Specific authentication of Hippocampus based on SNP markers

Seahorses, which are used as traditional Chinese medicine, have large market demands and premium price. Because of overfishing, many seahorse species are endangered and listed in IUCN Red List. The purpose of this study was to develop a simple, rapid and reliable molecular method for specific authentication of seahorse species. Cytochrome c oxidase subunit I (COI) sequences were analyzed for molecular authentication of seahorses in this study. Many single nucleotide polymorphism (SNP) sites were detected among COI sequences of nine seahorse species. Based on the SNP sites, four pairs of modified specific primers were designed respectively for authentication of Hippocampus kuda, Hippocampus japonicus, Hippocampus trimaculatus and Hippocampus kelloggi using allele-specific PCR. Moreover, a multiplex allele-specific PCR for simultaneous authentication of H. kuda, H. japonicus and H. trimaculatus was also developed. The study showed that allele-specific PCR required no sequencing and electrophoresis, and the only necessity is to add fluorochrome into PCR products. Hence the whole authentication procedure only takes about 1.5 h. This work provides an effective method for rapid and reliable identification of seahorse species.     

First record of the Pacific seahorse Hippocampus ingens in Guadalupe Island,Mexico

This is the first record of the Pacific seahorse Hippocampus ingens at a northern oceanic island from the eastern Pacific Ocean. The photographic record of the juvenile female H. ingens was made in November 2015 during a cage diving trip at Guadalupe Island, Mexico. The presence of H. ingens in this area could be related to long distance dispersal mechanisms, as has been observed in other species of seahorses.     

De Novo Transcriptome Analysis of Two Seahorse Species (Hippocampus erectus and H. mohnikei) and the Development of Molecular Markers for Population Genetics

Seahorse conservation has been performed utilizing various strategies for many decades, and the deeper understanding of genomic information is necessary to more efficiently protect the germplasm resources of seahorse species. However, little genetic information about seahorses currently exists in the public databases. In this study, high-throughput RNA sequencing for two seahorse species, Hippocampus erectus and H. mohnikei, was carried out, and de novo assembly generated 37,506 unigenes for H. erectus and 36,113 unigenes for H. mohnikei. Among them, 17,338 (46.23%) unigenes for H. erectus and 17,900 (49.57%) for H. mohnikei were successfully annotated based on the information available from the public databases. Through comparing the unigenes of two seahorse species, 7,802 candidate orthologous genes were identified and 5,268 genes among them could be annotated. In addition, gene ontology analysis of two species was similarly performed on biological processes, cellular components, and molecular functions. Twenty-four and twenty-one unigenes in H. erectus and H. mohnikei were annotated in the biosynthesis of unsaturated fatty acids pathways, and both seahorses lacked the Δ12 and Δ15 desaturases. Total of 8,992 and 9,116 SSR loci were obtained from H. erectus and H. mohnikei unigenes, respectively. Dozens of SSR were developed and then applied to assess the population genetic diversity, as well as cross-amplified in a related species, H. trimaculatus. The H_O and H_E values of the tested populations for H. erectus, H. mohnikei, and H. trimaculatus were medium. These resources would facilitate the conservation of the species through a better understanding of the genomics and comparative genome analysis within the Hippocampus genus.     

New records of the Japanese seahorse Hippocampus mohnikei in Southeast Asia lead to updates in range,habitat and threats

New records of the Japanese seahorse Hippocampus mohnikei from Cambodia, Malaysia, Thailand and Vietnam, along with recently published studies from India and Singapore, have greatly expanded the known range of H. mohnikei within Southeast Asia. These new records reveal novel habitat preferences and threats to H. mohnikei in the region. Although the global conservation status of H. mohnikei is classified as Data Deficient according to the IUCN Red List of Threatened Species, new sightings indicate that this species is found in similar habitats and faces similar threats as other Hippocampus species that are considered Vulnerable.     

