Introduction
The phylum Nemertea, commonly referred to as ‘ribbon worms,’ is mostly free-living (sometimes parasitic), elongate, soft-bodied, unsegmented worms possessing a coelomic cavity (rhynchocoel). The presence of rhynchocoel highlights the key feature of this phylum, for which they are often referred to as Rhynchocoela. They possess an eversible and protrusible proboscis also a diagnostic of this phylum (Kajihara, Reference Kajihara2020). The majority of nemerteans (∼99%) are marine, primarily inhabiting benthic environments (Agravat et al., Reference Agravat, Mankodi and Raval2022). The nemerteans have potent neurotoxins for defence and predation. The contractible body, eversible proboscis, and toxins help them to predate on a variety of invertebrates like polychaetes, crabs, and molluscs (Shrinivaasu et al., Reference Shrinivaasu, Venkataraman and Mohanraj2011). Additionally, some species exhibit scavenging behaviour, thereby contributing to organic matter cycling in benthic ecosystems (Thiel and Kruse, Reference Thiel and Kruse2001).
Phylum Nemertea comprises approximately 1,300 described species, although its classification has undergone repeated taxonomic revisions owing to the limited diagnostic value and high variability of many morphological characters (Sundberg, Reference Sundberg2015; Agravat et al., Reference Agravat, Mankodi and Raval2022). Early nemertean systematics followed the modified Stiasny–Wijnhoff framework, which recognized two classes (Anopla and Enopla) and four orders (Palaeonemertea, Heteronemertea, Hoplonemertea, and Bdellonemertea) (Chernyshev, Reference Chernyshev2021). Subsequent phylogenetic studies substantially revised this classification, and the phylum is currently recognized as comprising two superclasses (Pronemertea and Neonemertea), three classes (Palaeonemertea, Pilidiophora, and Hoplonemertea), and eight orders (Chernyshev, Reference Chernyshev2021). This persistent taxonomic instability has hindered comprehensive documentation of nemertean diversity, particularly in India, where research remains limited by the scarcity of robust taxonomic frameworks and specialist expertise (Mondal and Raghunathan, Reference Mondal and Raghunathan2024). To date, only 10 nemertean species represented by seven families and four orders have been reported from the Indian subcontinent. These are Prosadenoporus buergeri Punnett, 1903, Dinonemertes investigatoris Laidlaw, 1906, Drepanophorella rosea Punnett, 1903, Balionemertes australiensis Sundberg, Gibson & Olsson, 2003, Gorgonorhynchus repens Dakin & Fordham, 1931, Cerebratulus gardineri Punnett, 1903, Evelineus mcintoshii Langerhans, 1880, Baseodiscus hemprichii Ehrenberg, 1831, Notospermus tricuspidatus Quoy & Gaimard, 1833, Notospermus albovittatus Stimpson, 1855. These species have been recorded from the coastal regions of Gujarat, Tamil Nadu, Kerala, the Andaman and Nicobar Islands and Lakshadweep (Shrinivaasu et al., Reference Shrinivaasu, Venkataraman and Mohanraj2011; Agravat et al., Reference Agravat, Mankodi and Raval2022; Mondal and Raghunathan, Reference Mondal and Raghunathan2024). This apparent low diversity most likely reflects underexploration, taxonomic impediments, and the limited application of molecular approaches.
