Introduction
The genus Hippocampus corresponds to one of the most emblematic groups of species within the family Syngnathidae, presenting very distinct and peculiar apparent characteristics, where males incubate fertilized eggs in their brood pouch (Sreepada et al., Reference Sreepada, Desai and Naik2002; Chang et al., Reference Chang, Jang-Liaw, Lin, Fang and Shao2013; Sanaye et al., Reference Sanaye, Khandeparker, Rayadurga, Shivaramu, Kankonkar, Narvekar and Gauthankar2020). Many of the species in this family live in vulnerable areas, including marine environments, coral reefs and mangroves, which are environments prone to overfishing and consequently, the destruction of their habitats (Lai et al., Reference Lai, Sun, Chen, Lou, Qiu, Zhang and Cheng2019; Serite et al., Reference Serite, Ntshudisane, Swart, Simbine, Jaime and Teske2021).
Beyond this scenario, the number of seahorses is low due to certain biological and behavioural aspects, such as the reproduction rate of seahorses, which even under optimal conditions is low due to the small size of the offspring and prolonged parental care, associated with monogamy where some species stop reproducing when the partner dies or is captured (Kloc, Reference Kloc2023). These factors, associated with massive exploitation, capture of these species for traditional Chinese medicine (TCM), aquarium substrate and ornamentation, may be contributing to a population decline of species of the genus Hippocampus, with some species having vulnerable or threatened status (Vincent et al., Reference Vincent, Foster and Koldewey2011; Chang et al., Reference Chang, Jang-Liaw, Lin, Fang and Shao2013; Zhang et al., Reference Zhang, Ryu and Qian2017).
Currently, 52 species of Hippocampus seahorse are recognized worldwide (Eschmeyer et al., Reference Eschmeyer, Van der Laan and Fricke2023), of which the most common species in the Indian Ocean region include Hippocampus kuda, Hippocampus kelloggi, Hippocampus spinosissimus, Hippocampus trimaculatus, Hippocampus mohnikei, Hippocampus borboniensis and Hippocampus montebelloensis (Thangaraj and Lipton, Reference Thangaraj and Lipton2007; Yogeshkumar and Geetha, Reference Yogeshkumar and Geetha2012; Sanaye et al., Reference Sanaye, Khandeparker, Rayadurga, Shivaramu, Kankonkar, Narvekar and Gauthankar2020; Koning and Hoeksema, Reference Koning and Hoeksema2021). Recent research reveals the suspicion of the occurrence of other Hippocampus species in the Indian Ocean region such as Hippocampus histrix (Lourie et al., Reference Lourie, Vincent and Hall1999).
Most of the species mentioned in this study were identified using morphological analyses, which can make accurate identification difficult due to their varying morphotypes, including colours, camouflages, body proportions, tail length, filaments and snout length. This variability can lead to an underestimation of true species diversity. To overcome this challenge, researchers have increasingly used molecular tools, such as DNA barcoding, to aid in species identification (Guimarães-Costa et al., Reference Guimarães-Costa, Machado, Oliveira, Silva-Costa, Andrade, Giarrizzo, Ulrich, Sampaio and Schneider2019; Muhala et al., Reference Muhala, Guimarães-Costa, Macate, Tembe, Mula, Tóvela, Bessa-Silva, Valinotto and Sampaio2022; Tsoupas et al., Reference Tsoupas, Papavasileiou, Minoudi, Gkagkavouzis, Petriki, Bobori, Sapounidis, Koutrakis, Leonardos, Karaiskou and Triantafyllidis2022; Tang et al., Reference Tang, Deng, Luo, Duan, Wang and Zhang2023). In Mozambique, there is believed to be a large diversity of great seahorse species, including H. kuda and Hippocampus nalu, but concrete records of these species are currently lacking (Teske et al., Reference Teske, Hamilton, Palsboll, Choo, Gabr, Lourie, Santos, Sreepada, Cherry and Matthee2005; Lourie et al., Reference Lourie, Pollom and Foster2016; Short et al., Reference Short, Claassens, Smith, De Brauwer, Hamilton, Stat and Harasti2020).