Genetic Variations in Two Seahorse Species (Hippocampus mohnikei and Hippocampus trimaculatus): Evidence for Middle Pleistocene Population Expansion

Population genetic of seahorses is confidently influenced by their species-specific ecological requirements and life-history traits. In the present study, partial sequences of mitochondrial cytochrome b (cytb) and control region (CR) were obtained from 50 Hippocampus mohnikei and 92 H. trimaculatus from four zoogeographical zones. A total of 780 base pairs of cytb gene were sequenced to characterize mitochondrial DNA (mtDNA) diversity. The mtDNA marker revealed high haplotype diversity, low nucleotide diversity, and a lack of population structure across both populations of H. mohnikei and H. trimaculatus. A neighbour-joining (NJ) tree of cytb gene sequences showed that H. mohnikei haplotypes formed one cluster. A maximum likelihood (ML) tree of cytb gene sequences showed that H. trimaculatus belonged to one lineage. The star-like pattern median-joining network of cytb and CR markers indicated a previous demographic expansion of H. mohnikei and H. trimaculatus. The cytb and CR data sets exhibited a unimodal mismatch distribution, which may have resulted from population expansion. Mismatch analysis suggested that the expansion was initiated about 276,000 years ago for H. mohnikei and about 230,000 years ago for H. trimaculatus during the middle Pleistocene period. This study indicates a possible signature of genetic variation and population expansion in two seahorses under complex marine environments.     

Two new species of seahorses (Syngnathidae, Hippocampus) from the Middle Miocene (Sarmatian) Coprolitic Horizon in Tunjice Hills, Slovenia: The oldest fossil record of seahorses

Two new and the only known extinct seahorse species Hippocampus sarmaticus and Hippocampus slovenicus are described from the Middle Miocene beds (Lower Sarmatian) in Slovenia, representing the oldest known fossil record of seahorses. Hippocampus sarmaticus was most similar to the extant seahorse species Hippocampus trimaculatus, while H. slovenicus can be most easily compared to the extant pygmy seahorses H. bargibanti, H. denise, and H. colemani. These Sarmatian seahorses lived among seagrasses and macroalgae in the temperate shallow costal waters of the western part of the Central Paratethys Sea.     

Population dynamics of the long-snouted seahorse (Hippocampus guttulatus Cuvier, 1829) in the Mar Menor coastal lagoon

A 10-year monitoring program was developed to quantify the population dynamics of the long-snouted seahorse population in the Mar Menor coastal lagoon. Based on 985 underwater visual censuses, we estimated the long-snouted seahorse (Hippocampus guttulatus Cuvier, 1829) population size in the Mar Menor lagoon and its reduction in size in the last decades, as well as the effect of eutrophication crises in 2016 and 2019 on the species. The annual recruitment for the 2013–2020 period was estimated by comparing the relative abundance of early seahorse life stages in the ichthyoplankton. The density ranged from 0.0458 specimens/m³ at the beginning of the sampling period to 0.0004 at the end, showing a statistically significant difference between the three analyzed periods (H_gl=2 = 14.0, p = 0.001). The long-snouted seahorse population from the Mar Menor lagoon exemplifies the impact of fishing activities and human pressure, especially euxinic episodes and habitat destruction. As a result of this, the Mar Menor population has decreased from several million specimens to a few thousand, in only three decades. This species showed considerable resilience, the seahorse population began to recover once fishing activity stopped. In contrast, the long-snouted seahorse showed high vulnerability to habitat loss and an episodic flooding event. Adult seahorses showed preferences for highly complex habitats, especially Caulerpa prolifera–Cymodocea nodosa mixed meadows and habitats of high complexity and anthropogenic origin, such as harbors, jetties, or breakwaters. In contrast, juvenile seahorses preferred monotonous seabeds with low complexity, such as the sandy beds that are characteristic of the Mar Menor lagoon littoral.     

Home range behaviour of the monogamous Australian seahorse, <Emphasis Type="Italic">Hippocampus whitei</Emphasis>

We provide a quantitative account of local movements in the monogamous Australian species Hippocampus whitei, as a rare report of home range size in fishes living in seagrass habitats. Our study took place in shallow Posidonia seagrass beds in Port Jackson (Sydney Harbour), principally during January to March. Daily monitoring of individual seahorses during underwater observations revealed that both sexes maintained small and apparently undefended home ranges for several breeding cycles at least. Female home ranges were significantly larger than males, when analysed by both the minimum convex polygon and grid cell methods. Home range size was not correlated with either body size or seahorse density. Presumably, home ranges were small in H. whitei because camouflage (to avoid predation and to capture prey), mate fidelity and parental brooding meant they accrued little benefit (and potentially considerable cost) from moving more extensively. Sex differences in home range size may arise from constraints associated with male pregnancy. These fish are among the most sedentary of vertebrates, with relatively small home ranges equalled only by coral reef species. In terms of their conservation, relatively small protected areas may be sufficient to support breeding populations of H. whitei although that limited movement may result in considerable delays in the recolonisation of depleted areas.     

