First Integrative Taxonomic Insights into the Genus Hemiramphus (Family: Hemiramphidae) from the Indian Coast

First Integrative Taxonomic Insights into the Genus Hemiramphus (Family: Hemiramphidae) from the Indian Coast | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First Integrative Taxonomic Insights into the Genus Hemiramphus (Family: Hemiramphidae) from the Indian Coast Toji Thomas, E. M Abdussamad, Badarul Sijad This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7178444/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Dec, 2025 Read the published version in Thalassas: An International Journal of Marine Sciences → Version 1 posted 11 You are reading this latest preprint version Abstract The taxonomic status of Hemiramphus Cuvier, 1816 species along the Indian coast has been subject to frequent misidentifications, complicating ecological and fisheries research. This study provides a comprehensive assessment of the morphometric, meristic, and molecular characteristics of Hemiramphus far (Forsskål, 1775), H. lutkei (Valenciennes, 1847), and H. archipelagicus (Collette & Parin, 1978) to clarify their identification. Morphological analyses were conducted based on key diagnostic features, including eye shape, nostril position, body markings, beak length, pectoral fin length, body depth, vertebrae, and otolith shape. Additionally, molecular data from the COI and 16S rRNA genes were analysed using maximum likelihood phylogenetic methods to assess genetic divergence and species relationships. The findings refine the taxonomic delineation of Hemiramphus species in Indian waters, enhancing accuracy in species identification. This study provides critical baseline data for fisheries management, biodiversity conservation, and future ecological research. Halfbeaks morpho-meristic molecular 16SrRNA COI phylogenetic tree Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. INTRODUCTION The family Hemiramphidae (halfbeaks) is characterized by an elongated lower jaw, except in the genus Oxyporhamphus (Collette & Su, 1986 ). It comprises eight genera and 61 species globally. The genus Hemiramphus , derived from the Greek words hemi (half) and rhamphos (bill or beak), includes 11 marine species (Froese & Pauly, 2025), distributed across the Atlantic, Indian, and Pacific Oceans (Collette, 2004 ). Among them, H. balao , H. bermudensis , and H. brasiliensis are restricted to the Atlantic, while H. depauperatus , H. robustus , and H. saltator occur only in the Pacific. The remaining five species— H. archipelagicus , H. convexus , H. far , H. lutkei , and H. marginatus —are distributed throughout the Indo-Pacific region (Froese & Pauly, 2025). Hemiramphus archipelagicus is distributed across the Indo-Pacific, including Oman, Iran, Pakistan, Sri Lanka, Vietnam, Taiwan, Indonesia, and the Central Pacific (Behera et al. 2020 ; Natan et al. 2019 ; Tabassum et al. 2017 ). Originally described from the western Indian Ocean (Collette & Parin, 1978 ), it was initially believed to extend only as far north as the Philippines (Collette & Parin, 1978 ; Collette, 1999 ). However, a recent record from Taiwan represents its northernmost occurrence and marks the third Hemiramphus species recorded from the region (Hata et al. 2018 ). In Indian waters, H. archipelagicus has been confirmed from both the western and eastern coasts, including new records from the Bay of Bengal (Behera et al. 2020 ). Hemiramphus far has a wide Indo-Pacific distribution and is known to have migrated into the Mediterranean via the Suez Canal, a phenomenon referred to as Lessepsian migration. It has been recorded from Tunisian waters (Suissi et al. 2005), Güllük Bay in the southeastern Aegean Sea (Akyol & Ertosluk, 2019 ), and İzmir Bay in the northern Aegean Sea (Akyol & Tosunoğlu, 2020 ). Its westward expansion in the Mediterranean has been documented, though it has not yet reached the western basin (Kara et al. 2012 ), and an additional record has been reported from the Algerian coast. In Asia, H. far has been documented in the South China Sea, where its complete mitochondrial genome was sequenced for phylogenetic studies of Beloniformes (Zhu et al. 2018 ). It has also been reported from Jeju Island, Korea (Kim et al. 2014). Within Indian waters, H. far was first recorded by Behera et al. ( 2020 ), with additional occurrences from Tamil Nadu (Gopalakrishnan et al. 2010 ). Hemiramphus lutkei , originally considered a Pacific species, has now been recognized with a broader Indo-Pacific distribution. It has been recorded in Pakistan (Tabassum et al. 2014 ) and Indonesia (Talakua et al. 2022 ). In India, its occurrence has been confirmed from Odisha (Mohanty et al. 2020 ), Thoothukudi on the southeast coast (Kayalvizhi et al. 2018 ), and the Malabar coast (Jaleel et al. 2023), indicating its widespread presence in Indian coastal waters. These species primarily inhabit nearshore waters and play a crucial role in local ecosystems as prey for larger predatory fishes, as well as forming a significant part of small-scale fisheries. In India, Hemiramphus species contribute substantially to coastal fisheries; however, detailed taxonomic studies remain limited, with previous research primarily focused on distribution records and general morphological descriptions (Behera et al. 2020 ). Due to overlapping morphological traits, past studies have often faced challenges in correctly identifying species within this genus. Furthermore, there have been no studies exploring the evolutionary relationships among Indian Hemiramphus species. This study aims to clarify the taxonomy, distribution, and phylogenetic relationships of Hemiramphus species in Indian waters, providing new insights into their systematics and diversity. The findings are expected to contribute to future taxonomic research, ecological studies, and conservation efforts for these ecologically and economically important fishes. 2. MATERIALS AND METHODS 2.1. Sampling and collection Field sampling was conducted along the Indian coastline, covering all major maritime states including Lakshadweep and Andaman &Nicobar Islands. Specimens of Hemiramphus species were collected from fish landing centers across the Indian coast between 2021 and 2023(Fig. 1 & Table 1 ). A total of 150 specimens were collected from local fishermen operating gillnet at a depth of around 0–5 m. The specimens were photographed, and tissue samples were taken from the landing centre. The collected samples were brought to the laboratory for further analysis. 2.2. Morphometric and meristic analysis Standard morphometric measurements were taken using digital calipers with an accuracy of 0.1 mm (Hubbs and Lagler, 1958 ; Haedrich, 1967 ; Fischer, 2013 ). A total of 29 morphometric and 10 Meristic counts were recorded. These data were compared with previously published species descriptions to confirm identification (Collette and Parin, 1978 ; Mohanty et al. 2020 ; Behera et al. 2020 ). 2.3. Molecular analysis Tissue samples were subjected to DNA extraction using the genomic DNA kit (Origin), following the manufacturer’s protocol. The extracted DNA's concentration and purity were determined using a Nanodrop spectrophotometer (Eppendorf). Mitochondrial DNA fragments corresponding to the cytochrome oxidase subunit I (COI) and 16S rRNA genes were amplified through PCR using the primer sets WARD 1 (Ward et al. 2005 ) and Palumf-16s/Palumr-16s (Palumbi, 1996 ), respectively. Each PCR reaction was conducted in a total volume of 25 µl, comprising 50 ng of genomic DNA, 3 mM MgCl₂, and 0.3 mM of each primer. The thermal cycling conditions included an initial denaturation at 94°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 50 seconds for 16S rRNA or 30 seconds for COI, and extension at 72°C for 1 minute for COI or 90 seconds for 16S rRNA. The process concluded with a final extension at 72°C for 7 minutes. The PCR amplicons were examined using 1.2% agarose gel electrophoresis, with ethidium bromide staining for visualization. The quality of the sequences obtained from the PCR products was evaluated using the ABI Sequence Scanner (Version 1.0) after sequencing was conducted at Genspec, Cochin, India. Sequence alignment for COI and 16S rRNA genes, including those of closely related species retrieved from GenBank (Thompson et al. 2003), was performed using the Clustal W algorithm. Maximum likelihood (ML) analysis was conducted on the newly generated sequences using BIO Edit (Hall, 1999 ), while genetic distances were estimated using the Kimura 2-parameter model in MEGA 7 (Kumar et al. 2016 ). A bootstrap probability test with 1000 replicates (Felsenstein, 1985 ) was applied to assess the robustness of the phylogenetic analysis. ML trees were constructed to examine Hemiramphid species distributed along the Indian coast. The obtained COI and 16S rRNA sequences were submitted to NCBI GenBank, and accession numbers were assigned (Table 1 ). A Maximum Likelihood (ML) phylogenetic tree was constructed using the COI sequences obtained in this study along with reference sequences from GenBank. The analysis was performed in MEGA X with 1000 bootstrap replicates (Kumar et al. 2016 ). The Kimura 2-parameter model was used to calculate genetic distances, providing insights into the evolutionary relationships among Hemiramphus species in Indian waters. 3. RESULTS Morphometric, meristic, and molecular analyses were conducted on all the Hemiramphus specimens collected from the Indian coastline. The following section presents the results of our analyses, detailing the morphological variations, meristic counts, and genetic divergence observed among the examined specimens. 3.1 Hemiramphus archipelagicus ( Collette & Parin, 1978) 3.1.1 Systematics Order: Beloniformes Family: Hemiramphidae (Gill, 1859) Genus: Hemiramphus , Cuvier, 1816 Species: Hemiramphus archipelagicus, Collette & Parin, 1978 Common name: Jumping halfbeak Synonyms: No synonyms 3.1.2 Materials examined (a). DNR. No.GB.10.4.6.1.1, TL-265 mm, SL-223 mm, Collected from gill net catch, Tharuvaikulam Fishing Harbour, Tuticorin, Tamil Nadu (8º53´19.52304´´N,78º10´22.7838´´) Coromandel coast, Bay of Bengal Sea, around 0-5 m depth, Toji Thomas, March 2022. (Figure 2a) (b). DNR. No.GB.10.4.6.1.2, TL-310 mm, SL- 256 mm, Gillnet, collected from Kalamukk landing center, Kochi, Kerala (lat./long, 9.98° N, 76.24° E), Arabian Sea, West coast of India, around 0-5 m depth, by Toji Thomas, November 2022. (c). DNR. No.GB.10.4.6.1.3, TL-256mm, SL- 220 mm, Gillnet, Collected from Astaranga-Nuagarh fishing harbour, Odisha (lat./long, 19.98° N, 86.32° E), Bay of Bengal, East coast of India, around 0-5 m depth, by Toji Thomas, February 2020. (d). DNR. No.GB.10.4.6.1.4, TL-246 mm, SL- 211 mm, Gillnet, Collected from Sasson dock, Bombay, Maharashtra (lat./long, 18.90° N, 72.82° E), Arabian Sea, West coast of India, around 0-5 m depth, by Toji Thomas, August 2021. 3.1.3 Diagnosis The diagnosis of H. archipelagicus within the genus Hemiramphus is established based on key morphological and meristic characters. Compared to H. far and H. lutkei , H. archipelagicus possesses a relatively smaller head (4.0–4.4 times in SL) and a lower jaw that is 3.0–3.35 times in SL (Figure 3a). The species differs from H. far in having a deeper caudal peduncle (4.4–4.8% of SL vs. 5.4–6.0% in H. far ), a longer dorsal fin base (10.9–13.4% of SL vs. 9.4–11.9% in H. far ) (Figure 4a), and a greater number of gill rakers on the first gill arch (35–38 vs. 28–31 in H. far). The body is uniformly coloured without vertical bars, distinguishing it from H. far , which exhibits 3–8 vertical bars (Figure 5a). Additionally , H. archipelagicus lacks pigmentation in the fins, whereas H. far has pigmented fins (Figure 2). The anterior dorsal fin lobe is not well developed, whereas it is well developed in H. far and moderately developed in H. lutkei . 3.1.4 Description The body is elongated and laterally compressed. The lower jaw is prolonged (136–155% of HL), while the upper jaw is relatively short (15.4–19.7% of HL). The snout length ranges from 32.4–36.7% of HL. Head length is 17.2–18.7% of SL. Eye diameter is 4.0–4.9% of SL, and interorbital width is greater than ED. The body depth at the anal-fin origin is 9.4–11.8% of SL. The dorsal fin has 13–14 rays, the anal fin has 11–12 rays, the pelvic fin has 6 rays, and the pectoral fin has 11–12 rays. The species possesses 35–38 gill rakers on the first gill arch and 21–26 on the second gill arch. Lateral line scale count ranges from 54–56, and total vertebral count is 53–54 (Table 2). The dorsal fin originates at the 4th–5th ray of the anal fin (Figure4). 