Recently, specimens belonging to the genus Hippocampus were collected from the central coast of Mozambique in Quelimane. These specimens were found to be morphologically similar to H. kelloggi (Figure 1), which had not been previously recorded in this area. Given the importance of accurate species identification for effective management and the lack of available data on the occurrence of H. kelloggi in Mozambique, this study presents both morphological and molecular evidence for the first time of the occurrence of H. kelloggi on the central coast of Mozambique.
Figure 1. Study area, showing the sampling location of Hippocampus kelloggi specimens, first recorded in Quelimane city.
Materials and methods
Sample collection, DNA extraction, amplification and sequencing
Three specimens of Hippocampus were collected from the by-catch of artisanal fisheries at Zalala beach landing point in Quelimane, Mozambique (17°52′43.45″S, 36°51′24.89″E) (Figure 1). The fishes were captured using trawl net, the same used to capture other species. All the samples used were collected in June 2022.
Species were initially identified using the taxonomic keys of Lourie et al., (Reference Lourie, Pollom and Foster2016). The morphometric measurement and observation were taken from a single representative specimen at the Aquaculture Laboratory of the School of Marine and Coastal Science at the University of Eduardo Mondlane in Mozambique. Muscle tissue was removed and preserved in 96% ethanol for molecular analyses in the Evolution Laboratory at the Federal University of Pará, Brazil.
The genomic DNA was extracted using a Wizard Genomic DNA Purification kit (Promega, WI, USA), following the protocol for the extraction of muscle tissue defined by the manufacturer. The purity and concentration of the total DNA were evaluated in a Nanodrop 2000 spectrophotometer (Thermo Scientific, CA, USA). Partial sequences of the cytochrome oxidase subunit I (COI) gene were amplified by polymerase chain reaction (PCR) using the primers FishF1 and FishR1 (Ward et al., Reference Ward, Zemlak, Innes, Last and Hebert2005).
The PCRs were run in a final volume of 15 μl containing 2.5 μl of deoxynucleotide triphosphates (1.25 mM), 1.5 μl of 10× buffer solution, 0.7 μl of MgCl2 (50 mM), 0.5 μl of each primer (10 pmol μl−1), 1.0 μl of total genomic DNA (100 ng μl−1), 0.2 μl of Taq DNA polymerase (5 U μl−1) and pure water to complete the final volume of the reaction. The amplification protocol consisted of initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 40 s, hybridization at 50 °C for 40 s and then extension at 72 °C for 60 s, followed by a final extension at 72 °C for 5 min. The amplified PCR products were purified with polyethylene glycol 20% and sequenced using the Sanger method, with a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. Electrophoresis was run in an ABI 3500 XL (Thermo Fisher).
Data analyses
To assemble the final database, 39 sequences were obtained from GenBank and three sequences were generated in this study. From these, 38 sequences belonged to species of the genus Hippocampus, two sequences belonged to species of the genus Corythoichthys (Corythoichthys flavofasciatus and Corythoichthys schultzi) used as outgroups and another two sequences of Syngnathus were used as sister groups (Syngnathus abaster and Syngnathus schmidti).
The partial sequences of the COI gene were aligned and edited in BioEdit 7.1.9 (Hall, Reference Hall1999). Subsequently, for the identification of the species, the sequences were submitted to the Barcoding of Life Database (BOLDsystem) and the National Center for Biotechnology Information (GenBank). Inter and intraspecific genetic distances were calculated using Mega X software (Tamura et al., Reference Tamura, Stecher and Kumar2021) based on the K2P model. Maximum likelihood (ML) analysis was performed in the Iqtree software (Nguyen et al., Reference Nguyen, Schmidt, Haeseler and Minh2015), with 10,000 bootstrap pseudoreplicates, using the TN + F + I + G4 evolutionary model. The evolutionary models were selected based on the Bayesian information criterion using jModelTest2 software (Darriba et al., Reference Darriba, Taboada, Doallo and Posada2012). The trees were edited using the FigTree 1.4.3 (Rambaut, Reference Rambaut2017).
