocampus zosterae (40.7). The majority of TCM samples (85.7%) met the CITES size recommendation, in contrast to 4.8% of ECC samples. These results suggest non-size discriminatory bycatch is the most likely source of imported ECC specimens. In addition, CITES records indicate that approximately 602,275 dried specimens were imported into the U.S. from 2004-2020, but the exact species composition remains unknown as many U.S. imports records list one species or Hippocampus spp. from confiscated shipments due to difficulties in morphological identification and large numbers of individuals per shipment. Molecular identification was used to identify the species composition of confiscated shipment imports containing undesignated species, and similar to TCM, found H. trimaculatus and H. spinosissimus the most abundant. By combining DNA barcoding, size comparisons, and CITES database records, these results provide an important glimpse into the two primary dried U.S. seahorse end-markets, and may further inform the conservation status of several Hippocampus species.