Cerebratulus is a speciose genus under the family Lineidae containing 180 valid species globally (WoRMS, 2023) (accessed on 16 May, 2026). Of these, 27 species are currently reported from the Asia-Pacific (APAC) region. The genus Cerebratulus is represented by a single recorded species in the Indian subcontinent. However, owing to the limited availability of reliable taxonomic characters, accurate identification and differentiation among species remain challenging. Nemertine specimens exhibiting a slightly pointed head, horizontal lateral cephalic slits extending to the mouth, absence of eyes, and a dorsoventrally flattened body with translucent or white margins and a caudal cirrus (cc) are frequently assigned to the genus Cerebratulus. These external morphological features have long served as the primary diagnostic characters for distinguishing members of the genus. Nevertheless, many of these external characters are of limited diagnostic value, as they are either highly variable among populations and across different life stages or insufficiently distinct to discriminate among cryptic species (Verdes Gorín et al., Reference Verdes Gorín, Arias, Junoy Pintos, Schwartz and Kajihara2021). Samples seemingly identifiable as Cerebratulus sp. are not a single cosmopolitan species or a distinct unique evolutionary lineage, but rather a complex comprising several well-supported lineages that might represent cryptic species with narrower geographic ranges distributed in different areas. In addition, phylogenetic analyses show that some Cerebratulus species exist as polyphyletic groupings with several other species of the genus and species from other genera nesting within the clade (Verdes Gorín et al., Reference Verdes Gorín, Arias, Junoy Pintos, Schwartz and Kajihara2021). While internal morphology contributes to species identification, its utility is constrained by incomplete and often inadequate original descriptions (Sundberg et al., Reference Sundberg, Thuroczy Vodoti and Strand2010). Concurrently, increasing molecular evidence has revealed discordance between genetic data and morphology-based classifications. Therefore, reliance solely on morphological characters and conventional taxonomic keys is insufficient for accurate species-level delimitation within the genus Cerebratulus.
In recent years, mitochondrial markers, particularly the cytochrome c oxidase subunit I (mtCOI) gene, have become indispensable tools for species identification and phylogenetic analyses. This is especially relevant for nemerteans, a group in which species delimitation is often complicated by morphological simplicity, high interspecific variability, and a lack of consistent diagnostic characters. DNA barcoding based on the mtCOI region has therefore emerged as a reliable approach to complement traditional taxonomy, helping to resolve ambiguities that arise from morphology alone (Sundberg et al., Reference Sundberg, Kvist and Strand2016; Bhaumik and Mandal, Reference Bhaumik and Mandal2024). Compared with the more conserved 16S and 18S rRNA genes, mtCOI generally provides greater interspecific sequence variation and higher species-level resolution, making it the standard molecular marker in nemerteans (Sundberg et al., Reference Sundberg, Kvist and Strand2016). Nemertean taxonomy is increasingly adopting integrative frameworks that combine morphological and molecular evidence to achieve more robust species delimitation. Such approaches are particularly important for poorly studied taxa, where reliance on morphology alone may lead to misidentification or underestimation of diversity. Recent studies used DNA barcoding to detect cryptic diversity, support taxonomic revisions, and improve resolution of phylogenetic relationships within the genus Cerebratulus. The combined application of DNA barcoding and traditional morphological analysis is therefore essential for accurately resolving species boundaries and determining the taxonomic placement of members of the genus Cerebratulus.
Against this backdrop, the present study documents the first record of Cerebratulus cf. maldivensis from the Indian subcontinent, based on an integrative taxonomic approach combining morphological assessment and mtCOI-based DNA barcoding. Species identity was further established through detailed comparison of external morphological features with the original description of C. maldivensis provided by Punnett (Reference Punnett and Gardiner1903), alongside a critical evaluation of congeners using currently accepted taxonomic information available in the World Register of Marine Species (WoRMS). The species C. maldivensis was originally described by Punnett (Reference Punnett and Gardiner1903) from material collected from Miladhunmadulu Atoll, Maldives. C. maldivensis has remained poorly known, with only a limited description and no available molecular data. The mtCOI sequence generated in this study constitutes the first DNA barcode for C. cf. maldivensis, thereby providing a novel molecular reference for future taxonomic and phylogenetic investigations. The present work underscores the utility of integrative taxonomy, even when based on limited material, in resolving species identities, documenting distributional extensions, and enhancing baseline knowledge of understudied nemertean taxa.