3.1.5 Colouration : Characterized by a dark blue dorsal surface and a silvery white ventral side, with a red-tipped darkish beak. A dark lateral stripe widens under the dorsal fin, which is pigmented along the edges but transparent within. The upper caudal fin lobe is yellow, while the rest of the caudal fin is grey with darker edges. The anal, pectoral, and pelvic fins are fully transparent (Figure 2). 3.1.6 O tolith shape : The ventral margin is smoothly convex from the rostrum to the posterior tip, with only a few serrations. The anterior half of the dorsal margin is also smoothly convex (Figure 6a). 3.1.7 Distribution: Marine, distributed around the coastal waters of Tamil Nadu, Kerala, Odisha, Maharashtra and Lakshadweep. 3.1.8 Meristic formula: DF 13–14, AF 11–12, Pect F 11–12, Pel F 6, Gill rakers on first arch 35-38, Gill rakers on second arch 21-26, Vertebrae 53-54, Lateral line scales 54-56. 3.2 Hemiramphus far (Forsskål, 1775) 3.2.1 Systematics Order: Beloniformes Family: Hemiramphidae (Gill, 1859) Genus: Hemiramphus Cuvier, 1816 Species: Hemiramphus far, Forsskål, 1775 Synonyms: Esox far (Forsskål, 1775) , Esox gladius (Lacepède, 1803), Esox marginatus far (Forsskål, 1775), Hemiramphus commersonii (Cuvier, 1829), Hemiramphus mocquardianus (Thominot, 1886), Hemirhamphus commersonii (Cuvier, 1829), Hemirhamphus far (Forsskål, 1775), and Hemirhamphus obesus (Castelnau, 1861 ). 3.2.2 Materials examined (a).DNR.No. GB.10.4.6.6.1, TL-308 mm, SL- 262 mm, Gillnet, Collected from Pamban fishing harbour, Mandapam, Tamilnadu (lat./long, 9.28° N, 79.21° E), Bay of Bengal, East coast of India, around 0-5 m depth, by Toji Thomas, September 2019. (Figure 2b) (b). DNR.No. GB.10.4.6.6.2, TL-293 mm, SL- 249 mm, Gillnet, Collected from Neendakara landing center, Kollam, Kerala (lat./long, 8.94° N, 76.54° E), Arabian Sea, West coast of India, around 0-5 m depth, by Toji Thomas, November 2019. (e). DNR.No. GB.10.4.6.6.3, TL-722 mm, SL- 653mm, Gillnet, Collected from Kavaratti, Lakshadweep (lat./long, 10.57° N,72.63° E), Arabian Sea, West coast of India, around 0-100m depth, by Toji Thomas, in February 2023. 3.2.3 Diagnosis H. far is distinguished from H. archipelagicus and H. lutkei by its greater body depth at the anal-fin origin (10.6–13.0% of SL vs. 9.4–11.8% in H. archipelagicus and 9.3–12.6% in H. lutkei ). The species has a relatively longer lower jaw (132–151% of HL) compared to H. archipelagicus (136–155% of HL) and H. lutkei (132–154% of HL) (Figure 3b). It has a lower number of gill rakers on the first gill arch (28–31 vs. 35–38 in H. archipelagicus and 38–40 in H. lutkei ). The presence of 3–8 dark vertical bars on the body and pigmented fins differentiates H. far from H. archipelagicus and H. lutkei , both of which lack body bars and fin pigmentation (Figure 5b). The anterior dorsal fin lobe is well developed in H. far , whereas it is not well developed in H. archipelagicus and only moderately developed in H. lutkei . 3.2.4 Description The body is elongate, laterally compressed, and covered with 53–54 lateral line scales. The lower jaw is significantly elongated (132–151% of HL), while the upper jaw length ranges from 19.3–23.2% of HL. The snout length is 33.2–35.0% of HL. Head length is 16.8–18.9% of SL. Eye diameter is 4.3–4.9% of SL, and interorbital width is greater than ED. The dorsal fin has 12–13 rays, the anal fin has 10–11 rays, the pelvic fin has 6 rays, and the pectoral fin has 11–12 rays. The species possesses 28–31 gill rakers on the first gill arch and 20–25 on the second gill arch. Predorsal scale count is 35–38, and vertebrae number is 50–53. The dorsal fin originates at the 6th–8th ray of the anal fin (Figure 2b) (Table 2). 3.1.5 Colouration: Bluish dorsal side and silvery flanks, with dark vertical bars on the body. The dorsal fin is yellowish, the caudal fin has a blue lower lobe and a yellowish upper lobe, and the lower jaw tip is red. 3.1.6 Otolith shape: Comparatively broader, with a highly serrated convex ventral margin. The posterior margin is almost straight with distinct, tooth-like serrations. The dorsal margin has a few but distinct serrations(Figure 6b). 3.1.7 Distribution : Marine, distributed around the coasts of Tamil Nadu, Kerala, Odisha, Maharashtra, Lakshadweep and Andaman and Nicobar Islands. 3.1.8 Meristic formula: DF 12–13, AF 10–11, Pect F 11–12, Pel F 6, Gill rakers on first arch 28-31, Gill rakers on second arch 20-25, Vertebrae 50-53, Lateral line scales 53-54. 3.3 Hemiramphus lutkei (Valenciennes, 1847) 3.3.1 Systematics Order: Beloniformes Family: Hemiramphidae (Gill, 1859) Genus: Hemiramphus , Cuvier, 1816 Species: Hemiramphus lutkei, Valenciennes, 1847 Synonyms: Hemiramphus fasciatus (Bleeker, 1853), Hemiramphus japonicus (Brevoort, 1856), and Hemiramphus lukei (Valenciennes, 1847) 3.3.2 Materials examined (a).DNR.No. GB.10.4.6.7.1, TL-332 mm, SL- 284 mm, Collected from Mural net catch, Tharuvaikulam Fishing Harbour, Tuticorin, Tamil Nadu (8º53'19.52304"N,78º10'22.7838") Coromandel coast, Bay of Bengal Sea, around 0-5 m depth, Toji Thomas, March 2021. (Figure 2c) (b). DNR.No. GB.10.4.6.7.2, TL-317 mm, SL- 275 mm, Gillnet, collected from Kalamukk landing center, Kochi, Kerala (lat./long, 9.98° N, 76.24° E), Arabian Sea, West coast of India, around 0-5 m depth, by Toji Thomas, in November 2022. (c). DNR.No. GB.10.4.6.7.3, TL-345 mm, SL- 299 mm, Gillnet, Collected from Astaranga-Nuagarh fishing harbour, Odisha (lat./long, 19.98° N, 86.32° E), Bay of Bengal, East coast of India, around 0-5 m depth, by Toji Thomas, April 2023. (d). DNR.No. GB.10.4.6.7.4, TL-342 mm, SL- 293 mm, Gillnet, Collected from Junghalighat harbour, Port Blair, Andaman & Nicobar Islands (lat./long, 11.65° N, 92.72° E), Bay of Bengal, East coast of India, around 0-100m depth, by Toji Thomas, in March 2022. 3.3.3 Diagnosis H. lutkei is distinguished from H. archipelagicus and H. far by its relatively long pectoral fins, which are significantly longer than in the other two species (Figure 4c). The species has a higher number of gill rakers on the first gill arch (38–40 vs. 35–38 in H. archipelagicus and 28–31 in H. far ), as well as the highest count on the second gill arch (28–33 vs. 21–26 in H. archipelagicus and 20–25 in H. far ) (Table 2). The interorbital width in H. lutkei is less than ED, whereas it is greater than ED in H. archipelagicus and H. far . The species lacks vertical bars on the body and pigmentation in the fins, similar to H. archipelagicus but differing from H. far (Figure5c). The anterior lobe of the dorsal fin is moderately developed, in contrast to H. far , where it is well developed, and H. archipelagicus , where it is not well developed. 3.3.4 Description The body is slender, elongate, and laterally compressed, covered with 55–58 lateral line scales. The lower jaw is elongated (132–154% of HL), while the upper jaw length ranges from 15.0–21.0% of HL. The snout length is 31.0–35.0% of HL. Head length is 17.5–18.9% of SL. Eye diameter is 4.0–4.8% of SL, and interorbital width is less than ED. The dorsal fin has 12–14 rays, the anal fin has 11–13 rays, the pelvic fin has 6 rays, and the pectoral fin has 10–11 rays. The species possesses 38–40 gill rakers on the first gill arch and 28–33 on the second gill arch. Predorsal scale count is 38–42, and vertebrae number is 53–56 (Table 2). The dorsal fin originates at the 4th–5th ray of the anal fin (Figure4) 3.3.5 Colouration: The back is dark blue, transitioning to silvery white on the sides and ventral surface, with no spots or bars. The lower jaw's fleshy tip is bright red, and the upper lobe of the caudal fin has a bluish hue. 3.3.6 Otolith shape: Less deep than H. archipelagicus and H. far , with a less convex dorsal margin and nearly straight edges. The dorsal margin has fewer but comparatively larger and more distinct teeth(Figure 6c). 3.3.7 Distribution: Marine, distributed over the coasts of Tamil Nadu, Kerala, Odisha, Karnataka, Lakshadweep and Andaman and Nicobar Islands. 3.3.8 Meristic formula: DF 12–14, AF 11–13, Pect F 10–11, Pel F 6, Gill rakers on first arch 38-40, Gill rakers on second arch 28-33, Vertebrae 53-56, Lateral line scales 55-58. 3.4 Identification key for genus hemiramphus from Indian waters -Adapted from Collette, B.B. 1999. Hemiramphidae. Halfbeaks. p. 2180-2196. In K.E. Carpenter and V. Niem (eds.) FAO species identification guide for fishery purposes. The living marine resources of the Western Central Pacific. Vol. 4. Bony fishes part 2 (Mugilidae to Carangidae). FAO, Rome. 1a . Lower jaw not noticeably elongate in adults; anterior margin of upper jaw straight, not forming a prominent triangular anterior projection; pectoral fins long, 30 to 35% of standard length……… Oxyporhamphus 1b . Lower jaw elongate or not; anterior margin of upper jaw forming a prominent triangular anterior projection; pectoral fins not more than 28% of standard length……. 2 2a . Nasal papilla rounded, fan-shaped, or fimbriate; not projecting far beyond nasal fossa; caudal fin emarginate or forked, frequently with an elongate lower lobe; anal fin of males not different from those of females……… 3 2b . Nasal papilla elongate and pointed, not fimbriate; projecting well beyond nasal fossa; caudal fin rounded or truncate, with the longest rays in the middle of fin; anal fin of males modified, some rays widened and elongate. (freshwater and estuarine genera) …… 3a . Body compressed and ribbon-shaped; dorsal-fin rays 20 to 25; anal-fin rays 20 to 25; pectoral fins long, 25 to 28% of standard length; pectoral-fin rays usually 7 to 9…… Euleptorhamphus 3b . Body not ribbon-shaped; dorsal-fin rays 12 to 18; anal-fin rays 10 to 19; pectoral short, less than 20% of standard length; predorsal-fin rays 10 to 14…… 4 4a . Scales absent on upper jaw; preorbital ridge absent……. 5 4b . Scales present on upper jaw; preorbital ridge well developed…… Hyporhamphus and Rhynchorhamphus 5a . Presence of 3-9 vertical bars on the body, Dorsal fin is elongated 9.5-12% of standard length with a well-developed and pigmented anterior lobe; gill rakers 27-32; Caudal peduncle depth is high about 5.4-6 %. ……… Hemiramphus far 5b. Absence of vertical bars or spots on the body, dorsal fin short 7-9.5 % of standard length, not well-developed anterior lobe, presence or absence of pigmentation. Gill rakers 35-40, Caudal peduncle depth is less; 4.5-5% of standard length……... 6 6a . Pectoral fins relatively long, 4.5 to 5.4 times in standard length; not well-developed anterior lobe, absence of pigmentation; gill rakers on first arch 38 to 40, usually 38 or more; predorsal scales 35 to 43, usually more than 37…... Hemiramphus lutkei 6b. Pectoral fin relatively short, 4-4.5 times in standard length; not well-developed anterior lobe, presence of pigmentation; gill rakers on first arch 35 to 38, usually 36 or more; predorsal scales 35 to 43, usually more than 37…... Hemiramphus archipelagicus 3.4 Molecular analysis 3.4.1 Cytochrome oxidase I (COI) analysis DNA barcoding of the COI gene yielded clear differentiation among the three species. The Maximum Likelihood (ML) phylogenetic tree constructed from COI sequences (Figure 8) showed distinct clustering of H. lutkei , H. far , and H. archipelagicus . Genetic distances (K2P) calculated for COI sequences (Table 4) indicated moderate divergence between species, supporting morphological findings. H. archipelagicus shows a genetic difference of 7.5% with H. lutkei and 4.3% with H. far, while H. far and H. lutkei has a genetic difference of 6.1%.The H. far sequences form a distinct monophyletic clade, with present study sequences clustering closely with previously reported GenBank sequences (e.g., BU148547.1, MT888964.1). Similarly, H. archipelagicus appears as a separate lineage, with present study sequences grouping with reference sequences. H. lutkei also forms a well-defined cluster, with present study sequences showing close genetic similarity to GenBank entries such as OR113909.1 and MN855097.1 3.4.2 16S rRNA analysis Phylogenetic analysis of 16S rRNA sequences aligned with COI results, displaying clear separations among Hemiramphus species in the ML tree (Figure 9). The K2P distances for 16S rRNA (Table 5) reinforced the genetic distinctiveness of each species. H. archipelagicus shows a genetic difference of 2.5% with H. lutkei and 1% with H. far, while H. far and H. lutkei has a genetic difference of 2.1%. The sequence comparisons were supplemented by reference data from GenBank and BOLD, providing a comprehensive genetic profile (Supplementary Table 1). A detailed morphological examination of the specimens collected in this study confirmed their identification as H. lutkei . Following this confirmation, we performed BLAST analysis using the 16S sequences obtained from our specimens (Madden, 2013). The results revealed that the sequences of H. lutkei in our study closely aligned with sequences previously reported in GenBank under the names Hemiramphus balao (OP056959.1, AF243948.1) and Hemiramphus brasiliensis (NC_088002.1). This indicates that these GenBank sequences were misidentified in earlier studies and actually represent H. lutkei . The present study sequences of H. lutkei , H. archipelagicus , and H. far are positioned within their respective clades, showing close genetic affinity with reference sequences such as MK561619.1 for H. far . The high bootstrap values across major nodes strengthen the reliability of these phylogenetic relationships. Additionally, Ablennes hians serves as an outgroup, reinforcing the evolutionary framework of the Hemiramphus species. 