Genetic and taxonomic data corroborates the first report of lemon sharks, Negaprion brevirostris, in estuarine waters of Coiba National Park, Panama

Genetic and taxonomic data corroborates the first report of lemon sharks, Negaprion brevirostris, in estuarine waters of Coiba National Park, Panama | 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 Short Report Genetic and taxonomic data corroborates the first report of lemon sharks, Negaprion brevirostris, in estuarine waters of Coiba National Park, Panama Edgardo Enrique Diaz-Ferguson, Sara Ceciel Justo Riverol This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8183344/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract For almost two years community structure, characterization and identification of the main fish species from the largest estuaries of Coiba have been the focus of our research interest. Among selachii elasmobranchs (sharks), three common species ( Carcharhinus limbatus , Carcharhinus leucas and Sphyrna lewinii ) have been collected during our fish collection campaigns in San Juan, Rio Negro and Boca Grande estuaries. These three species plus the white tip reef shark, Triaenodon obesus , have been previously listed as frequent along the islands of Coiba archipelago base on visual census and environmental DNA list provided by UNESCO in collaboration with COIBA AIP Scientific Station. From February to October of 2025, seven young of the year (YOY) individuals of the lemon shark ( Negaprion brevirostris ), a species non-reported in this area before, were collected in Boca Grande estuary (two individuals during our dry season sampling campaign (February) and five individuals during the wet season (August-Cctober). Taxonomic and genetic data analysis support that collected individuals corresponded to lemon sharks, N. brevirostris . To corroborate taxonomic identification, genetic identification of two individuals was conducted using PCR amplification and product sequencing of two mitochondrial gene segments (COI and Control Region). Sequences from both genes showed a 99.17% and 99.83% similarity with GenBank referenced sequences for N. brevirostris . One unique haplotype (accession number: PX453691 and PX453712) was obtained from Boca Grande N. brevirostris samples. This is the first confirmed report of the presence of this species in waters of Coiba National Park. Therefore, these are the first reported DNA sequences deposited on GeneBank of this species for Panama Pacific waters. This information will allow scientists to compare these sequences with others deposited in GenBank as references for Eastern Tropical Pacific and unravel the existence of philopatry as well as local and regional connectivity patterns. These results will be key to establishing new management and surveillance strategies in Coiba National Park such as declaring Boca Grande estuary as nursery area and potentially a shark sanctuary. Eastern Tropical Pacific tropical estuaries Carcharhinidae demographic connectivity nursery areas in sharks shark conservation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Estuarine fish fauna is highly diverse in the tropics. This elevated diversity leads to the existence of more diverse habitats, larger species pool, stable and favorable conditions and a greater evolutionary speed (Baird & Elliot 2024). In these productive blue carbon ecotones, predators are abundant and serve as indicators of ecosystem health and complex trophic interactions (Whitfield & Elliot 2005; Blaber 2013 ). Shark species may be permanent or temporary residents of estuarine areas, using these ecosystems as nursery areas and feeding grounds (Roundtree and Able 1996 ; Heupel et al. 2007 ). Along the Eastern Tropical Pacific, shark estuarine species are frequently reported i.e., Carcharhinus leucas , Carcharinus limbatus , Rhizoprionodon longurio , Mustelus lunulatus , Mustelus henlei , and Sphyrna lewinii (Robles et al. 2015 ; Morales-Saldaña et al. 2025 ). The lemon shark, Negaprion brevirostris (Poey 1868), is a coastal tropical species distributed in the Eastern Pacific, Eastern Tropical Pacific, the Western Atlantic and the Caribbean Sea. This species prefers warm shallow estuarine waters fringed by mangroves and sandy bottoms (DeAngelis et al. 2007 ; Ruiz-Abierno et al. 2020 ; Forero-Bastidas et al. 2023 ). However, its presence appears to be rare in coastal and estuarine waters where the species is commonly reported, such as the Caribbean and Eastern Pacific (Forero-Bastidas, 2023). Coiba National Park, the largest marine protected area in Central America, hosts 814 recorded fish species (Coiba National Park Management Plan 2009). However, estuarine, mangrove and coral reef areas with the park harbor a reduced number of 166 fish species (Vega & Villarreal 2003 ). Within Coiba’s estuaries, fish species lists are limited to strictly estuarine areas like the San Juan, Rio Amarillo, Rio Negro and Boca Grande, largest rivers of Coiba archipelago, and mainly focused on teleost fish species (Nuñez et al. 2010 ). Published studies on shark species from Coiba National Park and other insular areas of Panama have so far reported