Materials and methods
Study site
The Sundarbans Estuarine System (SES) represents the southernmost part of the Indian sector of the Ganga–Brahmaputra delta and constitutes the largest monsoonal, macro-tidal, delta-front estuarine system along the northern margin of the Bay of Bengal (Nandy et al., Reference Nandy, Mandal, Deb, Ghosh, Nath and Chatterjee2018). The Thakuran River constitutes a principal riverine component of SES and is governed by the combined influence of tidal forcing and seasonal monsoonal freshwater influx. Within SES, the Thakuran River functions as a key hydrodynamic regulator, facilitating tidal exchange, sediment redistribution, and nutrient fluxes across the estuarine gradient. Its relatively balanced tidal–fluvial interaction sustains salinity gradients and enhances habitat heterogeneity, thereby supporting high biological productivity and diverse benthic communities (Nandy et al., Reference Nandy, Mandal, Deb, Ghosh, Nath and Chatterjee2018). Owing to its geomorphological connectivity and role in biogeochemical cycling, the Thakuran River is considered one of the most influential riverine systems structuring ecological processes within SES.
Sample collection and identification
Two complete specimens were collected from the Thakuran River, SES (21.83242° N, 88.53033° E) during a field survey conducted in March 2023 (Figure 1). Sediment samples were collected from a depth of 14 m using a Van-Veen grab (0.04 m2) and subsequently washed through a 0.5-mm sieve. Sieved samples were transported to the laboratory under refrigerated conditions. In the laboratory, the specimens were photographed and preserved in absolute ethanol. The specimens were then photographed using a stereoscope (Olympus SZX7) and a compound (Nikon ECLIPSE Ci) microscope. Taxonomic identification was performed through comparative analysis of morphological characters with published descriptions, keys, and revisions available in the literature for the genus Cerebratulus (McIntosh, Reference McIntosh1873; Hubrecht, Reference Hubrecht1887; Punnett, Reference Punnett1900, Reference Punnett and Gardiner1903; Coe, Reference Coe1901, Reference Coe1905; Punnett, Reference Punnett1901; Coe and Kunkel, Reference Coe and Kunkel1903; Punnett and Cooper, Reference Punnett and Cooper1909; Wijnhoff, Reference Wijnhoff1912; Gibson, Reference Gibson1981, Reference Gibson and Morton1990; Kajihara, Reference Kajihara2020; Chernyshev, Reference Chernyshev2021). The list of 27 Cerebratulus species along with their type localities currently reported from the APAC region are provided in Table 1. The validity and current taxonomic status of all species names were verified using the online databases WoRMS.
Sampling location of Cerebratulus cf. maldivensis specimens from Thakuran River, Sundarbans Estuarine System.
List of type localities of the accepted species of Cerebratulus Renier, 1804 from the Asia-Pacific (APAC) region
Table 1 Long description
Rows list 27 Cerebratulus species with columns for Sl. No., Species Name, Type Locality (Country), and References. Ambon Island, Indonesia appears for four species, Cerebratulus gracilis, latistomachus, laureolus, and profundifissus, all referenced to Staub, 1900. Eastern Indian Ocean appears for Cerebratulus multiporatus and zebra, referenced to Punnett and Cooper, 1909. Great Barrier Reef, Australia is listed for Cerebratulus magneticus, referenced to Gibson, 1981; Hokkaido, Japan for Cerebratulus orochi, referenced to Kajihara, 2020; and Hong Kong, China for Cerebratulus darvelli, referenced to Gibson, 1990. Kergeulen Islands is listed for Cerebratulus johnstoni, referenced to Wheeler, 1940; and Lakshwadeep, India for Cerebratulus gardineri, referenced to Punnett, 1903. Maldives is listed for five species, Cerebratulus haddoni, ischurus, maculatus, maldivensis, all referenced to Punnett, 1903. Japan appears again with Misaki for Cerebratulus arnosus, referenced to Takakura, 1898, and Sagami Bay for Cerebratulus formosus and superniger, both referenced to Iwata, 1957. Singapore is listed for seven species, Cerebratulus bedfordii, brunneus, erythrus, insignis, robustus, sordidus, and ulatiformius, all referenced to Punnett, 1900. Torres Strait, Australia is listed for Cerebratulus queenslandicus and torresianus, both referenced to Punnett, 1900.