4. DISCUSSION Integrative taxonomic approaches have proven crucial in resolving taxonomic ambiguities in various fish species. As seen in the recent reclassification of Scomberomorus guttatus using morphological, meristic, and genetic data (Abdussamad et al. 2024). Such methods have also been instrumental in revealing hidden diversity, as demonstrated in the goosefish genus Lophiomus , where the application of multigene phylogenetic inferences and morphometric analyses led to the identification of six species, including three new species and the resurrection of Chirolophius laticeps (Chen et al. 2024). Recent studies have increasingly highlighted the significance of integrative taxonomic approaches, which merge morphological and molecular data, for precise species identification and classification, especially in intricate groups such as the Beloniformes. For instance, the complete mitochondrial genome of H. far has been sequenced (Zhu et al. 2018), offering valuable molecular resources for phylogenetic studies. Moreover, the effectiveness of integrative taxonomy is evident in the recent description of new Belonidae species, as demonstrated by the identification of two new Ablennes needlefish species from the Indian Ocean (Toji et al. 2024). Additionally, the first report of H. archipelagicus from the western Bay of Bengal (Behera et al. 2020) has broadened our understanding of this species' distribution, further emphasizing the importance of detailed regional taxonomic studies. Studies on the length-weight relationship of nine Hemiramphidae species (Thomas et al. 2025) provide additional insights into the ecological diversity within this family. These findings emphasize that species complexes are more prevalent than previously recognized and underscore the necessity of detailed taxonomic work. This study delivers a detailed taxonomic analysis of three Hemiramphus species ( H. archipelagicus , H. far , and H. lutkei ) from Indian waters, employing an integrative approach that combines morphometric, meristic, and molecular data. This comprehensive strategy enables a more robust species delimitation and enhances our understanding of the relationships within this complex genus. The results corroborate the distinctness of these three species, emphasizing key morphological and meristic differences, and supporting these findings with molecular phylogenetic analyses. The three Hemiramphus species examined, H. archipelagicus , H. far , and H. lutkei , exhibit several distinguishing morpho-meristic characteristics beyond simple measurements. A key differentiating feature lies in their colouration and fin pigmentation. H. far is distinguished from the other two species by possessing 3-8 vertical bars on the body and yellowish or bluish colouration on the fins. Dorsal profile of H. lutkei is characterised by dark bluish or blackish colouration. Further distinctions can be observed in the development of the anterior dorsal fin lobe. In H. far , this lobe is well developed, whereas it is not well developed in H. archipelagicus and shows a moderate level of development in H. lutkei . Meristic counts also provide valuable characters for species delineation. H. lutkei possesses the highest number of gill rakers on the first arch (38–40), H. archipelagicus has an intermediate count (35–38), while H. far has the lowest number (28–31). Additionally, H. lutkei is unique in having an interorbital width that is less than its eye diameter, a feature that distinguishes it from both H. archipelagicus and H. far , where the interorbital width is greater than the eye diameter. Finally, H. lutkei is also characterised by its relatively longer pectoral fins compared to the other two species. Additional distinguishing characters are provided in Table 3. These subtle yet consistent differences support the species delimitation within the genus. The value of otolith morphology in distinguishing fish species has been increasingly recognized (Abdussamad et al. 2015, 2016, 2022; Joshi et al. 2012; Karahan et al. 2014; Libungan et al. 2015; Libungan & Pálsson, 2015). For example, Abdussamad et al. (2024) utilized otolith morphometry to establish taxonomic divergence within the Scomberomorus guttatus complex, demonstrating that otoliths can serve as a reliable alternative taxonomic tool. The differences in otolith shapes observed in this study offered extra characteristics that helped to distinguish between H. archipelagicus , H. far , and H. lutkei . Specifically, the otolith of H. archipelagicus is characterised by a smoothly convex ventral margin with few serrations and an anterior dorsal margin that is also smoothly convex. In contrast, H. far possesses a comparatively broader otolith with a highly serrated convex ventral margin and a nearly straight posterior margin exhibiting distinct, tooth-like serrations, along with a few distinct serrations on the dorsal margin. The otolith of H. lutkei is less deep than those of the other two species, displaying a less convex dorsal margin with nearly straight edges and fewer but comparatively larger and more distinct teeth on its dorsal margin. These distinct otolith morphologies further reinforce the taxonomic separation of these three Hemiramphus species. The molecular phylogenetic analyses, employing COI and 16S rRNA sequences, strongly corroborate the distinctness of all three Hemiramphus species. The clustering of individuals from each species in the respective clades on the maximum likelihood trees validates their identification based on morphological and meristic data. The incorporation of publicly available sequences in the phylogenetic analyses reinforces these results, enabling a broader comparison and confirmation of species boundaries. The genetic distances between H. archipelagicus and H. lutkei were 7.5% (COI) and 2.5% (16S rRNA); between H. archipelagicus and H. far , 4.3% (COI) and 1.0% (16S rRNA); and between H. far and H. lutkei , 6.1% (COI) and 2.1% (16S rRNA). These genetic distances align with established thresholds for species differentiation in marine fishes. These findings reinforce the morphological distinctions observed in meristic counts and morphometric ratios, validating the taxonomic status of the studied species. The consistency between morphological and molecular data highlights the importance of an integrative taxonomic approach. This study significantly enhances our understanding of the taxonomy of Hemiramphus fishes in Indian waters. It builds on the existing knowledge of Beloniformes diversity in the region (Thomas, 2022; Thomas et al. 2024; Thomas et al. 2025) by providing detailed descriptions and diagnoses of the three Hemiramphus species, along with supporting molecular evidence. This work clarifies the identification of these closely related species, which is essential for effective management and sustainable exploitation of fish resources, as well as for their use as effective biological indicators. Future research could explore the genetic diversity and population structure of these species across their geographic ranges and investigate the evolutionary relationships within the genus Hemiramphus on a broader scale. Further studies into the functional significance of the observed morphological differences, such as variations in fin length and gill raker number, would also be valuable. Declarations FUNDING This research has been carried out by a Junior Research Fellowship (JRF) jointly funded by Council of Scientific and Industrial Research (CSIR) and University Grants Commission (UGC), India. CONFLICT OF INTEREST The authors have no financial or proprietary interests in any material discussed in this article. The authors declare no competing interests. ACKNOWLEDGMENTS We express our sincere gratitude to Dr. Grinson George, the Director of the Central Marine Fisheries Research Institute -Kochi, for granting us access to the research space. The authors are deeply thankful to the University Grants Commission (UGC) for availing the research fellowship. The authors are greatly thankful to the faculties of the Department of Biosciences, Mangalore University for helping me to fulfill my Ph. D research. The authors are deeply thankful to Mr. Asish Gopi for assistance with the illustrations. We express our gratitude to the faculty and personnel of the CMFRI's finfish fisheries section for their invaluable assistance with this work. AUTHOR CONTRIBUTION T.T- Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, and Writing an original draft. E.M.A – Supervision and, Project administration, and Review and editing the original draft. 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Sci. , 360(1462), 1847-1857. https://doi.org/10.1098/rstb.2005.1716 Zhu, K., Lü, Z., Liu, B., Gong, L., Jiang, L., & Liu, L. (2018). The complete mitochondrial genome of Hemiramphus far (Beloniformes; Hemiramphidae) and phylogenetic studies of Beloniformes. Mitochondrial DNA Part B , 3(2), 1237-1238. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table 1. Sampling locations with coordinates, museum accession numbers (CMFRI), and GenBank accession numbers (NCBI) for specimens analyzed in the present study. Table2.docx Table 2. Morphometric (expressed as % of SL and % of HL) and meristic data for H. archipelagicus , H. far , and H. lutkei in the present study. Table3.docx Table 3. Morphometric measurements and meristic counts of Hemiramphus archipelagicus , Hemiramphus far , and Hemiramphus lutkei from the present study compared with data from previous studies. Table4.docx Table 4. Mean pairwise genetic distances (K2P) of COI sequences for Hemiramphus species, including sequences from the present study and GenBank. Table5.docx Table 5. Mean pairwise genetic distances (K2P) of 16S rRNA sequences for Hemiramphus species, including sequences from the present study and GenBank. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7178444","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":489165890,"identity":"d21b5239-15e5-465d-bd92-d3cf74ebe134","order_by":0,"name":"Toji Thomas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYHCChAOMDSCasfHBhwobMOMAPvU8IC0HwVqYmw1nnEkDaWkgpIWBAaKFvU2at+0wWBSvFnv2hoeHP+6wiebvP9gmzcN23m5t+2GgLTU20Tht4TkAdNiZtNwZNxKbLefw3E7ediYRqOVYWm4DLi0SCUAtbYdzG24wNt54I3E72ewAUAtjw2HCWuafP9ggwWNwLtns/EMitWw4kNgkyZNwwM7sBiFbzgD9crYtLXcj0C+GMw4kJ5jdANqSgMcv7O09yR8q22xy550//vDBx3929mbn0x8++FBjg1ML0J4EFG4iWGUCpjpkew6gcO3xKh4Fo2AUjIIRCQA9v3S2B+2RmQAAAABJRU5ErkJggg==","orcid":"","institution":"ICAR- Central Marine Fisheries Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Toji","middleName":"","lastName":"Thomas","suffix":""},{"id":489165891,"identity":"f913b335-e1c0-46d8-a5c6-39fafc8aebc8","order_by":1,"name":"E. 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10:38:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94791,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing sampling locations where specimens were collected for the present study.