only Carcharhinus limbatus , Carcharhinus leucas , Triaenodon obesus , and Ginglymostoma cirratum (Vega & Villarreal, 2003 ). Thus, none of the previous studies on the fish fauna of Coiba National Park have reported N. brevirostris , and its presence remains unconfirmed in waters of the largest marine national park and island in the Pacific of Central America. Therefore, the confirmation of the presence of Young of the Year (YOY) individuals of this species throughout the year provides strong evidence for declaring specific estuaries of Coiba National Park, such as Boca Grande, as nursery areas for the species. Fish surveys for Panama Pacific islands do not identify this species as common in these areas, and N. brevirostris has not been reported for Coiba archipelago or the Gulf of Chiriquí (Vega et al. 2003; Nuñez et al. 2010 ). The most recent list of commercial fish species for the Republic of Panama, based on samples from the Gulf of Panama samples, does includes N. brevirostris . However, this list is based on observational records and a taxonomic compilation provided by local NGOs, without genetic confirmation of captures or samples from other important fishery areas such as the Gulf of Chiriqui, Gulf of San Miguel and Gulf of Montijo (Garces et al. 2021). These findings confirm that N. brevirostris has not been reported in Coiba National Park and is not a common shark species in Panama Pacific waters. In fact, the species is absent from records of the most abundant shark species in Panama Pacific catches (Guzmán et al. 2020 , where 83% of the catch consisted of other species including: Sphyrna lewini, Carcharhinus falciformis, Carcharhinus porosus, Mustelus lunulatus and Alopias pelagicus (Morales-Saldaña et al. 2024). This short note provides the first documented report unequivocally confirming the presence of lemon sharks in waters of Coiba National Park. This finding is supported by taxonomic keys based on morphological features of seven YOY N. brevirostris as well as genetic analysis performed on two them using two mitochondrial gene segments (COI and Control Region) (Ward et al. 2005 ; Tillet et al. 2012 ). Material and Methods Sampling site: Coiba National Park is the largest marine protected area in Central America encompassing 216,000 hectares of marine surface (ANAM 2009; Botero et al. 2024 ). This conservation area contains extensive important tropical marine ecosystems (coral reef, rocky shores, mangroves, mud platforms, sand beaches and estuaries) along its 339 km coastal line. Within this marine seascape, Coiba is the largest island covering an area of 503 Km 2 . Eighteen rivers are registered on Coiba Island, of which the San Juan, Rio Negro, Barco Quebrado, Rio Amarillo and Boca Grande the largest in its territory (ANAM 2009). Fish samples (bony and elasmobranchs) were collected across all river estuaries following a salinity gradient as part of a fish inventory and monitoring program that Coiba Scientific Station currently leads and funded by the National Secretary of Science Technology and Innovation (SENACYT). Tissue samples were obtained during both dry- and wet-season field campaigns conducted in the largest estuaries of Coiba Island National Park—San Juan, Río Negro, and Boca Grande. Muscle tissue biopsies were collected from two juvenile sharks captured in the Boca Grande estuary (Fig. 2 ). DNA was extracted from tissues using DNA easy Blood and Tissue kit (QIAGEN, Inc., Valencia, CA, USA). DNA of extracted samples was checked for concentration and quality control using a nanodrop (Thermo Scientific, Waltham, MA, USA, https://www.thermofisher.com/pa/en/home.html accessed on September 2025. For COI a segment of 605bp was amplified by PCR reactions using FISH universal primers (Ward et al. 2005 ). PCR reactions for this gene were performed under the following conditions: an initial denaturation for 1 min at 95°C, follow by 5 cycles of denaturation at 95°C for 30 s, hybridization at 50°C for 40 s and extension at 72°C for 1 min. Followed by 35 cycles of denaturation at 95°C for 30 s, hybridization at 55°C for 40 s with and extension of 72°C for 1 min. For control region gene segment, PCR reactions were conducted at the following conditions: an initial denaturation step of 94◦ C for 1 min and 30 s followed by 35 cycles of a denaturation step at 94◦ C for 10 s, an annealing step at 59◦ C for 30 s followed by an extension step at 72◦ C for 1 min and a final extension step of 5 min at 72◦ C. Obtained sequences were aligned, edited and trimmed using Geneious Prime. Afterwards sequences ID were corroborated using BLAST and BOLD. Taxonomic identification was performed based on diagnostic morphological and meristic features described for the species (Compagno 1984 , 2002; Allen & Robertson 1994 ; Ebert et al. 2021 ). Specimens were characterized by a robust, fusiform body; short, broad snout; second dorsal fin slightly smaller but similar in shape to the first; and uniform yellow-brown coloration without distinctive markings (Fig. 3 ). This identification was further supported by morphometric comparisons with previously published juvenile datasets. YOY individuals