Sediment and bottom water samples were collected onboard for the assessment of environmental parameters, including salinity, temperature, sediment texture, and total organic carbon (TOC) content. All analyses were conducted in accordance with established standard protocols (El Wakeel and Riley, Reference El Wakeel and Riley1957; Buchanan, Reference Buchanan, Holme and Ad1984).
DNA extraction and PCRs
The two collected specimens were preserved in absolute ethanol for subsequent analyses and deposited in the Zoology Museum, Department of Life Sciences, Presidency University, Kolkata, India (Catalogue no. PUZ 522 and PUZ 523). Total genomic DNA was extracted using the QIAGEN DNeasy Blood and Tissue Kit following the manufacturer’s protocol. The mtCOI gene region was amplified using the universal primer pair LCO1490 and HCO2198 (Folmer et al., Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994).
Polymerase chain reaction (PCR) assays were conducted according to standard protocols (Bhaumik et al., Reference Bhaumik, Shahir, Mandal and Tripathy2025), with a total reaction mixture of 50 μL containing 50–100 ng of template DNA, 1× buffer, 2.5 mM MgCl₂, 0.2 mM dNTPs, 10 μM of each primer, and Taq polymerase. Thermal cycling conditions included an initial denaturation at 94 °C, followed by 30 cycles of denaturation, annealing, and extension, with a final extension step at 72 °C (Bhaumik and Mandal, Reference Bhaumik and Mandal2024). Amplified PCR products were resolved on 2% agarose gels stained with ethidium bromide, purified using the QIAquick Gel Purification Kit, and subsequently subjected to bidirectional Sanger sequencing using the same primer pair.
Data analysis
The mtCOI sequences obtained in this study were edited using BioEdit (v7.2.6) and aligned using the multiple sequence alignment program Clustal W (Thompson et al., Reference Thompson, Higgins and Gibson1994). The edited sequences were compared against nucleotide data available in GenBank using the ‘blastn’ algorithm to confirm species identification. The final barcode sequence was deposited in GenBank under the accession number PZ293175. The deposited and edited mtCOI sequence was then analysed along with 234 closely related sequences retrieved from the NCBI GenBank and BOLD databases, all belonging to the genus Cerebratulus (Table 2). The sequences were compiled using MEGA 11 (Tamura et al., Reference Tamura, Stecher and Kumar2021). Sequence alignment was performed using MUSCLE (Edgar, Reference Edgar2004). Model testing was performed on the dataset using the Best DNA/Protein Model function. The GTR + G + I substitution model was identified as the best-fit model (Tamura et al., Reference Tamura, Stecher and Kumar2021) with the lowest Bayesian Information Criterion (BIC) and corrected Akaike Information Criterion (AIC; Schwarz, Reference Schwarz1978). Accordingly, a Maximum Likelihood (ML) tree was constructed using the GTR + G + I model with 1000 bootstrap replicates to assess the phylogenetic relationships and to compare topology with the mtCOI dataset.