First records of Hippocampus algiricus in the Canary Islands (north‐east Atlantic Ocean) with an observation of hybridization with Hippocampus hippocampus

Morphometric and genetic analyses confirmed the first records of the West African seahorse Hippocampus algiricus at Gran Canaria Island (north‐east Atlantic Ocean), and also the first evidence of interspecific hybridization in seahorses. These results provide additional data on the distribution of H. algiricus that may help to establish future conservation strategies, and uncover a new potential sympatric scenario between H. algiricus and Hippocampus hippocampus.     

The endangered White's seahorse Hippocampus whitei chooses artificial over natural habitats

To explore whether the endangered White's seahorse Hippocampus whitei would choose to inhabit artificial over natural habitats, 10 adult H. whitei individuals were put through a series of binary choice trials in aquaria, during which they were offered different paired combinations of natural (different types of macroalga and seagrass) and artificial habitat (panels of swimming-net material). It was found that H. whitei displayed a significant choice for swimming-net material over all other available natural habitats and chose habitats according to the following rankings: (a) Net; (b) Sargassum sp.; (c) Posidonia australis; (d) Zostera muelleri. Hippocampus whitei’s choice of swimming net material over natural habitat suggests that these artificial structures could be a useful conservation measure for seahorses in areas where natural habitat is becoming less favourable due to declines in abundance or quality.     

Early life history of a seahorse, Hippocampus mohnikei, in Tokyo Bay, Japan

The early morphological development, seasonal and spatial occurrence patterns, and food habits of a seahorse, Hippocampus mohnikei, in offshore waters of Tokyo Bay, central Japan, were studied on the basis of 206 juvenile and young specimens (6.0–65.3 mm TL) collected between August 1995 and January 1999. All the specimens were collected within the period from May to January, inclusive, each year. In the least developed specimen (6.0 mm TL), the number of dorsal, anal, and pectoral fin rays had attained the adult complement, whereas the minute caudal fin, consisting of two rays, was present in juveniles of 6.0–26.4 mm TL. Hippocampus mohnikei≧35 mm TL, being larger than settlement size (ca. 30 mm TL), had very low gut fullness index values (GFI = 0, >70% of specimens), whereas those of 15–29 mm TL had higher values (GFI = 2–4, >80% of specimens). In addition, larger individuals selectively fed on larger planktonic animals (species of Brachyura), which occurred naturally at low densities, although smaller food items, such as Oithona davisae and Penilia avirostris, occurred abundantly, being consumed by smaller H. mohnikei individuals (15–34 mm TL). These results indicated that food availability for H. mohnikei in offshore waters of Tokyo Bay is significant for individuals larger than settlement size, because their food preference would shift from smaller food items to larger food items, which would be scarce in their environments. Received: January 12, 2001 / Revised: May 13, 2001 / Accepted: June 14, 2001     

Genetic monogamy despite social promiscuity in the pot-bellied seahorse (Hippocampus abdominalis)

Sexual selection theory predicts a positive correlation between relative parental investment and mate choice. In syngnathid fishes (seahorses and pipefish), males brood offspring in specialized brooding structures. While female-female mating competition has been demonstrated in some pipefishes, all seahorses (genus Hippocampus) studied to date have been found to have conventional sex roles with greater male-male competition for access to mates despite possessing the most complex brood structures in the family. Although multiple mating is common in pipefish, seahorses are again exceptional, exhibiting strict genetic monogamy. Both demographic and behavioural explanations have been offered to explain the lack of multiple mating in seahorse species, but these hypotheses have not yet been explicitly addressed. We investigated mating systems and brood parentage of the pot-bellied seahorse, Hippocampus abdominalis, a temperate-water species that is socially promiscuous with conventional sex roles in laboratory populations. We observed promiscuous courtship behaviour and sex-role reversal in high density, female-biased field populations of H. abdominalis. We hypothesize that sex roles are plastic in H. abdominalis, depending on local population density and sex ratio. Despite promiscuous courtship behaviour, all assayed male seahorses were genetically monogamous in both laboratory and wild populations. Physiological limitations associated with embryo incubation may explain the absence of multiple mating in seahorses and may have played an important role in the development of the unique reproductive behaviour typical in these species.     

