Introduction
Estuaries are dynamic transitional environments that support high levels of biodiversity and are essential habitats for a broad assemblage of marine and brackish water species (Elliott and McLusky, Reference Elliott and McLusky2002). As a result of their semi-enclosed environment, high organic content, and fluctuating salinity gradients, estuaries frequently function as both evolutionary hotspots and invasion gateways (Kennish, Reference Kennish2002; Costello et al., Reference Costello, Coll, Danovaro, Halpin, Ojaveer and Miloslavich2010). The introduction and documentation of non-native or range-expanding species into such environments have received considerable interest, since estuarine environments are especially susceptible to ecological changes initiated by new species introductions (Carlton and Geller, Reference Carlton and Geller1993; Occhipinti-Ambrogi and Savini, Reference Occhipinti-Ambrogi and Savini2003).
Among the taxa most frequently involved in range expansions and cryptic invasions are copepods due to their small size, high dispersal capability, and habitat specificity. A number of estuarine and coastal copepods have also extended their geographical distributions by natural dispersal or anthropogenic processes, such as Acartia tonsa, Oithona davisae, and Pseudodiaptomus marinus (Seuront, Reference Seuront2005; Cordell et al., Reference Cordell, Bollens, Draheim and Sytsma2008; Brylinski et al., Reference Brylinski, Antajan, Raud and Vincent2012). These expansions can go undetected without meticulous taxonomic and molecular analysis, especially for symbiotic copepods whose hosts and microhabitats can hinder detection (Boxshall and Defaye, Reference Boxshall and Defaye2008).
Hemicyclops tanakai, a poecilostomatoid copepod of the family Clausidiidae, was first described from the burrows of the mud shrimp Upogebia major in Tokyo Bay, Japan (Itoh and Nishida, Reference Itoh and Nishida2002). Members of the poecilostomatoid copepod genus Hemicyclops are distributed globally in intertidal and subtidal habitats, particularly in estuarine mudflats, but are absent from polar regions. It has since been recorded only from Korean coastal waters (Karanovic and Lee, Reference Karanovic and Lee2016), which shows a geographically restricted distribution. The genus Hemicyclops comprises nearly 40 described species of poecilostomatoid copepods distributed globally in intertidal and subtidal habitats, with records spanning the Indo-Pacific, Atlantic, and Mediterranean regions but absent from polar waters (Kim, Reference Kim2009). They are most frequently associated with sediment-rich estuarine mudflats and the burrows of benthic invertebrates, particularly decapod crustaceans such as thalassinid shrimps and crabs (Itoh and Nishida, Reference Itoh and Nishida2002, Reference Itoh and Nishida2007, Reference Itoh and Nishida2008). Other reported hosts include polychaetes, molluscs, and echiurans (Karanovic and Lee, Reference Karanovic and Lee2016). This ecological plasticity, ranging from commensal to symbiotic relationships, underlines the functional importance of Hemicyclops species in estuarine ecosystems, where they contribute to benthic–pelagic linkages, organic matter processing, and host–symbiont interactions. Their often cryptic lifestyle within burrows and sediments also makes them under-reported in biodiversity surveys, despite their potential role as indicators of estuarine ecosystem health.
Here, the first confirmed report of H. tanakai is reported from Indian waters, the Vellar estuary along the southeast coast of India. This discovery extends the previously known distribution of the species to the tropical Indian subcontinent and adds to the accumulating evidence of Indo-Pacific copepod range extensions. The Vellar estuary, characterized by its high detrital load and fluctuating salinity regime, is recognized for its biodiversity and aquaculture production, and is a possible hotspot for both indigenous and alien estuarine species (Rajasegar, Reference Rajasegar2003; Ajmal; Khan et al., Reference Khan, Manokaran and Lyla2014). Using a combination of morphological taxonomic keys and molecular information through sequencing of the 18S rDNA, the current study verifies the identity of H. tanakai and discusses the larger ecological and biogeographic implications of its finding in Indian estuarine waters. Against the backdrop of growing accounts of marine and estuarine invasions worldwide (Galil et al., Reference Galil, Marchini, Occhipinti-Ambrogi, Minchin, Narščius, Ojaveer and Olenin2014; Zhan et al., Reference Zhan, Zhang, Xia, Ni, Xiong, Chen, Haffner and MacIsaac2015), the current discovery highlights the need for regular biodiversity monitoring and the contribution of integrated taxonomy to the detection and comprehension of copepod dispersal and habitat colonization patterns.