Taxa names and GenBank/BOLD accession numbers of sequences used in the construction of the phylogenetic tree for genus Cerebratulus Renier, 1804
Table 2 Long description
Columns are Sl. No., Genus, Species, Similarity percent, and GenBank or BOLD Accession Number for 235 Cerebratulus sequences. Species names include maldivensis, lacteus, orochi, albifrons, formosus, leucopsis, mordukhovichi, montgomeryi, superniger, californiensis, herculeus, proboscis, lineolatus, fuscus, marginatus, longiceps, plus many rows labeled sp. and some with species shown as a dash. Similarity percent is missing for Sl. No. 1, Cerebratulus maldivensis, accession PZ293175, noted as present study. The highest similarity percent listed is 80.412 for Cerebratulus lacteus, accession OQ626346.1, and the lowest is 69.177 for Cerebratulus longiceps, accession GBSP3712 minus 12. Multiple entries share identical similarity values, including several Cerebratulus sp. rows at 78.053 with accessions OK081612.1 and OK081743.1, and many Cerebratulus sp. rows clustered at 72.745, 72.549, and 72.353 with GBSP accessions ending in minus 10. Cerebratulus californiensis appears repeatedly at 76.007 with ORNE accessions ending in minus 21, and also at 75.832 with additional ORNE accessions. Cerebratulus marginatus appears in a block of rows at 74.606 with multiple ORNE accessions, and also at 74.431, 74.327, 74.256, 74.081, and 73.929 with other accessions.
Results
Systematic position
Nemertea Schultz, 1851
Pilidiophora Tholesson and Norenburg, 2003
Heteronemertea Bürger, 1892
Lineidae McIntosh, 1874
Cerebratulus Renier, 1804
Cerebratulus cf. maldivensis Punnett, 1903
Environmental parameters of sampling site
The sediment samples collected from a depth of 14 m were also used to assess the environmental parameters of the sampling site. The bottom water salinity was recorded to be 27.89, and the sediment temperature at the time of sampling was 29.1°C. The sediment exhibited a TOC content of 0.87%. Granulometric analysis indicated a clayey silt composition, characterized by a high proportion of silt and low sand content (silt: 59.18%, clay: 24.76%, sand: 16.06%).
Morphological observations
Two complete specimens collected from sediment samples at a depth of 14 m in the Thakuran River, SES, were used to obtain the range of morphological characters. Specimens showed an average length of 32.4 mm (32.1–32.7 mm) (Figure 2a).
Cerebratulus cf. maldivensis (Punnett, Reference Punnett and Gardiner1903). (a) Dorso-lateral view (PUZ 522) of the entire specimen. (b) Anterior end, showing head with lateral cephalic grooves (cg). (c) Posterior end showing caudal cirrus (cc).
Proboscis: Proboscis moderate, eversible, and bears no stylets.
Head: Head bluntly pointed, set off from the rest of the body by a slight constriction. Lateral margins of head possess two lateral cephalic slits on either side (Figure 2b).
Rhynchopore: Small, midventral, longitudinal proboscis pore present. Located at the anteroventral surface of head.
Rhynchocoel: Short and does not extend to the posterior end of the body.
Mouth: Large, ventrally placed with a median, longitudinal slit. Opens posteriorly to the proboscis pore, located at about the level of the posterior end of the lateral cephalic grooves (cg) (Figure 2c).
Eyes: Eyes and ocelli are lacking.
Body: Long, dorso-ventrally flattened, lateral margins thin, adapted for swimming. Anterior region near the head comparatively rounded, gradually becoming more dorsolaterally flattened posteriorly. Body uniformly white, lacking any distinct markings. Both dorsoventral and longitudinal musculature well developed.
Gut: Anterior gut comprising a pharynx with distinct longitudinal ridges. Oesophageal region short and thick. Intestinal region broad and flattened, with thin, undulating margins. A small stomach present posterior to the oesophagus. Hindgut short, terminating in a terminal anus.
Anal end and caudal cirri: The anal end is extremely flattened. Posterior end is provided with an intact, short and thick cc (Figure 2c).
Remarks
Based on detailed morphological examination and comparison with established taxonomic keys, the specimen is identified as Cerebratulus cf. maldivensis. C. maldivensis is distinguished from other congeners by its uniformly white body, lacking any gradients, markings, spots, or splotches. It is comparatively small (∼3.3 cm) relative to species such as C. marginatus, which can exceed 50 cm in length (Hiebert, Reference Hiebert, Hiebert, Butler and Shanks2015). Unlike C. gardineri, C. maldivensis lacks eyes. Additionally, it possesses a relatively firm and stout cc, in contrast to other species that generally have longer, thinner, and more fragile cirri. The unique combination of body colour pattern, presence or absence of eyes, proboscis and cc morphology clearly distinguishes C. maldivensis from morphologically similar congeners reported from the Indian Ocean, all of which differ in one or more of these diagnostic characters (Table 3).