captured in previous comparable nursery studies of the species were species size range from 58 to 157 cm total length (TL), while neonates are characterizing by an open umbilical scar (Fig. 4 ) based on previous published size ranges for neonate and YOY of N. brevirostris (e.g. Freitas et al. 2006 ; Reyier et al. 2014 ; Ruiz-Abierno et al. 2020 ). Results Taxonomic ID: five females and two males (less than a year old) with a size range between 50.8 to 78 cm (average size of 2.21Kg) of length and weight between 1.26 to 4.0 kg (average weight of 68.54) were collected in different months of 2025 (February, August and October). All N.brevirostris were collected at Boca Grande estuary at the intermediate to high salinity transect of the estuary (Fig. 1 ). Taxonomic ID was based on external features and verified with taxonomic keys (Compagno 1984 , 1984 b, 2002; Ebert et al. 2021 ) (Figs. 3 and 4 ). Genetic results: DNA from two of the collected individuals was extracted and amplified using the COI gene primers. A sequence of 605 base pairs was obtained from each individual of N. brevirostris for COI. These same two samples were also amplified for an additional mitochondrial gene, the control gene with a size of 596 base pairs. Results showed more than 99.17% of similarity and 100% of query cover for referenced COI sequences (Wong et al. 2009 ). And 99.83% of similarity and 97% of query cover for referenced Control Region sequences (Schultz et al. 2008 ). One unique haplotype was obtained for both COI sequences. And accession numbers were generated for these sequences: PX453691 and PX453712. Molecular information together with morphological, meristic information confirms the occurrence of N. brevirostris in waters of Coiba National Park. Discussion The taxonomic identity of all collected Negaprion brevirostris specimens was verified using the key diagnostic characters of Allen & Robertson ( 1994 ) and corroborated with the morphological descriptions provided by Compagno ( 1984 , 1984 b, 2002). As illustrated in Fig. 3 , the first dorsal fin originates posterior to the rear margin of the pectoral fins, while the second dorsal fin is comparable in size or slightly smaller to the first dorsal fin. These features are consistent with published descriptions of the species (Cervigón et al. 1993 ; Compagno 1984 ). Additional diagnostic characteristics include a robust, fusiform body; a short and broadly rounded snout; and uniform yellow-brown coloration without distinctive markings, all in agreement with the descriptions by Compagno (2002) and Ebert et al. ( 2021 ). Ventral-view photographs of both individuals were also taken to document body profile, fin insertions, and other ventral characters that further support the identification as N. brevirostris (Fig. 4 ). Evidence of lemon shark presence in Coiba National Park has important implications for the management and conservation of shark species within this major marine protected area. Thus, despite we sampled other estuarine areas of Coiba National Park (i.e., San Juan and Rio Negro estuary) and always followed a salinity gradient during our sampling campaigns; all collected lemon sharks were found at Boca Grande estuary first transect (intermediate to high salinities) close to the mouth of the estuary in areas fringed by red mangrove ( Rhizophora mangle ) (Fig. 1 ). Bottom features of this estuary area corresponded to sandy bottoms with similar physiographic characteristics documented by other researchers as suitable habitat for N. brevirostris (Forero-Bastidas et al. 2023 ). The N. brevirostris specimens consisted of five females and two males (YOY) collected during three different months of the year (March, August and October). Taxonomic identification results were confirmed through genetic sequencing of tissue samples from two of these individuals. The obtained accession numbers represent the first deposited genetic sequences of lemon sharks observed and collected within Coiba National Park. The exclusive presence of YOY of N. brevirostris in the Boca Grande estuary throughout the year, and their absence from other estuarine systems of the Coiba National Park such as San Juan, Rio Negro, Barco Quebrado and Rio Amarillo, despite our sampling efforts during the year, suggests potential philopatric behavior in adult females lemon sharks to give birth in this area. Philopatric behavior has been reported in other coastal and estuarine shark species (Hueter et al. 2004). For instance, Sundstrom al. (2001) demonstrated through tagging studies, that adult female N. brevirostris exhibit philopatry in the Bahamas, returning to their birth nurseries to mate or give birth. Similarly, Feldheim et al. ( 2002 ), reported comparable findings using tagging and genotyping in adult females in the Bahamas. Another important finding indicating the ecological significance of this estuary for sharks is that the collected N. brevirostris individuals shared their habitat with neonates and YOY individuals from other shark species, including the blacktip shark Carcharhinus limbatus , the bull shark C. leucas , and the hammer head shark Sphyrna lewini. These