Materials and methods
Description of the study area
The Vellar Estuary, situated in Parangipettai, flows into the Bay of Bengal in southeastern India at approximately 11°29′N latitude and 79°46′E longitude. It is categorized into marine, brackish-tidal, and freshwater zones based on variations in salinity (Rajthilak et al., Reference Rajthilak, Santhanam, Raja, Suman, Rajasree, Ramkumar and Perumal2015). Estuaries typically act as both a source of pollution for coastal waters and a natural filter for river drainage. The Vellar estuary, in particular, is heavily impacted by human settlements, mechanized boating, agriculture, and aquaculture. These activities significantly contribute to increased metal contamination in the ecosystem, especially in edible fish species (Rajasegar, Reference Rajasegar2003) (Fig. 1).
Location map of the Vellar estuary, southeast coast of India, showing the sampling site of Hemicyclops tanakai. Insets indicate India with Tamil Nadu highlighted in red, Tamil Nadu state with Cuddalore district highlighted in yellow, and Cuddalore district showing Parangipettai region. The red marker pinpoints the sampling site (11°29′N, 79°46′E). The bottom panel shows a satellite image of the estuary with the exact sampling site indicated.
Figure 1 Long description
A composite figure with four map elements. A) An outline map of India with one area on the southeast coast shown in a contrasting fill. B) A zoomed regional map showing the same highlighted area with internal boundary lines. C) A small inset map with a scale bar labeled “0 50 100 200 Miles” above it. D) A satellite map of a coastal area with the text label “Sampling Site” placed near the shoreline and a red arrow pointing to the site. A north arrow with the letter “N” is shown to the left of the satellite map.
Plankton sampling and culturing
Zooplankton samples were collected once from the Vellar estuary, Parangipettai, southeast coast of India (11°29′N, 79°46′E), during March 2024. Plankton was sampled using a 158 µm mesh net (mouth diameter 0.35 m) towed at the subsurface level. At the time of collection, the surface water salinity was 27 ppt, and the temperature was 30 °C, values consistent with the mesohaline character of the estuary (Rajasegar, Reference Rajasegar2003). The samples were transported to the laboratory immediately and were relocated to a 5 L round-bottom plastic container containing 3 L of filtered seawater, which was vigorously aerated. To avoid contamination due to other zooplankton, the copepod samples were washed with fresh filtered seawater. Naupliar stages of copepod were isolated under a stereomicroscope and transferred into 5 L culture vessels containing 0.45 µm filtered seawater (salinity 25–28 ppt, temperature 28–32 °C). The cultures were maintained under laboratory conditions with continuous aeration and fed with a mixture of microalgae (Isochrysis galbana and Chaetoceros calcitrans) at a concentration of 8.98 × 106 cells mL−1.
Nauplii were reared through successive copepodid stages until adulthood. A total of 127 adults were obtained (83 females, 44 males). Of these, 87 individuals (♀: 57, ♂: 30) were preserved in 5% formalin for morphological analysis, while 40 individuals (♀: 20, ♂: 20) were preserved in 95% ethanol for genomic DNA extraction. Copepods were observed under a phase contrast microscope (Olympus CX41), and their appendages were photographed with a Nikon Eclipse Ci-S series camera mounted on a compound microscope. They were examined for morphological characteristics to determine their family, genus, and species using standard identification keys (Todd et al., Reference Todd, Laverack and Boxshall1996; Itoh and Nishida, Reference Itoh and Nishida2002).