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/076dd1ebb6ef12c5d7b9ea47.png"},{"id":87484065,"identity":"b3f5eb25-a056-422d-bd26-a9ae6f32bcdc","added_by":"auto","created_at":"2025-07-24 10:38:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115411,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative fresh specimens of halfbeak species used for in this study (scale bar = 3 cm): a) \u003cem\u003eH. archipelagicus\u003c/em\u003e(SL=223 mm), b) \u003cem\u003eH. far\u003c/em\u003e (SL=262 mm), and c) \u003cem\u003eH. lutkei\u003c/em\u003e (SL=242 mm).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/9cf1615dfb097ea200169006.png"},{"id":87484108,"identity":"2cfdf4b2-8db4-4ac1-99a6-ba7eb048b581","added_by":"auto","created_at":"2025-07-24 10:38:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116187,"visible":true,"origin":"","legend":"\u003cp\u003eHead and eye shape (scale bar = 2 cm) of: a) \u003cem\u003eH. archipelagicus\u003c/em\u003e, b) \u003cem\u003eH. far\u003c/em\u003e, and c) \u003cem\u003eH. lutkei\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/4c0443dfe294368a9b5750d2.png"},{"id":87484058,"identity":"1b2c0650-8bda-4db0-b577-efacf529fac2","added_by":"auto","created_at":"2025-07-24 10:38:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":171722,"visible":true,"origin":"","legend":"\u003cp\u003ePectoral fin length, and dorsal fin (DF) - anal fin (AF) origin (scale bar = 2 cm) of: a) \u003cem\u003eH. archipelagicus\u003c/em\u003e, b) \u003cem\u003eH. far\u003c/em\u003e, and c) \u003cem\u003eH. lutkei\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/34508da28bd5cfcbe6566032.png"},{"id":87484077,"identity":"5608b8b6-04ee-4c9d-bfae-e64f0b201214","added_by":"auto","created_at":"2025-07-24 10:38:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":103850,"visible":true,"origin":"","legend":"\u003cp\u003eBars on body and caudal shape (scale bar = 2 cm) of: a) \u003cem\u003eH. archipelagicus\u003c/em\u003e, b) \u003cem\u003eH. far\u003c/em\u003e, and c) \u003cem\u003eH. lutkei\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/28b43b7c7d0da43f88506ec1.png"},{"id":87484032,"identity":"81ff4350-85f2-4d7e-81ef-b092a22b4d87","added_by":"auto","created_at":"2025-07-24 10:38:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":94491,"visible":true,"origin":"","legend":"\u003cp\u003eOtolith shape (scale bar = 1 mm) of: a) \u003cem\u003eH. archipelagicus\u003c/em\u003e, b) \u003cem\u003eH. far\u003c/em\u003e, and c) \u003cem\u003eH. lutkei\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/c252213a03f71134627fa125.png"},{"id":87484064,"identity":"64e51c44-873a-489e-a52c-9b979517373b","added_by":"auto","created_at":"2025-07-24 10:38:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":59957,"visible":true,"origin":"","legend":"\u003cp\u003eVertebrae. a) \u003cem\u003eH. archipelagicus\u003c/em\u003e, b) \u003cem\u003eH. far\u003c/em\u003e, and c) \u003cem\u003eH. lutkei\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/efd2211c1d9b301082713eef.png"},{"id":87485026,"identity":"b18786cc-b104-44ad-a66a-043bacd97740","added_by":"auto","created_at":"2025-07-24 10:46:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":180311,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood tree of \u003cem\u003eHemiramphus\u003c/em\u003e species based on COI sequences from this study and GenBank, with \u003cem\u003eAblennes hians\u003c/em\u003e as the outgroup.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/5c50b9bda4a8d1bb474d2a85.png"},{"id":87484113,"identity":"ef8996bc-e73a-446c-a579-0ec0e7bca097","added_by":"auto","created_at":"2025-07-24 10:38:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":64482,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood tree of \u003cem\u003eHemiramphus\u003c/em\u003e species based on 16S rRNA sequences from this study and GenBank, with \u003cem\u003eAblennes hians\u003c/em\u003e as the outgroup.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/f2f6103d2a98fce39a5ea3a3.png"},{"id":97723770,"identity":"920eb001-9b6a-4ef6-b7b1-eba2333f3117","added_by":"auto","created_at":"2025-12-08 16:05:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1979283,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/cea75e63-f7de-409f-a6e2-b9df128a06e9.pdf"},{"id":87485021,"identity":"953b0072-410c-4a46-a5f0-6d63693c5397","added_by":"auto","created_at":"2025-07-24 10:46:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28141,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. \u003c/strong\u003eSampling locations with coordinates, museum accession numbers (CMFRI), and GenBank accession numbers (NCBI) for specimens analyzed in the present study.\u003c/p\u003e","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/29ef88a46ac15d0a23ed2b85.docx"},{"id":87484102,"identity":"66f3eb66-cf8d-4b1e-9889-4329065958b7","added_by":"auto","created_at":"2025-07-24 10:38:25","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":41979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 2. \u003c/strong\u003eMorphometric (expressed as % of SL and % of HL) and meristic data for \u003cem\u003eH. archipelagicus\u003c/em\u003e, \u003cem\u003eH. far\u003c/em\u003e, and \u003cem\u003eH. lutkei\u003c/em\u003e in the present study.\u003c/p\u003e","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/6a4e49f75cb16241be3b97fb.docx"},{"id":87484079,"identity":"dd40c57b-a2b4-4b50-bc88-aff8ee704a30","added_by":"auto","created_at":"2025-07-24 10:38:24","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":38167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e. Morphometric measurements and meristic counts of \u003cem\u003eHemiramphus archipelagicus\u003c/em\u003e, \u003cem\u003eHemiramphus far\u003c/em\u003e, and \u003cem\u003eHemiramphus lutkei\u003c/em\u003e from the present study compared with data from previous studies.\u003c/p\u003e","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/e5f7f9dc0cbc0d928be2f21e.docx"},{"id":87485025,"identity":"b8d32250-f7c5-4009-922a-84fcd7b69b50","added_by":"auto","created_at":"2025-07-24 10:46:25","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 4. \u003c/strong\u003eMean pairwise genetic distances (K2P) of COI sequences for \u003cem\u003eHemiramphus\u003c/em\u003e species, including sequences from the present study and GenBank.\u003c/p\u003e","description":"","filename":"Table4.docx","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/b1b327eb8b74eb3b0bfde338.docx"},{"id":87484100,"identity":"40dd38b8-d534-444a-984d-216e1f2b30fd","added_by":"auto","created_at":"2025-07-24 10:38:25","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":17105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 5. \u003c/strong\u003eMean pairwise genetic distances (K2P) of 16S rRNA sequences for \u003cem\u003eHemiramphus\u003c/em\u003e species, including sequences from the present study and GenBank.\u003c/p\u003e","description":"","filename":"Table5.docx","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/ca233ee3ad2434424eeab273.docx"},{"id":87485874,"identity":"4eab25fe-7e55-4dc5-a40a-e309d839c65c","added_by":"auto","created_at":"2025-07-24 10:54:26","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":26351,"visible":true,"origin":"","legend":"","description":"","filename":"SupplimentaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7178444/v1/1564e042863837cce18382c9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"First Integrative Taxonomic Insights into the Genus Hemiramphus (Family: Hemiramphidae) from the Indian Coast","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe family Hemiramphidae (halfbeaks) is characterized by an elongated lower jaw, except in the genus \u003cem\u003eOxyporhamphus\u003c/em\u003e (Collette \u0026amp; Su, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). It comprises eight genera and 61 species globally. The genus \u003cem\u003eHemiramphus\u003c/em\u003e, derived from the Greek words \u003cem\u003ehemi\u003c/em\u003e (half) and \u003cem\u003erhamphos\u003c/em\u003e (bill or beak), includes 11 marine species (Froese \u0026amp; Pauly, 2025), distributed across the Atlantic, Indian, and Pacific Oceans (Collette, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Among them, \u003cem\u003eH. balao\u003c/em\u003e, \u003cem\u003eH. bermudensis\u003c/em\u003e, and \u003cem\u003eH. brasiliensis\u003c/em\u003e are restricted to the Atlantic, while \u003cem\u003eH. depauperatus\u003c/em\u003e, \u003cem\u003eH. robustus\u003c/em\u003e, and \u003cem\u003eH. saltator\u003c/em\u003e occur only in the Pacific. The remaining five species\u0026mdash;\u003cem\u003eH. archipelagicus\u003c/em\u003e, \u003cem\u003eH. convexus\u003c/em\u003e, \u003cem\u003eH. far\u003c/em\u003e, \u003cem\u003eH. lutkei\u003c/em\u003e, and \u003cem\u003eH. marginatus\u003c/em\u003e\u0026mdash;are distributed throughout the Indo-Pacific region (Froese \u0026amp; Pauly, 2025).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHemiramphus archipelagicus\u003c/em\u003e is distributed across the Indo-Pacific, including Oman, Iran, Pakistan, Sri Lanka, Vietnam, Taiwan, Indonesia, and the Central Pacific (Behera et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Natan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tabassum et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Originally described from the western Indian Ocean (Collette \u0026amp; Parin, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1978\u003c/span\u003e), it was initially believed to extend only as far north as the Philippines (Collette \u0026amp; Parin, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Collette, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). However, a recent record from Taiwan represents its northernmost occurrence and marks the third \u003cem\u003eHemiramphus\u003c/em\u003e species recorded from the region (Hata et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In Indian waters, \u003cem\u003eH. archipelagicus\u003c/em\u003e has been confirmed from both the western and eastern coasts, including new records from the Bay of Bengal (Behera et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHemiramphus far\u003c/em\u003e has a wide Indo-Pacific distribution and is known to have migrated into the Mediterranean via the Suez Canal, a phenomenon referred to as Lessepsian migration. It has been recorded from Tunisian waters (Suissi et al. 2005), G\u0026uuml;ll\u0026uuml;k Bay in the southeastern Aegean Sea (Akyol \u0026amp; Ertosluk, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and İzmir Bay in the northern Aegean Sea (Akyol \u0026amp; Tosunoğlu, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Its westward expansion in the Mediterranean has been documented, though it has not yet reached the western basin (Kara et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and an additional record has been reported from the Algerian coast. In Asia, \u003cem\u003eH. far\u003c/em\u003e has been documented in the South China Sea, where its complete mitochondrial genome was sequenced for phylogenetic studies of Beloniformes (Zhu et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It has also been reported from Jeju Island, Korea (Kim et al. 2014). Within Indian waters, \u003cem\u003eH. far\u003c/em\u003e was first recorded by Behera et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), with additional occurrences from Tamil Nadu (Gopalakrishnan et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHemiramphus lutkei\u003c/em\u003e, originally considered a Pacific species, has now been recognized with a broader Indo-Pacific distribution. It has been recorded in Pakistan (Tabassum et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and Indonesia (Talakua et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In India, its occurrence has been confirmed from Odisha (Mohanty et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Thoothukudi on the southeast coast (Kayalvizhi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and the Malabar coast (Jaleel et al. 2023), indicating its widespread presence in Indian coastal waters.