Results
The mitochondrial DNA dataset consisted of 593 bp for the COI gene. The phylogenetic analysis demonstrated a clear separation of species, however few nodes with high support values (Figure 2). The generated sequences clustered together with the other species of H. kelloggi, being a monophyletic group with a support value for the clade of 97.
Figure 2. Maximum likelihood tree with the COI gene sequences. The sequences of Hippocampus spp. in red correspond to the species found in Mozambique. The vertical bar highlights the group formed by Hippocampus kelloggi species.
The genetic divergence within the Mozambique Hippocampus species was 0%, and the distance between Hippocampus from Mozambique and the H. kelloggi group was 0.2%. This grouping of Hippocampus from Mozambique with the H. kelloggi group indicates a close relationship, whereas the distance between the H. kelloggi group and other species ranged from 7% (H. kelloggi × H. spinosissimus) to 22% (H. kelloggi × H. nalu). Genetic distances between other species in the genus varied from 1% (Hippocampus reidi × Hippocampus algiricus) to 24.8% (H. nalu × Hippocampus breviceps) (see Table S1).
These genetic findings confirmed the morphological characteristics of the specimens, including a narrow body with thick rings, tiny white spots running vertically on the body, low and rounded spines, medium–high coronet with five short spines and a high plate in front, prominent cheek and eye spines, which are consistent with the description of H. kelloggi, which has distinct characteristics from other Hippocampus species that occur in the region of Mozambique (Table 1).
Table 1. Morphological characteristics of the seahorse, Hippocampus kelloggi used in this study and others species that occur in Mozambique
Discussion
Seahorses are a group of iconic and heavily exploited fish species, with over 20 million individuals captured annually for use in TCM and aquarium (Chang et al., Reference Chang, Jang-Liaw, Lin, Fang and Shao2013). The high rate of capture is a concern for seahorse conservation, particularly as many species have yet to be described for certain distribution areas, as is the case with H. kelloggi, the focus of this study, which was found in estuarine regions of Mozambique. This observation represents the first record of the species in this region and was confirmed through molecular data using the partial mitochondrial COI gene, which indicates that the species is widely distributed in the Indo-Pacific region. The broad distribution of relatively sedentary species such as H. kelloggi can be explained by rafting, where individuals are carried by ocean currents, and by their ability to rapidly adapt to new environments (Li et al., Reference Li, Olave, Hou, Qin, Schneider, Gao, Tu, Wang, Qi, Nater, Kautt, Wan, Zhang, Liu, Zhang, Zhang, Zhang, Qu, Liu, Chen, Zhong, Zhang, Meng, Wang, Yin, Huang, Venkatesh, Meyer, Lu and Lin2021).
Several studies have shown that diversification of this species group occurred via dispersal routes, for example, Li et al. (Reference Li, Olave, Hou, Qin, Schneider, Gao, Tu, Wang, Qi, Nater, Kautt, Wan, Zhang, Liu, Zhang, Zhang, Zhang, Qu, Liu, Chen, Zhong, Zhang, Meng, Wang, Yin, Huang, Venkatesh, Meyer, Lu and Lin2021) using genomic analysis showed that around 13 Ma, the ancestors of H. kelloggi and H. spinosissimus emerged as a new lineage dispersing across the Indo-Australian archipelago after the closure of the Tethys Sea, increasing diversity in the original centre of seahorse species.