Comparative diagnostic morphological characters of Cerebratulus maldivensis and morphologically similar Cerebratulus species reported from the Indian Ocean
Table 3 Long description
Diagnostic Characters are compared across seven species columns: C. gardineri, C. haddoni, C. ischurus, C. maculatus, C. multiporatus, C. zebra, and C. maldivensis. Rows list Cephalic furrow, Eyes, Colour pattern, Proboscis, and Anal cirrus. For Cephalic furrow, C. gardineri and C. ischurus are described as deep and reaching almost or nearly to the brain; C. maculatus reaches nearly to the brain and extends backward to the level where the cerebral organ terminates; C. multiporatus extends about halfway to the middle line and is not prolonged beyond the brain region; C. zebra extends about halfway to the middle line and is prolonged past the mouth; C. haddoni is lateral, horizontal, and distinct on both sides of the head; C. maldivensis has two lateral cephalic slits on either side on the lateral margins of the head. For Eyes, C. gardineri and C. maculatus have numerous small eyes; C. haddoni has eyes indistinct or absent; C. ischurus and C. maldivensis have eyes absent; C. multiporatus has eyes and frontal organ absent; C. zebra has a small frontal organ present and eyes absent. For Colour pattern, C. gardineri is white; C. haddoni is more or less uniformly coloured or mottled without a distinct colour pattern; C. ischurus is uniform pale dirty brown; C. maculatus is pale brownish yellow with black spots; C. multiporatus is colourless; C. zebra has irregular transverse bands of dark greyish brown on a pale grey ground; C. maldivensis is whitish without any markings. For Proboscis, C. gardineri is long and fairly stout; C. ischurus is slender; C. maculatus is fairly well developed; C. multiporatus is small; C. zebra is deficient or lacking; C. maldivensis is moderate and eversible; the C. haddoni proboscis entry is given as N. A. For Anal cirrus, C. gardineri is delicate and thread like; C. haddoni is short, filamentous, transparent, and fragile; C. ischurus is short, conical, and slender; C. maculatus is listed as posterior end missing; C. multiporatus and C. zebra are listed as present; C. maldivensis is firm and stout.
Molecular analysis
The ML method complemented with GTR + G + I model was used to construct the ML tree (Figure 3). The GTR + G + I was found to be the best model as it showed the lowest BIC (39936.345) and corrected AIC (AICc: 35220.373). The constructed phylogenetic tree with the highest log likelihood (−16844.91) was selected. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. A discrete Gamma distribution was used to model evolutionary rate differences among sites (3 categories (+G, parameter = 0.5956)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 39.27% sites). The ML phylogenetic tree constructed for C. cf. maldivensis showed a clear differentiation between the described species, and a high genetic divergence was obtained between the other species (Figure 3). The overall mean interspecific p-distance between the sequenced Cerebratulus cf. maldivensis and other Cerebratulus species was 18.0% ± 1.0% (mean ± SE), with individual pairwise distances ranging from 0.1574 to 0.3766 (Supplementary Table 1).
Cerebratulus cf. maldivensis (Punnett, Reference Punnett and Gardiner1903). Maximum Likelihood phylogenetic tree constructed using mtCOI gene sequences and the most similar sequences from NCBI GenBank and BOLD database.
Figure 3 Long description
A circular phylogenetic tree with numerous branches radiating from a central point represents evolutionary relationships among various species. Each branch ends with a label indicating a species name or identifier. The tree is organized in a circular layout, with branches extending outward and the labels are arranged around the perimeter. The central point represents the common ancestor and the branches illustrate the divergence of species over time. The tree includes a highlighted branch in red, indicating a specific species or group of interest. The labels are densely packed, showing a wide diversity of species included in the analysis.