\u003c/p\u003e\u003cp\u003eThese species primarily inhabit nearshore waters and play a crucial role in local ecosystems as prey for larger predatory fishes, as well as forming a significant part of small-scale fisheries. In India, \u003cem\u003eHemiramphus\u003c/em\u003e species contribute substantially to coastal fisheries; however, detailed taxonomic studies remain limited, with previous research primarily focused on distribution records and general morphological descriptions (Behera et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Due to overlapping morphological traits, past studies have often faced challenges in correctly identifying species within this genus. Furthermore, there have been no studies exploring the evolutionary relationships among Indian \u003cem\u003eHemiramphus\u003c/em\u003e species. This study aims to clarify the taxonomy, distribution, and phylogenetic relationships of \u003cem\u003eHemiramphus\u003c/em\u003e species in Indian waters, providing new insights into their systematics and diversity. The findings are expected to contribute to future taxonomic research, ecological studies, and conservation efforts for these ecologically and economically important fishes.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Sampling and collection\u003c/h2\u003e\n \u003cp\u003eField sampling was conducted along the Indian coastline, covering all major maritime states including Lakshadweep and Andaman \u0026amp;Nicobar Islands. Specimens of \u003cem\u003eHemiramphus\u003c/em\u003e species were collected from fish landing centers across the Indian coast between 2021 and 2023(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026amp; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). A total of 150 specimens were collected from local fishermen operating gillnet at a depth of around 0\u0026ndash;5 m. The specimens were photographed, and tissue samples were taken from the landing centre. The collected samples were brought to the laboratory for further analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Morphometric and meristic analysis\u003c/h2\u003e\n \u003cp\u003eStandard morphometric measurements were taken using digital calipers with an accuracy of 0.1 mm (Hubbs and Lagler, \u003cspan class=\"CitationRef\"\u003e1958\u003c/span\u003e; Haedrich, \u003cspan class=\"CitationRef\"\u003e1967\u003c/span\u003e; Fischer, \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). A total of 29 morphometric and 10 Meristic counts were recorded. These data were compared with previously published species descriptions to confirm identification (Collette and Parin, \u003cspan class=\"CitationRef\"\u003e1978\u003c/span\u003e; Mohanty et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Behera et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Molecular analysis\u003c/h2\u003e\n \u003cp\u003eTissue samples were subjected to DNA extraction using the genomic DNA kit (Origin), following the manufacturer\u0026rsquo;s protocol. The extracted DNA\u0026apos;s concentration and purity were determined using a Nanodrop spectrophotometer (Eppendorf). Mitochondrial DNA fragments corresponding to the cytochrome oxidase subunit I (COI) and 16S rRNA genes were amplified through PCR using the primer sets WARD 1 (Ward et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e) and Palumf-16s/Palumr-16s (Palumbi, \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e), respectively. Each PCR reaction was conducted in a total volume of 25 \u0026micro;l, comprising 50 ng of genomic DNA, 3 mM MgCl₂, and 0.3 mM of each primer. The thermal cycling conditions included an initial denaturation at 94\u0026deg;C for 3 minutes, followed by 30 cycles of denaturation at 94\u0026deg;C for 30 seconds, annealing at 50\u0026deg;C for 50 seconds for 16S rRNA or 30 seconds for COI, and extension at 72\u0026deg;C for 1 minute for COI or 90 seconds for 16S rRNA. The process concluded with a final extension at 72\u0026deg;C for 7 minutes. The PCR amplicons were examined using 1.2% agarose gel electrophoresis, with ethidium bromide staining for visualization.\u003c/p\u003e\n \u003cp\u003eThe quality of the sequences obtained from the PCR products was evaluated using the ABI Sequence Scanner (Version 1.0) after sequencing was conducted at Genspec, Cochin, India. Sequence alignment for COI and 16S rRNA genes, including those of closely related species retrieved from GenBank (Thompson et al. 2003), was performed using the Clustal W algorithm. Maximum likelihood (ML) analysis was conducted on the newly generated sequences using BIO Edit (Hall, \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e), while genetic distances were estimated using the Kimura 2-parameter model in MEGA 7 (Kumar et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). A bootstrap probability test with 1000 replicates (Felsenstein, \u003cspan class=\"CitationRef\"\u003e1985\u003c/span\u003e) was applied to assess the robustness of the phylogenetic analysis. ML trees were constructed to examine Hemiramphid species distributed along the Indian coast. The obtained COI and 16S rRNA sequences were submitted to NCBI GenBank, and accession numbers were assigned (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). A Maximum Likelihood (ML) phylogenetic tree was constructed using the COI sequences obtained in this study along with reference sequences from GenBank. The analysis was performed in MEGA X with 1000 bootstrap replicates (Kumar et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The Kimura 2-parameter model was used to calculate genetic distances, providing insights into the evolutionary relationships among \u003cem\u003eHemiramphus\u003c/em\u003e species in Indian waters.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003eMorphometric, meristic, and molecular analyses were conducted on all the \u003cem\u003eHemiramphus\u003c/em\u003e specimens collected from the Indian coastline. The following section presents the results of our analyses, detailing the morphological variations, meristic counts, and genetic divergence observed among the examined specimens. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1\u003cem\u003e Hemiramphus archipelagicus \u003c/em\u003e(\u003c/strong\u003e\u003cstrong\u003eCollette \u0026amp; Parin, 1978)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.1 Systematics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOrder: Beloniformes\u003c/p\u003e\n\u003cp\u003eFamily: Hemiramphidae (Gill, 1859)\u003c/p\u003e\n\u003cp\u003eGenus: \u003cem\u003eHemiramphus\u003c/em\u003e, Cuvier, 1816\u003c/p\u003e\n\u003cp\u003eSpecies:\u003cem\u003e Hemiramphus archipelagicus, \u003c/em\u003eCollette \u0026amp; Parin, 1978\u003c/p\u003e\n\u003cp\u003eCommon name: Jumping halfbeak\u003c/p\u003e\n\u003cp\u003eSynonyms: No synonyms\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.2 Materials examined\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a). DNR. No.GB.10.4.6.1.1, TL-265 mm, SL-223 mm, Collected from gill net catch, Tharuvaikulam Fishing Harbour, Tuticorin, Tamil Nadu (8º53´19.52304´´N,78º10´22.7838´´) Coromandel coast, Bay of Bengal Sea, around 0-5 m depth, Toji Thomas, March 2022. (Figure 2a)\u003c/p\u003e\n\u003cp\u003e(b). DNR. No.GB.10.4.6.1.2, TL-310 mm, SL- 256 mm, Gillnet, collected from Kalamukk landing center, Kochi, Kerala (lat./long, 9.98° N, 76.24° E), Arabian Sea, West coast of India, around 0-5 m depth, by Toji Thomas, November 2022.\u003c/p\u003e\n\u003cp\u003e(c). DNR. No.GB.10.4.6.1.3, TL-256mm, SL- 220 mm, Gillnet, Collected from Astaranga-Nuagarh fishing harbour, Odisha (lat./long, 19.98° N, 86.32° E), Bay of Bengal, East coast of India, around 0-5 m depth, by Toji Thomas, February 2020.\u003c/p\u003e\n\u003cp\u003e(d). DNR. No.GB.10.4.6.1.4, TL-246 mm, SL- 211 mm, Gillnet, Collected from Sasson dock, Bombay, Maharashtra (lat./long, 18.90° N, 72.82° E), Arabian Sea, West coast of India, around 0-5 m depth, by Toji Thomas, August 2021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.3 Diagnosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe diagnosis of H. archipelagicus within the genus \u003cem\u003eHemiramphus\u003c/em\u003e is established based on key morphological and meristic characters. Compared to \u003cem\u003eH. far\u003c/em\u003e and \u003cem\u003eH. lutkei\u003c/em\u003e, \u003cem\u003eH. archipelagicus\u003c/em\u003e possesses a relatively smaller head (4.0–4.4 times in SL) and a lower jaw that is 3.0–3.35 times in SL (Figure 3a). The species differs from \u003cem\u003eH. far\u003c/em\u003e in having a deeper caudal peduncle (4.4–4.8% of SL vs. 5.4–6.0% in \u003cem\u003eH. far\u003c/em\u003e), a longer dorsal fin base (10.9–13.4% of SL vs. 9.4–11.9% in \u003cem\u003eH. far\u003c/em\u003e) (Figure 4a), and a greater number of gill rakers on the first gill arch (35–38 vs. 28–31 in H. far). The body is uniformly coloured without vertical bars, distinguishing it from \u003cem\u003eH. far\u003c/em\u003e, which exhibits 3–8 vertical bars (Figure 5a). Additionally\u003cem\u003e, H. archipelagicus\u003c/em\u003e lacks pigmentation in the fins, whereas \u003cem\u003eH. far\u003c/em\u003e has pigmented fins (Figure 2). The anterior dorsal fin lobe is not well developed, whereas it is well developed in \u003cem\u003eH. far\u003c/em\u003e and moderately developed in \u003cem\u003eH. lutkei\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.4 Description\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe body is elongated and laterally compressed. The lower jaw is prolonged (136–155% of HL), while the upper jaw is relatively short (15.4–19.7% of HL). The snout length ranges from 32.4–36.7% of HL. Head length is 17.2–18.7% of SL. Eye diameter is 4.0–4.9% of SL, and interorbital width is greater than ED. The body depth at the anal-fin origin is 9.4–11.8% of SL. The dorsal fin has 13–14 rays, the anal fin has 11–12 rays, the pelvic fin has 6 rays, and the pectoral fin has 11–12 rays. The species possesses 35–38 gill rakers on the first gill arch and 21–26 on the second gill arch. Lateral line scale count ranges from 54–56, and total vertebral count is 53–54 (Table 2). The dorsal fin originates at the 4th–5th ray of the anal fin (Figure4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.5 Colouration\u003c/strong\u003e: Characterized by a dark blue dorsal surface and a silvery white ventral side, with a red-tipped darkish beak. A dark lateral stripe widens under the dorsal fin, which is pigmented along the edges but transparent within. The upper caudal fin lobe is yellow, while the rest of the caudal fin is grey with darker edges. The anal, pectoral, and pelvic fins are fully transparent (Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.6 O\u003c/strong\u003e\u003cstrong\u003etolith shape\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eThe ventral margin is smoothly convex from the rostrum to the posterior tip, with only a few serrations. The anterior half of the dorsal margin is also smoothly convex (Figure 6a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.7 Distribution: \u003c/strong\u003eMarine, distributed around the coastal waters of Tamil Nadu, Kerala, Odisha, Maharashtra and Lakshadweep.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.8 Meristic formula: \u003c/strong\u003eDF 13–14, AF 11–12, Pect F 11–12, Pel F 6, Gill rakers on first arch 35-38, Gill rakers on second arch 21-26, Vertebrae 53-54, Lateral line scales 54-56.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2\u003cem\u003e Hemiramphus far \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e(Forsskål, 1775)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.1 Systematics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOrder: Beloniformes\u003c/p\u003e\n\u003cp\u003eFamily: Hemiramphidae (Gill, 1859)\u003c/p\u003e\n\u003cp\u003eGenus: \u003cem\u003eHemiramphus\u003c/em\u003e Cuvier, 1816\u003c/p\u003e\n\u003cp\u003eSpecies:\u003cem\u003e Hemiramphus far, \u003c/em\u003eForsskål, 1775\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynonyms: \u003c/strong\u003e\u003cbr\u003e \u003cem\u003eEsox far\u003c/em\u003e (Forsskål, 1775) , \u003cem\u003eEsox gladius\u003c/em\u003e (Lacepède, 1803), \u003cem\u003eEsox marginatus far\u003c/em\u003e (Forsskål, 1775), \u003cem\u003eHemiramphus commersonii\u003c/em\u003e (Cuvier, 1829), \u003cem\u003eHemiramphus mocquardianus\u003c/em\u003e (Thominot, 1886), \u003cem\u003eHemirhamphus commersonii\u003c/em\u003e (Cuvier, 1829), \u003cem\u003eHemirhamphus far\u003c/em\u003e (Forsskål, 1775), and \u003cem\u003eHemirhamphus obesus\u003c/em\u003e (Castelnau, 1861\u003cstrong\u003e).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.2 Materials examined\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a).DNR.No. GB.10.4.6.6.1, TL-308 mm, SL- 262 mm, Gillnet, Collected from Pamban fishing harbour, Mandapam, Tamilnadu (lat./long, 9.28° N, 79.21° E), Bay of Bengal, East coast of India, around 0-5 m depth, by Toji Thomas, September 2019. (Figure 2b)\u003c/p\u003e\n\u003cp\u003e(b). DNR.No. GB.10.4.6.6.2, TL-293 mm, SL- 249 mm, Gillnet, Collected from Neendakara landing center, Kollam, Kerala (lat./long, 8.94° N, 76.54° E), Arabian Sea, West coast of India, around 0-5 m depth, by Toji Thomas, November 2019.