observations support the designation of the Boca Grande estuary as a potential nursery area and, more broadly, as a candidate site for shark sanctuary status. Rohner et al. 2025 emphasized that the quality and functionality of habitats, i.e., the presence of critical habitats that support life history functions, should be prioritized instead of over species range when developing effective shark conservation strategies. Declarations Acknowledgements: We want to thank SENACYT for supporting this research through a research and development grant for sustainable development of project IDDS 2024-063 entitle “Diversity, Connectivity and Environmental Quality of Estuaries: Towards and National Strategy of Conservation for Panama Estuaries” granted to the first author. We thank Coiba AIP Scientific Station for the administrative support and for the research logistics support during sampling campaigns to Coiba estuaries. We also thank Dr. Ginger Pocock for edition and final comments to the present manuscript. 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00:06:20","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124756,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8183344/v1/f0d9d7e8f2fc5a5abfaac5f9.png"},{"id":98421792,"identity":"bf83cb02-ff7a-458f-9941-feaa8a2905dc","added_by":"auto","created_at":"2025-12-17 16:29:28","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69478,"visible":true,"origin":"","legend":"","description":"","filename":"MARBD25003370structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8183344/v1/e95e97683b64669c6fedb10f.xml"},{"id":97930721,"identity":"e0d3f51b-89c5-4a3c-8d56-78fcabe0cb48","added_by":"auto","created_at":"2025-12-11 00:06:20","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":75683,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8183344/v1/095c1f17461bc90ea4f02bda.html"},{"id":98422692,"identity":"0924a93d-0c5b-4de4-aa9b-f5862f3e7610","added_by":"auto","created_at":"2025-12-17 16:31:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":444918,"visible":true,"origin":"","legend":"\u003cp\u003eBoca Grande Estuary at Coiba National Park, Panama. Color dots indicate the studied transects for determining fish composition. All \u003cem\u003eN. brevirostris\u003c/em\u003e were collected at T1 intermediate to high salinity 16 to 29ppt.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8183344/v1/e6af73e6de5b7d688bffc5ad.png"},{"id":98422088,"identity":"e57478b5-f58c-43cc-b689-ae8234505400","added_by":"auto","created_at":"2025-12-17 16:30:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":438961,"visible":true,"origin":"","legend":"\u003cp\u003eTissue biopsy for DNA conducted on N. brevirostris individuals.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8183344/v1/53c75d5b21c1eda66707530c.png"},{"id":97930707,"identity":"601a6f86-d1f3-4e02-8474-15b86bab851d","added_by":"auto","created_at":"2025-12-11 00:06:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1290192,"visible":true,"origin":"","legend":"\u003cp\u003eYoung of the Year (YOY) of \u003cem\u003eN. brevirostris \u003c/em\u003ecollected in Boca Grande estuary, Coiba National Park\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8183344/v1/84516bbb973c8015e6943577.png"},{"id":97930710,"identity":"962bd87b-f4b6-4e1e-aa78-c82510245c3c","added_by":"auto","created_at":"2025-12-11 00:06:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":200617,"visible":true,"origin":"","legend":"\u003cp\u003eVentral features of mouth, teeth and open umbilical scar of \u003cem\u003eN. brevirostris\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8183344/v1/b095588c24e62aaee94a4081.png"},{"id":100379807,"identity":"1716eed7-5aa3-47bb-995c-a5a9cc1fdcd7","added_by":"auto","created_at":"2026-01-16 09:35:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2881465,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8183344/v1/72cdb5ae-6c41-475e-88f4-f7675c3bcd4a.pdf"},{"id":97930719,"identity":"2dc51807-439d-475d-a936-f062e130966d","added_by":"auto","created_at":"2025-12-11 00:06:20","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1366602,"visible":true,"origin":"","legend":"","description":"","filename":"firstreportNbrevirostrissupplementarymaterialDF7.docx","url":"https://assets-eu.researchsquare.com/files/rs-8183344/v1/1b8ac77264f406ab9ca83adb.docx"}],"financialInterests":"","formattedTitle":"Genetic and taxonomic data corroborates the first report of lemon sharks, Negaprion brevirostris, in estuarine waters of Coiba National Park, Panama","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEstuarine fish fauna is highly diverse in the tropics. This elevated diversity leads to the existence of more diverse habitats, larger species pool, stable and favorable conditions and a greater evolutionary speed (Baird \u0026amp; Elliot 2024). In these productive blue carbon ecotones, predators are abundant and serve as indicators of ecosystem health and complex trophic interactions (Whitfield \u0026amp; Elliot 2005; Blaber \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Shark species may be permanent or temporary residents of estuarine areas, using these