The use of molecular tools and morphological data has been widely used for species identification and delineation. The COI gene is a powerful gene for the genetic identification of species, including in forensic work that helps in the identification of dried seahorses, due to their morphological changes after the drying process (Chang et al., Reference Chang, Jang-Liaw, Lin, Fang and Shao2013; Wang et al., Reference Wang, Zhong, Guo and Hou2020; Serite et al., Reference Serite, Ntshudisane, Swart, Simbine, Jaime and Teske2021).
In addition to molecular identification, the ML tree generated from partial COI sequences of H. kelloggi with other representatives of the genus showed a clear separation of species and monophyly in all clades, corroborating with previous studies (Chang et al., Reference Chang, Jang-Liaw, Lin, Fang and Shao2013; Wang et al., Reference Wang, Zhong, Guo and Hou2020; Li et al., Reference Li, Olave, Hou, Qin, Schneider, Gao, Tu, Wang, Qi, Nater, Kautt, Wan, Zhang, Liu, Zhang, Zhang, Zhang, Qu, Liu, Chen, Zhong, Zhang, Meng, Wang, Yin, Huang, Venkatesh, Meyer, Lu and Lin2021). The data also reveal recent separation between some groups, such as the complex involving the species of H. algiricus and H. reidi, in addition to Hippocampus capensis and Hippocampus fuscus, with low genetic divergence values. Furthermore, the morphological data are in agreement with the measurements described in other studies that characterized H. kelloggi, reinforcing the results obtained through the COI gene (Lourie et al., Reference Lourie, Pollom and Foster2016; Behera et al., Reference Behera, Mahari and Mishira2023).
The great seahorse has been extensively explored within the genus, as it is one of the largest representatives in size and has been found in several studies involving the identification of dry-marketed seahorses (Chang et al., Reference Chang, Jang-Liaw, Lin, Fang and Shao2013; Wang et al., Reference Wang, Zhong, Guo and Hou2020; Serite et al., Reference Serite, Ntshudisane, Swart, Simbine, Jaime and Teske2021). The lack of recognition of many taxa included in species complexes, in addition to underestimating diversity in various classes, leads to misinterpretation of ecological patterns and geographic distribution. For instance, nothing is known about the distribution patterns and evolutionary aspects of the Hippocampus species in the coastal areas of Mozambique. This is even more worrying because one of the most important criteria for species conservation is the geographic distribution.
Although H. kelloggi is a widely distributed species throughout the Indo-Pacific, new records on the occurrence of H. kelloggi in the coastal waters of Mozambique suggest that its distribution is much wider than what is known, which in turn increases the need for greater monitoring of these environments for better conservation and management of this seahorse species.
Conclusions
In conclusion, this study increases the extension area of occurrence of the H. kelloggi species, confirming once again the efficiency of DNA barcoding associated with morphological data for species identification, which can be used for studies involving the conservation of seahorses and their habitat.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0025315423000668.
Acknowledgements
We are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, the Conselho Nacional de Desenvolvimento Científico e Tecnológico, and the Programa de Apoio a Publicação Qualificada of the Pró-Reitoria de Pesquisa e Pós-Graduação for providing financial support.
Author contributions
V. Muhala and A. Guimarães-Costa made the genomic DNA extraction, conceptualization and draft preparation of the paper; G. Rocha and I. E. Macate collected the samples and made field morphological and meristic identification; R. P. Corrêa de Sousa carried out the bioinformatic analysis; J. Carneiro performed the data curation and validation and supervised the study; V. Muhala, L. Watanabe and R. P. Corrêa de Sousa led the original paper writing; M. Vallinoto reviewed and edited the paper; I. Sampaio administrated the project and made funding acquisition. All authors have read and agreed to the published version of the manuscript.
Financial support
This research was financed by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) through a research project 407536/2021-3, 309916/2021-6 and the APC was funded by Pro-Reitoria de Pesquisa e Pós-Graduação of the Universidade Federal do Pará.
Competing interest
None.
Ethical standards
The present study did not involve any experimentation with live specimens. Therefore, no live specimen was caught for the study or kept in captivity. Therefore, there was no need for ethical approval.
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