\u003c/p\u003e\n\u003cp\u003e(e). DNR.No. GB.10.4.6.6.3, TL-722 mm, SL- 653mm, Gillnet, Collected from Kavaratti, Lakshadweep (lat./long, 10.57° N,72.63° E), Arabian Sea, West coast of India, around 0-100m depth, by Toji Thomas, in February 2023.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.3 Diagnosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eH. far\u003c/em\u003e is distinguished from \u003cem\u003eH. archipelagicus\u003c/em\u003e and \u003cem\u003eH. lutkei\u003c/em\u003e by its greater body depth at the anal-fin origin (10.6–13.0% of SL vs. 9.4–11.8% in \u003cem\u003eH. archipelagicus\u003c/em\u003e and 9.3–12.6% in \u003cem\u003eH. lutkei\u003c/em\u003e). The species has a relatively longer lower jaw (132–151% of HL) compared to \u003cem\u003eH. archipelagicus\u003c/em\u003e (136–155% of HL) and \u003cem\u003eH. lutkei\u003c/em\u003e (132–154% of HL) (Figure 3b). It has a lower number of gill rakers on the first gill arch (28–31 vs. 35–38 in \u003cem\u003eH. archipelagicus\u003c/em\u003e and 38–40 in \u003cem\u003eH. lutkei\u003c/em\u003e). The presence of 3–8 dark vertical bars on the body and pigmented fins differentiates \u003cem\u003eH. far\u003c/em\u003e from \u003cem\u003eH. archipelagicus\u003c/em\u003e and \u003cem\u003eH. lutkei\u003c/em\u003e, both of which lack body bars and fin pigmentation (Figure 5b). The anterior dorsal fin lobe is well developed in \u003cem\u003eH. far\u003c/em\u003e, whereas it is not well developed in \u003cem\u003eH. archipelagicus\u003c/em\u003e and only moderately developed in \u003cem\u003eH. lutkei\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.4 Description\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe body is elongate, laterally compressed, and covered with 53–54 lateral line scales. The lower jaw is significantly elongated (132–151% of HL), while the upper jaw length ranges from 19.3–23.2% of HL. The snout length is 33.2–35.0% of HL. Head length is 16.8–18.9% of SL. Eye diameter is 4.3–4.9% of SL, and interorbital width is greater than ED. The dorsal fin has 12–13 rays, the anal fin has 10–11 rays, the pelvic fin has 6 rays, and the pectoral fin has 11–12 rays. The species possesses 28–31 gill rakers on the first gill arch and 20–25 on the second gill arch. Predorsal scale count is 35–38, and vertebrae number is 50–53. The dorsal fin originates at the 6th–8th ray of the anal fin (Figure 2b) (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.5 Colouration: \u003c/strong\u003eBluish dorsal side and silvery flanks, with dark vertical bars on the body. The dorsal fin is yellowish, the caudal fin has a blue lower lobe and a yellowish upper lobe, and the lower jaw tip is red.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.6 Otolith shape: \u003c/strong\u003eComparatively broader, with a highly serrated convex ventral margin. The posterior margin is almost straight with distinct, tooth-like serrations. The dorsal margin has a few but distinct serrations(Figure 6b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.7 Distribution\u003c/strong\u003e: Marine, distributed around the coasts of Tamil Nadu, Kerala, Odisha, Maharashtra, Lakshadweep and Andaman and Nicobar Islands.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.8 Meristic formula: \u003c/strong\u003eDF 12–13, AF 10–11, Pect F 11–12, Pel F 6, Gill rakers on first arch 28-31, Gill rakers on second arch 20-25, Vertebrae 50-53, Lateral line scales 53-54.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 \u003cem\u003eHemiramphus lutkei \u003c/em\u003e(Valenciennes, 1847)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.1 Systematics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOrder: Beloniformes\u003c/p\u003e\n\u003cp\u003eFamily: Hemiramphidae (Gill, 1859)\u003c/p\u003e\n\u003cp\u003eGenus: \u003cem\u003eHemiramphus\u003c/em\u003e, Cuvier, 1816\u003c/p\u003e\n\u003cp\u003eSpecies:\u003cem\u003e Hemiramphus lutkei, \u003c/em\u003eValenciennes, 1847\u003c/p\u003e\n\u003cp\u003eSynonyms: \u003cem\u003eHemiramphus fasciatus\u003c/em\u003e (Bleeker, 1853), \u003cem\u003eHemiramphus japonicus\u003c/em\u003e (Brevoort, 1856), and \u003cem\u003eHemiramphus lukei\u003c/em\u003e (Valenciennes, 1847) \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.2 Materials examined\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a).DNR.No. GB.10.4.6.7.1, TL-332 mm, SL- 284 mm, Collected from Mural net catch, Tharuvaikulam Fishing Harbour, Tuticorin, Tamil Nadu (8º53'19.52304\"N,78º10'22.7838\") Coromandel coast, Bay of Bengal Sea, around 0-5 m depth, Toji Thomas, March 2021. (Figure 2c)\u003c/p\u003e\n\u003cp\u003e(b). DNR.No. GB.10.4.6.7.2, TL-317 mm, SL- 275 mm, Gillnet, collected from Kalamukk landing center, Kochi, Kerala (lat./long, 9.98° N, 76.24° E), Arabian Sea, West coast of India, around 0-5 m depth, by Toji Thomas, in November 2022.\u003c/p\u003e\n\u003cp\u003e(c). DNR.No. GB.10.4.6.7.3, TL-345 mm, SL- 299 mm, Gillnet, Collected from Astaranga-Nuagarh fishing harbour, Odisha (lat./long, 19.98° N, 86.32° E), Bay of Bengal, East coast of India, around 0-5 m depth, by Toji Thomas, April 2023.\u003c/p\u003e\n\u003cp\u003e(d). DNR.No. GB.10.4.6.7.4, TL-342 mm, SL- 293 mm, Gillnet, Collected from Junghalighat harbour, Port Blair, Andaman \u0026amp; Nicobar Islands (lat./long, 11.65° N, 92.72° E), Bay of Bengal, East coast of India, around 0-100m depth, by Toji Thomas, in March 2022.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.3 Diagnosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eH. lutkei\u003c/em\u003e is distinguished from \u003cem\u003eH. archipelagicus\u003c/em\u003e and \u003cem\u003eH. far\u003c/em\u003e by its relatively long pectoral fins, which are significantly longer than in the other two species (Figure 4c). The species has a higher number of gill rakers on the first gill arch (38–40 vs. 35–38 in \u003cem\u003eH. archipelagicus\u003c/em\u003e and 28–31 in \u003cem\u003eH. far\u003c/em\u003e), as well as the highest count on the second gill arch (28–33 vs. 21–26 in \u003cem\u003eH. archipelagicus\u003c/em\u003e and 20–25 in \u003cem\u003eH. far\u003c/em\u003e) (Table 2). The interorbital width in \u003cem\u003eH. lutkei\u003c/em\u003e is less than ED, whereas it is greater than ED in \u003cem\u003eH. archipelagicus\u003c/em\u003e and \u003cem\u003eH. far\u003c/em\u003e. The species lacks vertical bars on the body and pigmentation in the fins, similar to \u003cem\u003eH. archipelagicus\u003c/em\u003e but differing from \u003cem\u003eH. far\u003c/em\u003e (Figure5c). The anterior lobe of the dorsal fin is moderately developed, in contrast to \u003cem\u003eH. far\u003c/em\u003e, where it is well developed, and \u003cem\u003eH. archipelagicus\u003c/em\u003e, where it is not well developed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.4 Description\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe body is slender, elongate, and laterally compressed, covered with 55–58 lateral line scales. The lower jaw is elongated (132–154% of HL), while the upper jaw length ranges from 15.0–21.0% of HL. The snout length is 31.0–35.0% of HL. Head length is 17.5–18.9% of SL. Eye diameter is 4.0–4.8% of SL, and interorbital width is less than ED. The dorsal fin has 12–14 rays, the anal fin has 11–13 rays, the pelvic fin has 6 rays, and the pectoral fin has 10–11 rays. The species possesses 38–40 gill rakers on the first gill arch and 28–33 on the second gill arch. Predorsal scale count is 38–42, and vertebrae number is 53–56 (Table 2). The dorsal fin originates at the 4th–5th ray of the anal fin (Figure4)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.5 Colouration: \u003c/strong\u003eThe back is dark blue, transitioning to silvery white on the sides and ventral surface, with no spots or bars. The lower jaw's fleshy tip is bright red, and the upper lobe of the caudal fin has a bluish hue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.6 Otolith shape: \u003c/strong\u003eLess deep than \u003cem\u003eH. archipelagicus\u003c/em\u003e and \u003cem\u003eH. far\u003c/em\u003e, with a less convex dorsal margin and nearly straight edges. The dorsal margin has fewer but comparatively larger and more distinct teeth(Figure 6c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.7 Distribution: \u003c/strong\u003eMarine, distributed over the coasts of Tamil Nadu, Kerala, Odisha, Karnataka, Lakshadweep and Andaman and Nicobar Islands.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.8 Meristic formula: \u003c/strong\u003eDF 12–14, AF 11–13, Pect F 10–11, Pel F 6, Gill rakers on first arch 38-40, Gill rakers on second arch 28-33, Vertebrae 53-56, Lateral line scales 55-58.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Identification key for genus \u003cem\u003ehemiramphus\u003c/em\u003e from Indian waters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e-Adapted from Collette, B.B. 1999. Hemiramphidae. Halfbeaks. p. 2180-2196. In K.E. Carpenter and V. Niem (eds.) FAO species identification guide for fishery purposes. The living marine resources of the Western Central Pacific. Vol. 4. Bony fishes part 2 (Mugilidae to Carangidae). FAO, Rome. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1a\u003c/strong\u003e. Lower jaw not noticeably elongate in adults; anterior margin of upper jaw straight, not forming a prominent triangular anterior projection; pectoral fins long, 30 to 35% of standard length……… \u003cstrong\u003e\u003cem\u003eOxyporhamphus\u003c/em\u003e\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1b\u003c/strong\u003e. Lower jaw elongate or not; anterior margin of upper jaw forming a prominent triangular anterior projection; pectoral fins not more than 28% of standard length……. \u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2a\u003c/strong\u003e. Nasal papilla rounded, fan-shaped, or fimbriate; not projecting far beyond nasal fossa; caudal fin emarginate or forked, frequently with an elongate lower lobe; anal fin of males not different from those of females………\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2b\u003c/strong\u003e. Nasal papilla elongate and pointed, not fimbriate; projecting well beyond nasal fossa; caudal fin rounded or truncate, with the longest rays in the middle of fin; anal fin of males modified, some rays widened and elongate. (freshwater and estuarine genera) ……\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3a\u003c/strong\u003e. Body compressed and ribbon-shaped; dorsal-fin rays 20 to 25; anal-fin rays 20 to 25; pectoral fins long, 25 to 28% of standard length; pectoral-fin rays usually 7 to 9…… \u003cstrong\u003e\u003cem\u003eEuleptorhamphus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3b\u003c/strong\u003e. Body not ribbon-shaped; dorsal-fin rays 12 to 18; anal-fin rays 10 to 19; pectoral short, less than 20% of standard length; predorsal-fin rays 10 to 14…… \u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4a\u003c/strong\u003e. Scales absent on upper jaw; preorbital ridge absent…….\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4b\u003c/strong\u003e. Scales present on upper jaw; preorbital ridge well developed…… \u003cstrong\u003e\u003cem\u003eHyporhamphus\u003c/em\u003e\u003c/strong\u003e and \u003cstrong\u003e\u003cem\u003eRhynchorhamphus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5a\u003c/strong\u003e. Presence of 3-9 vertical bars on the body, Dorsal fin is elongated 9.5-12% of standard length with a well-developed and pigmented anterior lobe; gill rakers 27-32; Caudal peduncle depth is high about 5.4-6 %. ……… \u003cstrong\u003e\u003cem\u003eHemiramphus far\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5b. \u003c/strong\u003eAbsence of vertical bars or spots on the body, dorsal fin short 7-9.5 % of standard length, not well-developed anterior lobe, presence or absence of pigmentation. Gill rakers 35-40, Caudal peduncle depth is less; 4.5-5% of standard length……...\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6a\u003c/strong\u003e. Pectoral fins relatively long, 4.5 to 5.4 times in standard length; not well-developed anterior lobe, absence of pigmentation; gill rakers on first arch 38 to 40, usually 38 or more; predorsal scales 35 to 43, usually more than 37…... \u003cstrong\u003e\u003cem\u003eHemiramphus lutkei\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6b.