Discussion
The phylogenetic placement of Nemertea within Metazoa remains unresolved and continues to represent a persistent challenge in invertebrate systematics (Sundberg et al., Reference Sundberg, Kvist and Strand2016). Although nearly half of the currently recognized nemertean species were described by the early 20th century, these classifications were primarily based on subjective evaluations of morphological characters rather than explicit phylogenetic frameworks (Sundberg, Reference Sundberg2015). Consequently, a substantial proportion of historical taxa remain taxonomically ambiguous, frequently lacking traceable type material or clearly defined diagnostic criteria. This issue is further compounded by the limited availability of comprehensive identification keys (Sundberg, Reference Sundberg2015), as well as the extremely small number of active specialists worldwide. In addition, the widespread occurrence of cryptic species and the presence of non-monophyletic groupings within Nemertea continue to complicate species delimitation and phylogenetic inference (Verdes Gorín et al., Reference Verdes Gorín, Arias, Junoy Pintos, Schwartz and Kajihara2021). In India, these constraints are particularly pronounced, resulting in significant underrepresentation of nemertean diversity, largely due to limited research focus on the group (Dhara et al., Reference Dhara, Mandal and Ghorai2022).
DNA barcoding has been increasingly applied as an auxiliary tool for nemertean species identification (Kvist et al., Reference Kvist, Laumer, Junoy and Giribet2014). However, its application requires careful interpretation. Sundberg et al. (Reference Sundberg, Kvist and Strand2016) demonstrated that the mtCOI gene does not consistently exhibit a universal barcoding gap across Nemertea, although a degree of resolution may be achieved at the genus level. For the genus Cerebratulus, a divergence threshold of approximately 14% (uncorrected p-distance) has been proposed. Notably, intraspecific divergence exceeding this value, as observed in C. marginatus (17.5%), indicates the presence of cryptic lineages and complicates the delineation of species boundaries. These observations highlight that the effectiveness of mtCOI barcoding is dependent on the availability of well-curated reference datasets, which remain insufficient for most nemertean taxa.
In the present study, an integrative taxonomic approach was employed to identify a specimen of genus Cerebratulus collected from the SES, constituting the first record of C. cf. maldivensis from the Indian subcontinent. Morphological examination revealed a combination of characters consistent with the original description of C. maldivensis Punnett, 1903, supporting its identification. The observed morphological features correspond closely with those reported for the species and allow it to be distinguished from other congeners. Ecological data from the sampling locality further support this assignment. The sediment was characterized by a high proportion of silt, consistent with the habitat preferences of the genus Cerebratulus, which are typically associated with fine-grained substrates in shallow marine and subtidal environments (Thiel and Kruse, Reference Thiel and Kruse2001). Comparable environmental conditions have been described from the type locality of C. maldivensis in the Miladhunmadulu Atoll, northern Maldives (Punnett, Reference Punnett and Gardiner1903), where mangrove-associated wetland systems are dominated by silt-rich, sand-deficient substrates (MEE, 2015; Saha et al., Reference Saha, Mitra, Pal, Zaman and Mitra2019; Sreelekshmi et al., Reference Sreelekshmi, Nisha and Ratheesh2025). Both regions also experience semidiurnal tidal regimes with moderate amplitudes and relatively low-energy hydrodynamic conditions conducive to fine sediment deposition (Rasheed et al., Reference Rasheed, Warder, Plancherel and Piggott2021; Karim et al., Reference Karim, Nahiduzzaman, Ahmmad, Mainuddin, Wahid and Alam2026). The similarity in environmental characteristics between these regions supports the ecological plausibility of the occurrence of C. cf. maldivensis in the SES. The occurrence of C. cf. maldivensis in the SES may be explained by more than one mechanism. Oceanographic connectivity, particularly via the eastward-flowing Southwest Monsoon Current, may facilitate dispersal from the Maldives towards the Bay of Bengal (Su et al., Reference Su, Wijeratne and Pattiaratchi2021). Alternatively, the species may possess a broader distribution across Indo–West Pacific mangrove ecosystems and remain unrecorded due to limited sampling and taxonomic expertise in the region. The latter explanation is consistent with the general underreporting of nemertean diversity in India (Sundberg, Reference Sundberg2015; Dhara et al., Reference Dhara, Mandal and Ghorai2022). However, this currently represents a hypothesis requiring future validation through population genetic, larval dispersal, or broader phylogeographic studies. Future investigations integrating extensive geographic sampling coupled with population genetic analyses are needed to determine the geographical distribution, connectivity, and potential cryptic diversity of C. maldivensis.