ecosystems as nursery areas and feeding grounds (Roundtree and Able \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Heupel et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Along the Eastern Tropical Pacific, shark estuarine species are frequently reported i.e., \u003cem\u003eCarcharhinus leucas\u003c/em\u003e, \u003cem\u003eCarcharinus limbatus\u003c/em\u003e, \u003cem\u003eRhizoprionodon longurio\u003c/em\u003e, \u003cem\u003eMustelus lunulatus\u003c/em\u003e, \u003cem\u003eMustelus henlei\u003c/em\u003e, and \u003cem\u003eSphyrna lewinii\u003c/em\u003e (Robles et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Morales-Salda\u0026ntilde;a et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe lemon shark, \u003cem\u003eNegaprion brevirostris\u003c/em\u003e (Poey 1868), is a coastal tropical species distributed in the Eastern Pacific, Eastern Tropical Pacific, the Western Atlantic and the Caribbean Sea. This species prefers warm shallow estuarine waters fringed by mangroves and sandy bottoms (DeAngelis et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ruiz-Abierno et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Forero-Bastidas et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, its presence appears to be rare in coastal and estuarine waters where the species is commonly reported, such as the Caribbean and Eastern Pacific (Forero-Bastidas, 2023). Coiba National Park, the largest marine protected area in Central America, hosts 814 recorded fish species (Coiba National Park Management Plan 2009). However, estuarine, mangrove and coral reef areas with the park harbor a reduced number of 166 fish species (Vega \u0026amp; Villarreal \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Within Coiba\u0026rsquo;s estuaries, fish species lists are limited to strictly estuarine areas like the San Juan, Rio Amarillo, Rio Negro and Boca Grande, largest rivers of Coiba archipelago, and mainly focused on teleost fish species (Nu\u0026ntilde;ez et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Published studies on shark species from Coiba National Park and other insular areas of Panama have so far reported only \u003cem\u003eCarcharhinus limbatus\u003c/em\u003e, \u003cem\u003eCarcharhinus leucas\u003c/em\u003e, \u003cem\u003eTriaenodon obesus\u003c/em\u003e, and \u003cem\u003eGinglymostoma cirratum\u003c/em\u003e (Vega \u0026amp; Villarreal, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThus, none of the previous studies on the fish fauna of Coiba National Park have reported \u003cem\u003eN. brevirostris\u003c/em\u003e, and its presence remains unconfirmed in waters of the largest marine national park and island in the Pacific of Central America. Therefore, the confirmation of the presence of Young of the Year (YOY) individuals of this species throughout the year provides strong evidence for declaring specific estuaries of Coiba National Park, such as Boca Grande, as nursery areas for the species.\u003c/p\u003e\u003cp\u003eFish surveys for Panama Pacific islands do not identify this species as common in these areas, and N. brevirostris has not been reported for Coiba archipelago or the Gulf of Chiriqu\u0026iacute; (Vega et al. 2003; Nu\u0026ntilde;ez et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The most recent list of commercial fish species for the Republic of Panama, based on samples from the Gulf of Panama samples, does includes \u003cem\u003eN. brevirostris\u003c/em\u003e. However, this list is based on observational records and a taxonomic compilation provided by local NGOs, without genetic confirmation of captures or samples from other important fishery areas such as the Gulf of Chiriqui, Gulf of San Miguel and Gulf of Montijo (Garces et al. 2021). These findings confirm that \u003cem\u003eN. brevirostris\u003c/em\u003e has not been reported in Coiba National Park and is not a common shark species in Panama Pacific waters. In fact, the species is absent from records of the most abundant shark species in Panama Pacific catches (Guzm\u0026aacute;n et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, where 83% of the catch consisted of other species including: \u003cem\u003eSphyrna lewini, Carcharhinus falciformis, Carcharhinus porosus, Mustelus lunulatus\u003c/em\u003e and \u003cem\u003eAlopias pelagicus\u003c/em\u003e (Morales-Salda\u0026ntilde;a et al. 2024).\u003c/p\u003e\u003cp\u003eThis short note provides the first documented report unequivocally confirming the presence of lemon sharks in waters of Coiba National Park. This finding is supported by taxonomic keys based on morphological features of seven YOY \u003cem\u003eN. brevirostris\u003c/em\u003e as well as genetic analysis performed on two them using two mitochondrial gene segments (COI and Control Region) (Ward et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Tillet et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eSampling site:\u003c/p\u003e\u003cp\u003eCoiba National Park is the largest marine protected area in Central America encompassing 216,000 hectares of marine surface (ANAM 2009; Botero et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This conservation area contains extensive important tropical marine ecosystems (coral reef, rocky shores, mangroves, mud platforms, sand beaches and estuaries) along its 339 km coastal line. Within this marine seascape, Coiba is the largest island covering an area of 503 Km\u003csup\u003e2\u003c/sup\u003e. Eighteen rivers are registered on Coiba Island, of which the San Juan, Rio Negro, Barco Quebrado, Rio Amarillo and Boca Grande the largest in its territory (ANAM 2009). Fish samples (bony and elasmobranchs) were collected across all river estuaries following a salinity gradient as part of a fish inventory and monitoring program that Coiba Scientific Station currently leads and funded by the National Secretary of Science Technology and Innovation (SENACYT).