Novel microsatellite loci in the threatened European long-snouted seahorse (<Emphasis Type="Italic">Hippocampus guttulatus</Emphasis>) for genetic diversity and parentage analysis

The long-snouted seahorse Hippocampus guttulatus is one of the two European seahorse species. We describe the isolation of the first 12 microsatellite loci in this threatened species. These new markers were tested in non-invasive samples of 32 seahorses from NW Spain. The number of alleles ranged from 2 to 15 (mean: 6.3) and expected heterozygosity from 0.031 to 0.912 (mean: 0.500). All loci conformed to Hardy–Weinberg expectations and no genotypic disequilibrium was observed between any pair of loci. The theoretical exclusion probabilities for this set of loci, when no parental information exists or when one parent is known, were 0.973 and 0.998, respectively. This study indicates the usefulness of these novel loci for population analysis and kinship studies in Hippocampus guttulatus. Their potential application is extended to the other European seahorse species, since all loci were successfully cross-amplified in H. hippocampus.     

Life history and ecology of the elusive European short‐snouted seahorse Hippocampus hippocampus

To improve the understanding of the life history and ecology of one of Europe's most elusive fishes, the short‐snouted seahorse Hippocampus hippocampus, data from wild populations in a shallow coastal lagoon in southern Portugal were analysed. The data were collected from 17 tagged seahorses on a focal‐study grid as well as from >350 seahorses encountered during underwater visual surveys and a fishery‐independent study using beach seines. These populations of settled juveniles and adults had a mean population density of 0·009 m^?2. During the study period (2000–2004), reproduction peaked in July and August. Juveniles recruited to the lagoon at c. 66 mm standard length (L_S) and 0·5 years of age and established small home ranges (0·8 to 18·2 m²). First reproduction was estimated at 100 mm and 1 year of age. Based on a fitted von Bertalanffy model, H. hippocampus grew quickly (growth coefficient K = 0·93) to a maximum theoretical size L_∞ = 150 mm and have a maximum lifespan of c. 3·2 years. Courtship behaviours were consistent with the maintenance of pair bonds and males brooded multiple batches of young per year. Estimated annual reproductive output averaged 871 young (±632). Together these analyses provide the first life‐history parameters for this species and indicate that H. hippocampus bears characteristics of opportunist and intermediate strategists. Such populations are predicted to exhibit large fluctuations in abundance, making them vulnerable to extended periods of poor recruitment.     

Biology of a seahorse species, Hippocampus comes in the central Philippines

Based on 16 months of field observations on tagged seahorses Hippocampus comes in the Philippines, adults were found to be nocturnal, to maintain small home ranges, and to live mostly among corals. Prolonged pair associations suggested that H. comes , like many other seahorse species, were probably monogamous, a conclusion consistent with their low density and sparse distribution. Site and mate fidelity suggest that H. comes populations may fare poorly under current high levels of exploitation.     

Conservation value of Codium tenue habitat for the endangered Knysna seahorse Hippocampus capensis

We investigated the density and size structure of the endangered Knysna seahorse Hippocampus capensis in the macroalga Codium tenue at three sites in a residential marina estate in the Knysna Estuary, South Africa, over an 18 month period from March 2017 to August 2018. Seahorses were present in C. tenue throughout the year. Average (± SE) densities of 0.33 (± 0.03) and 0.23 (± 0.03) seahorses per kg of C. tenue were found for 2017 and 2018 respectively. Density did not vary across sites but varied significantly across months with a decreasing trend from summer to spring. The average (± SE) adult seahorse height was 75.16 mm (± 0.63 mm) and 69.09 mm (± 0.64 mm) and mass was 2.05 g (± 0.04 g) and 1.65 g (± 0.04 g) for males and females, respectively, and an even sex ratio was observed throughout the sampling period. Height varied across months, with significantly smaller seahorses found in June and August of 2017. Tail length was highly correlated with height and therefore can serve as a potential proxy for determining the height of H. capensis. This study showed that C. tenue is consistently utilised by Knysna seahorse and should therefore be considered an important habitat to protect and facilitate the ongoing conservation of this endangered seahorse species.     