\u003c/strong\u003e Pectoral fin relatively short, 4-4.5 times in standard length; not well-developed anterior lobe, presence of pigmentation; gill rakers on first arch 35 to 38, usually 36 or more; predorsal scales 35 to 43, usually more than 37…... \u003cstrong\u003e\u003cem\u003eHemiramphus archipelagicus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Molecular analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1 \u003cem\u003eCytochrome oxidase I\u003c/em\u003e (COI) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA barcoding of the COI gene yielded clear differentiation among the three species. The Maximum Likelihood (ML) phylogenetic tree constructed from COI sequences (Figure 8) showed distinct clustering of \u003cem\u003eH. lutkei\u003c/em\u003e, \u003cem\u003eH. far\u003c/em\u003e, and \u003cem\u003eH. archipelagicus\u003c/em\u003e. Genetic distances (K2P) calculated for COI sequences (Table 4) indicated moderate divergence between species, supporting morphological findings.\u003cem\u003e H. archipelagicus\u003c/em\u003e shows a genetic difference of 7.5% with \u003cem\u003eH. lutkei\u003c/em\u003e and 4.3% with \u003cem\u003eH. far,\u003c/em\u003e while \u003cem\u003eH. far \u003c/em\u003eand \u003cem\u003eH. lutkei \u003c/em\u003ehas a genetic difference of 6.1%.The \u003cem\u003eH. far\u003c/em\u003e sequences form a distinct monophyletic clade, with present study sequences clustering closely with previously reported GenBank sequences (e.g., BU148547.1, MT888964.1). Similarly, \u003cem\u003eH. archipelagicus\u003c/em\u003e appears as a separate lineage, with present study sequences grouping with reference sequences. \u003cem\u003eH. lutkei\u003c/em\u003e also forms a well-defined cluster, with present study sequences showing close genetic similarity to GenBank entries such as OR113909.1 and MN855097.1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2 16S rRNA analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis of 16S rRNA sequences aligned with COI results, displaying clear separations among \u003cem\u003eHemiramphus\u003c/em\u003e species in the ML tree (Figure 9). The K2P distances for 16S rRNA (Table 5) reinforced the genetic distinctiveness of each species. \u003cem\u003eH. archipelagicus\u003c/em\u003e shows a genetic difference of 2.5% with \u003cem\u003eH. lutkei\u003c/em\u003e and 1% with \u003cem\u003eH. far,\u003c/em\u003e while \u003cem\u003eH. far \u003c/em\u003eand \u003cem\u003eH. lutkei \u003c/em\u003ehas a genetic difference of 2.1%. The sequence comparisons were supplemented by reference data from GenBank and BOLD, providing a comprehensive genetic profile (Supplementary Table 1). \u003c/p\u003e\n\u003cp\u003eA detailed morphological examination of the specimens collected in this study confirmed their identification as \u003cem\u003eH. lutkei\u003c/em\u003e. Following this confirmation, we performed BLAST analysis using the 16S sequences obtained from our specimens (Madden, 2013). The results revealed that the sequences of \u003cem\u003eH. lutkei\u003c/em\u003e in our study closely aligned with sequences previously reported in GenBank under the names \u003cem\u003eHemiramphus balao\u003c/em\u003e (OP056959.1, AF243948.1) and \u003cem\u003eHemiramphus brasiliensis\u003c/em\u003e (NC_088002.1). This indicates that these GenBank sequences were misidentified in earlier studies and actually represent \u003cem\u003eH. lutkei\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe present study sequences of \u003cem\u003eH. lutkei\u003c/em\u003e, \u003cem\u003eH. archipelagicus\u003c/em\u003e, and \u003cem\u003eH. far\u003c/em\u003e are positioned within their respective clades, showing close genetic affinity with reference sequences such as MK561619.1 for \u003cem\u003eH. far\u003c/em\u003e. The high bootstrap values across major nodes strengthen the reliability of these phylogenetic relationships. Additionally, \u003cem\u003eAblennes hians\u003c/em\u003e serves as an outgroup, reinforcing the evolutionary framework of the \u003cem\u003eHemiramphus\u003c/em\u003e species.\u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eIntegrative taxonomic approaches have proven crucial in resolving taxonomic ambiguities in various fish species. As seen in the recent reclassification of \u003cem\u003eScomberomorus guttatus\u003c/em\u003e using morphological, meristic, and genetic data (Abdussamad et al. 2024). Such methods have also been instrumental in revealing hidden diversity, as demonstrated in the goosefish genus \u003cem\u003eLophiomus\u003c/em\u003e, where the application of multigene phylogenetic inferences and morphometric analyses led to the identification of six species, including three new species and the resurrection of \u003cem\u003eChirolophius laticeps\u003c/em\u003e (Chen et al. 2024). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRecent studies have increasingly highlighted the significance of integrative taxonomic approaches, which merge morphological and molecular data, for precise species identification and classification, especially in intricate groups such as the Beloniformes. For instance, the complete mitochondrial genome of \u003cem\u003eH. far\u003c/em\u003e has been sequenced (Zhu et al. 2018), offering valuable molecular resources for phylogenetic studies. Moreover, the effectiveness of integrative taxonomy is evident in the recent description of new Belonidae species, as demonstrated by the identification of two new \u003cem\u003eAblennes\u003c/em\u003e needlefish species from the Indian Ocean (Toji et al. 2024). Additionally, the first report of \u003cem\u003eH. archipelagicus\u003c/em\u003e from the western Bay of Bengal (Behera et al. 2020) has broadened our understanding of this species\u0026apos; distribution, further emphasizing the importance of detailed regional taxonomic studies. Studies on the length-weight relationship of nine Hemiramphidae species (Thomas et al. 2025) provide additional insights into the ecological diversity within this family. These findings emphasize that species complexes are more prevalent than previously recognized and underscore the necessity of detailed taxonomic work. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study delivers a detailed taxonomic analysis of three \u003cem\u003eHemiramphus\u003c/em\u003e species (\u003cem\u003eH. archipelagicus\u003c/em\u003e, \u003cem\u003eH. far\u003c/em\u003e, and \u003cem\u003eH. lutkei\u003c/em\u003e) from Indian waters, employing an integrative approach that combines morphometric, meristic, and molecular data. This comprehensive strategy enables a more robust species delimitation and enhances our understanding of the relationships within this complex genus. The results corroborate the distinctness of these three species, emphasizing key morphological and meristic differences, and supporting these findings with molecular phylogenetic analyses.\u003c/p\u003e\n\u003cp\u003eThe three Hemiramphus species examined, \u003cem\u003eH. archipelagicus\u003c/em\u003e, \u003cem\u003eH. far\u003c/em\u003e, and \u003cem\u003eH. lutkei\u003c/em\u003e, exhibit several distinguishing morpho-meristic characteristics beyond simple measurements. A key differentiating feature lies in their colouration and fin pigmentation. \u003cem\u003eH. far\u0026nbsp;\u003c/em\u003eis distinguished from the other two species by possessing 3-8 vertical bars on the body and yellowish or bluish colouration on the fins. Dorsal profile of \u003cem\u003eH. lutkei\u0026nbsp;\u003c/em\u003eis characterised by dark bluish or blackish colouration. Further distinctions can be observed in the development of the anterior dorsal fin lobe. In \u003cem\u003eH. far\u003c/em\u003e, this lobe is well developed, whereas it is not well developed in \u003cem\u003eH. archipelagicus\u003c/em\u003e and shows a moderate level of development in \u003cem\u003eH. lutkei\u003c/em\u003e. Meristic counts also provide valuable characters for species delineation. \u003cem\u003eH. lutkei\u003c/em\u003e possesses the highest number of gill rakers on the first arch (38\u0026ndash;40), \u003cem\u003eH. archipelagicus\u003c/em\u003e has an intermediate count (35\u0026ndash;38), while \u003cem\u003eH. far\u003c/em\u003e has the lowest number (28\u0026ndash;31). Additionally, \u003cem\u003eH. lutkei\u003c/em\u003e is unique in having an interorbital width that is less than its eye diameter, a feature that distinguishes it from both \u003cem\u003eH. archipelagicus\u003c/em\u003e and \u003cem\u003eH. far\u003c/em\u003e, where the interorbital width is greater than the eye diameter. Finally, \u003cem\u003eH. lutkei\u003c/em\u003e is also characterised by its relatively longer pectoral fins compared to the other two species. Additional distinguishing characters are provided in Table 3. These subtle yet consistent differences support the species delimitation within the genus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe value of otolith morphology in distinguishing fish species has been increasingly recognized (Abdussamad et al. 2015, 2016, 2022; Joshi et al. 2012; Karahan et al. 2014; Libungan et al. 2015; Libungan \u0026amp; P\u0026aacute;lsson, 2015). For example, Abdussamad et al. (2024) utilized otolith morphometry to establish taxonomic divergence within the \u003cem\u003eScomberomorus guttatus\u003c/em\u003e complex, demonstrating that otoliths can serve as a reliable alternative taxonomic tool. \u0026nbsp;The differences in otolith shapes observed in this study offered extra characteristics that helped to distinguish between \u003cem\u003eH. archipelagicus\u003c/em\u003e, \u003cem\u003eH. far\u003c/em\u003e, and \u003cem\u003eH. lutkei\u003c/em\u003e. Specifically, the otolith of \u003cem\u003eH. archipelagicus\u003c/em\u003e is characterised by a smoothly convex ventral margin with few serrations and an anterior dorsal margin that is also smoothly convex. In contrast, \u003cem\u003eH. far\u003c/em\u003e possesses a comparatively broader otolith with a highly serrated convex ventral margin and a nearly straight posterior margin exhibiting distinct, tooth-like serrations, along with a few distinct serrations on the dorsal margin. The otolith of \u003cem\u003eH. lutkei\u003c/em\u003e is less deep than those of the other two species, displaying a less convex dorsal margin with nearly straight edges and fewer but comparatively larger and more distinct teeth on its dorsal margin. These distinct otolith morphologies further reinforce the taxonomic separation of these three Hemiramphus species.\u003c/p\u003e\n\u003cp\u003eThe molecular phylogenetic analyses, employing COI and 16S rRNA sequences, strongly corroborate the distinctness of all three \u003cem\u003eHemiramphus\u0026nbsp;\u003c/em\u003especies. The clustering of individuals from each species in the respective clades on the maximum likelihood trees validates their identification based on morphological and meristic data. The incorporation of publicly available sequences in the phylogenetic analyses reinforces these results, enabling a broader comparison and confirmation of species boundaries. The genetic distances between \u003cem\u003eH. archipelagicus\u003c/em\u003e and \u003cem\u003eH. lutkei\u003c/em\u003e were 7.5% (COI) and 2.5% (16S rRNA); between \u003cem\u003eH. archipelagicus\u003c/em\u003e and \u003cem\u003eH. far\u003c/em\u003e, 4.3% (COI) and 1.0% (16S rRNA); and between \u003cem\u003eH. far\u003c/em\u003e and \u003cem\u003eH. lutkei\u003c/em\u003e, 6.1% (COI) and 2.1% (16S rRNA). These genetic distances align with established thresholds for species differentiation in marine fishes. These findings reinforce the morphological distinctions observed in meristic counts and morphometric ratios, validating the taxonomic status of the studied species. The consistency between morphological and molecular data highlights the importance of an integrative taxonomic approach.