\u003c/p\u003e\u003c/p\u003e\u003cp\u003eTissue samples were obtained during both dry- and wet-season field campaigns conducted in the largest estuaries of Coiba Island National Park\u0026mdash;San Juan, R\u0026iacute;o Negro, and Boca Grande. Muscle tissue biopsies were collected from two juvenile sharks captured in the Boca Grande estuary (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). DNA was extracted from tissues using DNA easy Blood and Tissue kit (QIAGEN, Inc., Valencia, CA, USA). DNA of extracted samples was checked for concentration and quality control using a nanodrop (Thermo Scientific, Waltham, MA, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.thermofisher.com/pa/en/home.html\u003c/span\u003e\u003cspan address=\"https://www.thermofisher.com/pa/en/home.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e accessed on September 2025. For COI a segment of 605bp was amplified by PCR reactions using FISH universal primers (Ward et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). PCR reactions for this gene were performed under the following conditions: an initial denaturation for 1 min at 95\u0026deg;C, follow by 5 cycles of denaturation at 95\u0026deg;C for 30 s, hybridization at 50\u0026deg;C for 40 s and extension at 72\u0026deg;C for 1 min. Followed by 35 cycles of denaturation at 95\u0026deg;C for 30 s, hybridization at 55\u0026deg;C for 40 s with and extension of 72\u0026deg;C for 1 min. For control region gene segment, PCR reactions were conducted at the following conditions: an initial denaturation step of 94◦ C for 1 min and 30 s followed by 35 cycles of a denaturation step at 94◦ C for 10 s, an annealing step at 59◦ C for 30 s followed by an extension step at 72◦ C for 1 min and a final extension step of 5 min at 72◦ C. Obtained sequences were aligned, edited and trimmed using Geneious Prime. Afterwards sequences ID were corroborated using BLAST and BOLD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTaxonomic identification was performed based on diagnostic morphological and meristic features described for the species (Compagno \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, 2002; Allen \u0026amp; Robertson \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Ebert et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Specimens were characterized by a robust, fusiform body; short, broad snout; second dorsal fin slightly smaller but similar in shape to the first; and uniform yellow-brown coloration without distinctive markings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This identification was further supported by morphometric comparisons with previously published juvenile datasets. YOY individuals captured in previous comparable nursery studies of the species were species size range from 58 to 157 cm total length (TL), while neonates are characterizing by an open umbilical scar (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) based on previous published size ranges for neonate and YOY of \u003cem\u003eN. brevirostris\u003c/em\u003e (e.g. Freitas et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Reyier et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ruiz-Abierno et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTaxonomic ID: five females and two males (less than a year old) with a size range between 50.8 to 78 cm (average size of 2.21Kg) of length and weight between 1.26 to 4.0 kg (average weight of 68.54) were collected in different months of 2025 (February, August and October). All \u003cem\u003eN.brevirostris\u003c/em\u003e were collected at Boca Grande estuary at the intermediate to high salinity transect of the estuary (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Taxonomic ID was based on external features and verified with taxonomic keys (Compagno \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1984\u003c/span\u003eb, 2002; Ebert et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGenetic results: DNA from two of the collected individuals was extracted and amplified using the COI gene primers. A sequence of 605 base pairs was obtained from each individual of \u003cem\u003eN. brevirostris\u003c/em\u003e for COI. These same two samples were also amplified for an additional mitochondrial gene, the control gene with a size of 596 base pairs. Results showed more than 99.17% of similarity and 100% of query cover for referenced COI sequences (Wong et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). And 99.83% of similarity and 97% of query cover for referenced Control Region sequences (Schultz et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). One unique haplotype was obtained for both COI sequences. And accession numbers were generated for these sequences: PX453691 and PX453712. Molecular information together with morphological, meristic information confirms the occurrence of \u003cem\u003eN. brevirostris\u003c/em\u003e in waters of Coiba National Park.