Life history and population dynamics of an endangered seahorse (Hippocampus capensis) within an artificial habitat

Species-specific life-history information is critical for successful conservation, particularly in establishing an accurate baseline status. Obtaining such information is challenging for most species, but in particular for rare and threatened marine species. To facilitate future conservation of the endangered Knysna seahorse (Hippocampus capensis) this study aimed to determine important life-history information for this species. Visible implant fluorescent elastomer (VIFE) tags were used to mark 78 seahorses within a residential marina estate in the Knysna estuary, South Africa, in February 2018. Using a mark-resight approach, the size and movement patterns of the population and growth rate of seahorses were determined over a 14-month period. The closed population estimate for H. capensis, within Thesen Islands Marina, was estimated to be 134 (118–152 95% C.I. ) in February 2018 compared to only 72 (48–108 95% C.I. ) in February 2019. The species showed rapid initial growth with males and females having similar rates of growth based on the specialised von Bertalanffy growth function model. The importance of Reno mattresses as a habitat for H. capensis was confirmed based on the high abundance and site fidelity of the population, which emphasises the conservation potential of heavily modified environments for threatened seahorse species. The use of VIFE tags was deemed effective in studying this endangered seahorse and allowed the collection of important information for this species which can be used in future Red List assessments and conservation actions.     

Does a No-Take Marine Protected Area Benefit Seahorses?

Seahorses are iconic charismatic species that are often used to ‘champion’ marine conservation causes around the world. As they are threatened in many countries by over-exploitation and habitat loss, marine protected areas (MPAs) could help with their protection and recovery. MPAs may conserve seahorses through protecting essential habitats and removing fishing pressures. Populations of White''s seahorse, Hippocampus whitei, a species endemic to New South Wales, Australia, were monitored monthly from 2006 to 2009 using diver surveys at two sites within a no-take marine protected areas established in 1983, and at two control sites outside the no-take MPA sites. Predators of H. whitei were also identified and monitored. Hippocampus whitei were more abundant at the control sites. Seahorse predators (3 species of fish and 2 species of octopus) were more abundant within the no-take MPA sites. Seahorse and predator abundances were negatively correlated. Substantial variability in the seahorse population at one of the control sites reinforced the importance of long-term monitoring and use of multiple control sites to assess the outcomes of MPAs for seahorses. MPAs should be used cautiously to conserve seahorse populations as there is the risk of a negative impact through increased predator abundance.     

The first known fossil record of pipehorses (Teleostei: Syngnathidae: Haliichthyinae) from the Miocene Coprolitic Horizon from the Tunjice Hills,Slovenia

The first known fossil specimens of pipehorses (Haliichthyinae) were unearthed from the Middle Miocene (Sarmatian) beds of the Coprolitic Horizon in the Tunjice Hills, Slovenia. These fossil pipehorses belong to a new genus and species Hippohaliichthys edis, which was similar to the extant species Haliichthys taeniophorus. The body morphology indicates that the described fossil pipehorses were also closely related to the pygmy pipehorse Hippotropiscis frenki and the seahorse Hippocampus sarmaticus, two taxa which were also found in the Coprolitic Horizon. The described fossil material of pipehorses indicates that seahorses evolved from a group of pipehorses that were similar in size and shape to extant and fossil pipehorses of the Haliichthyinae subfamily.     

The origin and evolution of seahorses (genus Hippocampus): a phylogenetic study using the cytochrome b gene of mitochondrial DNA

Phylogenetic relationships among 93 specimens of 22 species of seahorses (genus Hippocampus) from the Atlantic and Indo-Pacific Oceans were analysed using cytochrome b gene sequence data. A maximum sequence divergence of 23.2% (Kimura 2-parameter model) suggests a pre-Tethyan origin for the genus. Despite a greater number of seahorse species in the Indo-Pacific than in the Atlantic Ocean, there was no compelling genetic evidence to support an Indo-Pacific origin for the genus Hippocampus. The phylogenetic data suggest that high diversity in the Indo-Pacific results from speciation events dating from the Pleistocene to the Miocene, or earlier. Both vicariance and dispersal events in structuring the current global distribution of seahorses. The results suggested that several species designations need re-evaluating, and further phylogeographic studies are required to determine patterns and processes of seahorse dispersal.