\u003c/p\u003e\n\u003cp\u003eThis study significantly enhances our understanding of the taxonomy of \u003cem\u003eHemiramphus\u003c/em\u003e fishes in Indian waters. It builds on the existing knowledge of Beloniformes diversity in the region (Thomas, 2022; Thomas et al. 2024; Thomas et al. 2025) by providing detailed descriptions and diagnoses of the three \u003cem\u003eHemiramphus\u003c/em\u003e species, along with supporting molecular evidence. This work clarifies the identification of these closely related species, which is essential for effective management and sustainable exploitation of fish resources, as well as for their use as effective biological indicators. Future research could explore the genetic diversity and population structure of these species across their geographic ranges and investigate the evolutionary relationships within the genus \u003cem\u003eHemiramphus\u003c/em\u003e on a broader scale. Further studies into the functional significance of the observed morphological differences, such as variations in fin length and gill raker number, would also be valuable.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has been carried out by a Junior Research Fellowship (JRF) jointly funded by Council of Scientific and Industrial Research (CSIR)\u0026nbsp;and University Grants Commission (UGC), India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no financial or proprietary interests in any material discussed in this article. The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our sincere gratitude to Dr. Grinson George, the Director of the Central Marine Fisheries Research Institute -Kochi, for granting us access to the research space. The authors are deeply thankful to the University Grants Commission (UGC) for availing the research fellowship. The authors are greatly thankful to the faculties of the Department of Biosciences, Mangalore University for helping me to fulfill my Ph. D research. The authors are deeply thankful to Mr. Asish Gopi for assistance with the illustrations. We express our gratitude to the faculty and personnel of the CMFRI's finfish fisheries section for their invaluable assistance with this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.T- Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, and Writing an original draft.\u003c/p\u003e\n\u003cp\u003eE.M.A – Supervision and, Project administration, and Review and editing the original draft.\u003c/p\u003e\n\u003cp\u003eB.S –Methodology, Software, Visualization, Writing original draft and Writing - review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e Abdussamad, E. M., Gopalakrishnan, A., Mini, K. G., Sukumaran, S., Divya, P. R., Retheesh, T. B., Muhammed, A. A., Dipti, N. V., Akhil, A. R., Thomas, T., \u0026amp; Jacob, K. D. (2022). 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First record of \u003cem\u003eHemiramphus archipelagicus\u003c/em\u003e (Beloniformes: Hemiramphidae) from Taiwan. \u003cem\u003ePlatax\u003c/em\u003e, 15, 65-71.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Hubbs, C. L., \u0026amp; Lagler, K. L. (1958). \u003cem\u003eFishes of the Great Lakes region\u003c/em\u003e (2nd ed.). Cranbrook Institute of Science Bulletin.\u003c/li\u003e\n \u003cli\u003eJaleel, K. A., Mumthaz, T. M. V., Vinayan, T., \u0026amp; George, T. (2022). Ichthyofaunal diversity of Kavvayi Backwaters, Malabar Coast of India: A preliminary study. \u003cem\u003eAsian J. Fish. Aquat. Res.\u003c/em\u003e, 19(2), 12-19.\u003c/li\u003e\n \u003cli\u003eJoshi, K. K., Abdussamad, E. M., Koya, K. P., Rohit, P., Ghosh, S., Elayathu, M. N. K., Prakasan, D., Sebastine, M., Beni, N., \u0026amp; Rao, G. S. (2012). Taxonomy and key for the identification of tuna species exploited from the Indian EEZ. \u003cem\u003eIndian J. Fish.\u003c/em\u003e, 59(3), 53\u0026ndash;60.\u003c/li\u003e\n \u003cli\u003eKara, M. H., Rouag, F., \u0026amp; Laouira, L. (2012). Westward range expansion of the Lessepsian spotted halfbeak \u003cem\u003eHemiramphus far\u003c/em\u003e (Hemiramphidae) in the Mediterranean Sea. \u003cem\u003eMar. Biodivers. Rec.\u003c/em\u003e, 5, e45.\u003c/li\u003e\n \u003cli\u003eKarahan, A., Borsa, P., Gucu, A. C., Kandemir, I., Ozkan, E., Orek, Y. A., Acan, S. C., Koban, E., \u0026amp; Togan, I. (2014). Geometric morphometrics, Fourier analysis of otolith shape, and nuclear-DNA markers distinguish two anchovy species (\u003cem\u003eEngraulis\u003c/em\u003e spp.) in the eastern Mediterranean Sea. \u003cem\u003eFish. Res.\u003c/em\u003e, 159, 45\u0026ndash;55.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Kayalvizhi, E., Jayakumar, N., Jawahar, P., \u0026amp; Srinivasan, A. (2018). Checklist of beloniform fishes of coastal waters of Thoothukudi, southeast coast of India. \u003cem\u003eJ. Entomol. Zool. Stud.\u003c/em\u003e, 6(6), 306-309.\u003c/li\u003e\n \u003cli\u003eKumar, S., Stecher, G., \u0026amp; Tamura, K. (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e, 33(7), 1870-1874. https://doi.org/10.1093/molbev/msw054\u003c/li\u003e\n \u003cli\u003eLibungan, L. A., \u0026Oacute;skarsson, G. J., Slotte, A., Jacobsen, J. A., \u0026amp; P\u0026aacute;lsson, S. (2015). Otolith shape: A population marker for Atlantic herring, \u003cem\u003eClupea harengus\u003c/em\u003e. \u003cem\u003eJ. Fish Biol.\u003c/em\u003e, 86(4), 1377\u0026ndash;1395.\u003c/li\u003e\n \u003cli\u003eLibungan, L. A., \u0026amp; P\u0026aacute;lsson, S. (2015). Shape R: An R package to study otolith shape variation among fish populations. \u003cem\u003ePLoS One\u003c/em\u003e, 10(3), e0121102.\u003c/li\u003e\n \u003cli\u003eMohanty, S. R., Seth, J. K., Mohapatra, A., \u0026amp; Mishra, S. S. (2020). Note on occurrence of Lutke\u0026rsquo;s halfbeak \u003cem\u003eHemiramphus lutkei\u003c/em\u003e Valenciennes, 1847 (Beloniformes: Hemiramphidae), along Odisha Coast. \u003cem\u003eJ. Fish.\u003c/em\u003e, 8(2), 865-867.\u003c/li\u003e\n \u003cli\u003eNatan, Y., Pattikawa, J. A., \u0026amp; Tomia, B. (2019). Biological aspects of jumping halfbeak (\u003cem\u003eHemiramphus archipelagicus\u003c/em\u003e) in the waters of Kelang Island, Western Seram, Indonesia. \u003cem\u003eAquac. Aquar. Conserv. Legis.\u003c/em\u003e, 12(2), 629-635.\u003c/li\u003e\n \u003cli\u003ePalumbi, S. R. (1996). The polymerase chain reaction. In D. M. Hillis, C. Moritz, \u0026amp; B. K. Mable (Eds.), \u003cem\u003eMolecular systematics\u003c/em\u003e (pp. 205-247). Sinauer Associates.\u003c/li\u003e\n \u003cli\u003eSouissi, J. B., El Mnif, N. T., Mahjoub, M. S., Mejri, H., Quignard, J. P., Capap\u0026eacute;, C., \u0026amp; Zaouali, J. (2005). On the recent occurrence of marine exotic species in the Tunisian waters. In \u003cem\u003eProceedings of the Seventh International Conference on the Mediterranean Coastal Environment, Medcoast 05\u003c/em\u003e (Vol. 1, pp. 529-540). Kusadasi, Turkey.\u003c/li\u003e\n \u003cli\u003eTabassum, S., Hossen, M. A., Yousuf, F., Elahi, N., Hossain, M. Y., Pramanik, M. N. U., ... \u0026amp; Elgorban, A. M. (2017). Temporal variations of condition and prey-predator status for two Halfbeaks (\u003cem\u003eHemiramphus archipelagicus\u003c/em\u003e and \u003cem\u003eH. lutkei\u003c/em\u003e) in the Karachi Coast of Pakistan through multi-model inference. \u003cem\u003eJ. Coast. Life Med.\u003c/em\u003e, 5(2), 85-88.\u003c/li\u003e\n \u003cli\u003eTabassum, S., Yousuf, F., Elahi, N., Rahman, M. M., \u0026amp; Hossain, M. Y. (2014). Coast, Pakistan. \u003cem\u003eJ. Coast. 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An integrative taxonomic study on needlefishes (Belonidae) unveils two new species within the genus \u003cem\u003eAblennes\u003c/em\u003e from the Indian Ocean. \u003cem\u003eReg. Stud. Mar. Sci.\u003c/em\u003e, 74, 103522.\u003c/li\u003e\n \u003cli\u003eWard, R. D., Zemlak, T. S., Innes, B. H., Last, P. R., \u0026amp; Hebert, P. D. (2005). DNA barcoding Australia\u0026apos;s fish species. \u003cem\u003ePhilos. Trans. R. Soc. B Biol. Sci.\u003c/em\u003e, 360(1462), 1847-1857. https://doi.org/10.1098/rstb.2005.1716\u003c/li\u003e\n \u003cli\u003eZhu, K., L\u0026uuml;, Z., Liu, B., Gong, L., Jiang, L., \u0026amp; Liu, L. (2018). The complete mitochondrial genome of \u003cem\u003eHemiramphus far\u003c/em\u003e (Beloniformes; Hemiramphidae) and phylogenetic studies of Beloniformes. \u003cem\u003eMitochondrial DNA Part B\u003c/em\u003e, 3(2), 1237-1238.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"thalassas-an-international-journal-of-marine-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"thal","sideBox":"Learn more about [Thalassas: An International Journal of Marine Sciences](http://link.springer.com/journal/41208)","snPcode":"41208","submissionUrl":"https://submission.nature.com/new-submission/41208/3","title":"Thalassas: An International Journal of Marine Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Halfbeaks, morpho-meristic, molecular, 16SrRNA, COI, phylogenetic tree","lastPublishedDoi":"10.21203/rs.3.rs-7178444/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7178444/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe taxonomic status of \u003cem\u003eHemiramphus\u003c/em\u003e Cuvier, 1816 species along the Indian coast has been subject to frequent misidentifications, complicating ecological and fisheries research. This study provides a comprehensive assessment of the morphometric, meristic, and molecular characteristics of \u003cem\u003eHemiramphus far\u003c/em\u003e(Forsskål, 1775), \u003cem\u003eH. lutkei\u003c/em\u003e (Valenciennes, 1847), and \u003cem\u003eH. archipelagicus\u003c/em\u003e (Collette \u0026amp; Parin, 1978) to clarify their identification. Morphological analyses were conducted based on key diagnostic features, including eye shape, nostril position, body markings, beak length, pectoral fin length, body depth, vertebrae, and otolith shape. Additionally, molecular data from the COI and 16S rRNA genes were analysed using maximum likelihood phylogenetic methods to assess genetic divergence and species relationships. The findings refine the taxonomic delineation of \u003cem\u003eHemiramphus\u003c/em\u003e species in Indian waters, enhancing accuracy in species identification. This study provides critical baseline data for fisheries management, biodiversity conservation, and future ecological research.\u003c/p\u003e","manuscriptTitle":"First Integrative Taxonomic Insights into the Genus Hemiramphus (Family: Hemiramphidae) from the Indian Coast","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 10:38:12","doi":"10.21203/rs.3.rs-7178444/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-18T09:45:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-03T10:37:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-03T09:02:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267165002220017442793420830439473037681","date":"2025-08-03T08:50:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275656555282164977431945807202115723671","date":"2025-08-01T04:34:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213613858486290210918472427959703396701","date":"2025-07-24T03:51:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283181546545377180196073114039728343198","date":"2025-07-22T11:53:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-22T11:48:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-22T11:39:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-22T10:52:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Thalassas: An International Journal of Marine Sciences","date":"2025-07-21T14:13:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"thalassas-an-international-journal-of-marine-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"thal","sideBox":"Learn more about [Thalassas: An International Journal of Marine Sciences](http://link.springer.com/journal/41208)","snPcode":"41208","submissionUrl":"https://submission.nature.com/new-submission/41208/3","title":"Thalassas: An International Journal of Marine Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a2a14fa2-7939-46b6-9d11-3da3f6398b4e","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T15:59:28+00:00","versionOfRecord":{"articleIdentity":"rs-7178444","link":"https://doi.org/10.1007/s41208-025-01004-0","journal":{"identity":"thalassas-an-international-journal-of-marine-sciences","isVorOnly":false,"title":"Thalassas: An International Journal of Marine Sciences"},"publishedOn":"2025-12-05 15:56:55","publishedOnDateReadable":"December 5th, 2025"},"versionCreatedAt":"2025-07-24 10:38:12","video":"","vorDoi":"10.1007/s41208-025-01004-0","vorDoiUrl":"https://doi.org/10.1007/s41208-025-01004-0","workflowStages":[]},"version":"v1","identity":"rs-7178444","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7178444","identity":"rs-7178444","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}