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe taxonomic identity of all collected \u003cem\u003eNegaprion brevirostris\u003c/em\u003e specimens was verified using the key diagnostic characters of Allen \u0026amp; Robertson (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and corroborated with the morphological descriptions provided by Compagno (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1984\u003c/span\u003eb, 2002). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the first dorsal fin originates posterior to the rear margin of the pectoral fins, while the second dorsal fin is comparable in size or slightly smaller to the first dorsal fin. These features are consistent with published descriptions of the species (Cervig\u0026oacute;n et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Compagno \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Additional diagnostic characteristics include a robust, fusiform body; a short and broadly rounded snout; and uniform yellow-brown coloration without distinctive markings, all in agreement with the descriptions by Compagno (2002) and Ebert et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ventral-view photographs of both individuals were also taken to document body profile, fin insertions, and other ventral characters that further support the identification as \u003cem\u003eN. brevirostris\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEvidence of lemon shark presence in Coiba National Park has important implications for the management and conservation of shark species within this major marine protected area. Thus, despite we sampled other estuarine areas of Coiba National Park (i.e., San Juan and Rio Negro estuary) and always followed a salinity gradient during our sampling campaigns; all collected lemon sharks were found at Boca Grande estuary first transect (intermediate to high salinities) close to the mouth of the estuary in areas fringed by red mangrove (\u003cem\u003eRhizophora mangle\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Bottom features of this estuary area corresponded to sandy bottoms with similar physiographic characteristics documented by other researchers as suitable habitat for \u003cem\u003eN. brevirostris\u003c/em\u003e (Forero-Bastidas et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eN. brevirostris\u003c/em\u003e specimens consisted of five females and two males (YOY) collected during three different months of the year (March, August and October). Taxonomic identification results were confirmed through genetic sequencing of tissue samples from two of these individuals. The obtained accession numbers represent the first deposited genetic sequences of lemon sharks observed and collected within Coiba National Park.\u003c/p\u003e\u003cp\u003eThe exclusive presence of YOY of \u003cem\u003eN. brevirostris\u003c/em\u003e in the Boca Grande estuary throughout the year, and their absence from other estuarine systems of the Coiba National Park such as San Juan, Rio Negro, Barco Quebrado and Rio Amarillo, despite our sampling efforts during the year, suggests potential philopatric behavior in adult females lemon sharks to give birth in this area. Philopatric behavior has been reported in other coastal and estuarine shark species (Hueter et al. 2004). For instance, Sundstrom al. (2001) demonstrated through tagging studies, that adult female \u003cem\u003eN. brevirostris exhibit\u003c/em\u003e philopatry in the Bahamas, returning to their birth nurseries to mate or give birth. Similarly, Feldheim et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), reported comparable findings using tagging and genotyping in adult females in the Bahamas.\u003c/p\u003e\u003cp\u003eAnother important finding indicating the ecological significance of this estuary for sharks is that the collected \u003cem\u003eN. brevirostris\u003c/em\u003e individuals shared their habitat with neonates and YOY individuals from other shark species, including the blacktip shark \u003cem\u003eCarcharhinus limbatus\u003c/em\u003e, the bull shark \u003cem\u003eC. leucas\u003c/em\u003e, and the hammer head shark \u003cem\u003eSphyrna lewini.