The molecular data generated in this study provide additional support for species identification. The mtCOI sequence of C. cf. maldivensis is reported here for the first time, and no comparable sequences were found in BOLD or NCBI databases. The overall mean p-distance between C. cf. maldivensis and other species of Cerebratulus available in these databases (C. fuscus, C. albifrons, C. orochi, C. marginatus, C. herculeus, C. lacteus, C. formosus, C. mordukhovichi, C. leucopsis, C. californiensis, C. lineolatus, C. longiceps, C. montgomeryi) was found to be 18% ± 1% (mean ± SE). According to Sundberg et al. (Reference Sundberg, Kvist and Strand2016), the observed genetic divergence exceeds both the proposed genus-level threshold (14%) and the reported intraspecific variation within Cerebratulus. Such elevated interspecific p-distances strongly indicate that the present specimen is not conspecific with any currently barcoded species. These findings support the distinct taxonomic status of C. cf. maldivensis and are consistent with its morphological differentiation. The limited availability of specimens precluded destructive examination of internal anatomical characters. Nevertheless, the integrative taxonomic approach adopted here, combining external morphology, comparative diagnosis, and mtCOI sequence data, provides robust support for the identification of C. cf. maldivensis.
Conclusions
The results of the present study demonstrate the importance of integrating morphological and molecular approaches in nemertean taxonomy. While morphological characters remain essential for species recognition, molecular data provide an independent line of evidence that can resolve ambiguities associated with phenotypic variability and cryptic diversity. The addition of mtCOI sequence data for C. cf. maldivensis contributes to the currently limited molecular dataset for Cerebratulus. Expansion of such datasets will be necessary for improving species delimitation and for resolving phylogenetic relationships within the genus. In conclusion, the present study documents the first record of C. cf. maldivensis in the SES and also provides the first molecular barcode for the species. The combined morphological and molecular evidence supports its identification and highlights the utility of integrative taxonomy in addressing long-standing challenges in nemertean systematics. These findings not only expand the known distribution of the species but also highlight the critical need for intensified sampling and molecular characterization of nemerteans in underexplored regions to resolve broader patterns of diversity and evolution within the phylum.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0025315426101738
Acknowledgements
The authors are thankful to research boat personnel Mr Nihar Mondal and his entire team for their help and co-operation during sample collections in challenging situations. The authors would like to express their heartfelt appreciation to the members of the Marine Ecology Laboratory for their excellent teamwork. All necessary permits for sampling and observational field studies have been obtained by the authors from the competent authorities.
Author contributions
S.B. collected the specimens, studied morphological characters, performed and analysed the molecular data, wrote the manuscript, and designed illustrations. S.G. mined crucial information and sequences from databases, generated tables, collected and structured information for writing the manuscript. S.M. acquired funding, conceived and designed research, collected the specimens, studied morphological characters, analysed the molecular data, revised the manuscript critically, designed illustrations, and gave final approval for publication.
Declaration of interest
The authors declare no competing interests.
Data availability statement
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Ethics and approach
No animal testing was performed during the study.
Funding statement
This work is supported by a grant awarded to S.M. from DBT, India (BT/INF/22/ SP45088/2022 dated 17.02.2022).