\u003c/em\u003e These observations support the designation of the Boca Grande estuary as a potential nursery area and, more broadly, as a candidate site for shark sanctuary status. Rohner et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e emphasized that the quality and functionality of habitats, i.e., the presence of critical habitats that support life history functions, should be prioritized instead of over species range when developing effective shark conservation strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to thank SENACYT for supporting this research through a research and development grant for sustainable development of project IDDS 2024-063 entitle \u0026ldquo;Diversity, Connectivity and Environmental Quality of Estuaries: Towards and National Strategy of Conservation for Panama Estuaries\u0026rdquo; granted to the first author. We thank Coiba AIP Scientific Station for the administrative support and for the research logistics support during sampling campaigns to Coiba estuaries. We also thank Dr. Ginger Pocock for edition and final comments to the present manuscript. Collection permit for this research ARG-002-2025 was approved by the Direction of Protected Areas and Biodiversity of the Ministry of Environment, Panama.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAllen G, Robertson R (1994) Fishes of the Tropical Eastern Pacific, 332p edn. University of Hawaii\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePlan de manejo del Parque Nacional Coiba. ANAM, Compiladores JL, Mat\u0026eacute; (2009) D. Tovar y E. Arcia, Y. Hidalgo, STRI. 168p\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAngelis B, McCandless C, Kohler N, Recksiek C, Skomal G (2008) First characterization of shark nursery habitat in the United States Virgin Islands: evidence of habitat partitioning by two shark species. 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Journal of Fish Biology\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWong EH, Shivji M, Hanner R (2009) Identifying sharks with DNA barcodes. 2009. Molecular Ecology Resources 9 (1): 243\u0026ndash;256\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Eastern Tropical Pacific, tropical estuaries, Carcharhinidae, demographic connectivity, nursery areas in sharks, shark conservation","lastPublishedDoi":"10.21203/rs.3.rs-8183344/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8183344/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFor almost two years community structure, characterization and identification of the main fish species from the largest estuaries of Coiba have been the focus of our research interest. Among selachii elasmobranchs (sharks), three common species (\u003cem\u003eCarcharhinus limbatus\u003c/em\u003e, \u003cem\u003eCarcharhinus leucas\u003c/em\u003e and \u003cem\u003eSphyrna lewinii\u003c/em\u003e) have been collected during our fish collection campaigns in San Juan, Rio Negro and Boca Grande estuaries. These three species plus the white tip reef shark, \u003cem\u003eTriaenodon obesus\u003c/em\u003e, have been previously listed as frequent along the islands of Coiba archipelago base on visual census and environmental DNA list provided by UNESCO in collaboration with COIBA AIP Scientific Station. From February to October of 2025, seven young of the year (YOY) individuals of the lemon shark (\u003cem\u003eNegaprion brevirostris\u003c/em\u003e), a species non-reported in this area before, were collected in Boca Grande estuary (two individuals during our dry season sampling campaign (February) and five individuals during the wet season (August-Cctober). Taxonomic and genetic data analysis support that collected individuals corresponded to lemon sharks, \u003cem\u003eN. brevirostris\u003c/em\u003e. To corroborate taxonomic identification, genetic identification of two individuals was conducted using PCR amplification and product sequencing of two mitochondrial gene segments (COI and Control Region). Sequences from both genes showed a 99.17% and 99.83% similarity with GenBank referenced sequences for \u003cem\u003eN. brevirostris\u003c/em\u003e. One unique haplotype (accession number: PX453691 and PX453712) was obtained from Boca Grande \u003cem\u003eN. brevirostris\u003c/em\u003e samples. This is the first confirmed report of the presence of this species in waters of Coiba National Park. Therefore, these are the first reported DNA sequences deposited on GeneBank of this species for Panama Pacific waters. This information will allow scientists to compare these sequences with others deposited in GenBank as references for Eastern Tropical Pacific and unravel the existence of philopatry as well as local and regional connectivity patterns. These results will be key to establishing new management and surveillance strategies in Coiba National Park such as declaring Boca Grande estuary as nursery area and potentially a shark sanctuary.\u003c/p\u003e","manuscriptTitle":"Genetic and taxonomic data corroborates the first report of lemon sharks, Negaprion brevirostris, in estuarine waters of Coiba National Park, Panama","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 00:06:15","doi":"10.21203/rs.3.rs-8183344/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"77c9f185-75c0-4ec6-bbdf-8ea43e810f65","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T08:01:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 00:06:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8183344","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8183344","identity":"rs-8183344","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}