Genomic DNA isolation, PCR, and DNA sequencing
The copepod samples were processed at the Central Aquaculture Genetics, Pathology, and Food Testing Laboratory, Rajiv Gandhi Centre for Aquaculture. Total genomic DNA was extracted using the phenol–chloroform method (Sambrook and Russell, Reference Sambrook and Russell2001), standardized for small crustaceans by CAGL. DNA quality was verified by electrophoresis on a 0.7% agarose gel.
PCR (Polymerase Chain Reaction) amplification of partial 18S rDNA was carried out using universal eukaryotic primers 18SF (5′-AAC CTG GTT GAT CCT GCC AGT-3′) and 18SR (5′-TGA TCC TTC TGC AGG TTC ACC TAC-3′) (Medlin et al., Reference Medlin, Elwood, Stickel and Sogin1988). Each 25 µL PCR reaction contained 12.5 µL of 2 × EmeraldAmp GT PCR Master Mix (Takara Bio), 0.2 µM of each primer, and approximately 75 ng of genomic DNA. The amplification profile consisted of an initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 50 s, annealing at 58 °C for 50 s, and extension at 72 °C for 50 s, with a final extension at 72 °C for 10 min.
PCR products were purified and subjected to bidirectional sequencing using the Sanger sequencing method with the BigDye® Terminator v3.1 Cycle Sequencing Kit on an Applied Biosystems 3730XL DNA Analyzer. Sequence chromatograms were inspected, edited, and assembled using MEGA v.11 (Tamura et al., Reference Tamura, Stecher and Kumar2021). The final sequence was deposited in GenBank under accession number [PQ845087.1]. As only the 18S rDNA locus was sequenced in this study, future work incorporating mitochondrial COI or ITS regions will provide greater resolution among closely related Hemicyclops species.
Results
H. tanakai (Itoh and Nishida, 2002)
Based on morphological characteristics described by Itoh and Nishida (Reference Itoh and Nishida2002), the collected copepod was provisionally identified as H. tanakai. The specific identification required the observation of leg morphology (P1–P5) and genital somite.
Description
Dorsoventrally compressed poecilostomatoid copepod, with adult body lengths ranging from 1900 to 2100 µm (including caudal setae). Females are typically slightly longer than males. The body is robust, with an ovoid prosome widest at the posterior margin of the cephalosome. The prosome is approximately 1.6 times as long as wide and 1.4 times the length of the urosome. In dorsal view, the prosome width exceeds that of the genital segment by 2.2 times in males and 2.8 times in females. The urosome tapers posteriorly. In females, the genital double-somite is distinctly swollen anteriorly, about 2 times the width of the anal somite and 1.3 times longer than its maximum width. The genital openings are situated laterally on this swelling. In males, the genital somite is posteriorly expanded and heart-shaped, with a width-to-length ratio of 1.25 (0.8 times as long as wide) and 2.7 times the width of the anal somite. The caudal rami are cylindrical, measuring 2.1–2.2 times longer than wide, and bear a combination of terminal and lateral setae of varied thickness. Antennules are 7-segmented in both sexes, with the fourth segment being the longest. Males possess an additional seta on the third and fourth segments. Antennae are 4-segmented, ornamented with species-specific spinules and setae; notably, males lack long spinules on the outer edge of the second segment, replaced by fine hairs. The labrum is trapezoidal, with a convex cutting edge and a ventral row of setules present only in females. Mouthparts, including the mandibles, maxillules, and paragnaths, are similar in both sexes. The male maxilla differs by having a robust claw instead of an inner seta on the second segment. Maxillipeds are sexually dimorphic: females exhibit strong spinulose setae, while males have a large, terminal claw adapted for grasping. Swimming legs (P1–P4) are biramous, with 3-segmented exopods and endopods; endopods are slightly longer than exopods. The spine and seta formulae are consistent between sexes. The fifth leg is 2-segmented in both sexes; the terminal segment bears three spines and one seta. In males, the sixth leg is reduced to a small claw located on the posteroventral flap of the genital somite and is unrecognizable in females (Figs. 2 and 3). Key morphometric parameters for males and females of H. tanakai are summarized in Table 1. These values represent descriptive measurements (mean ± SD) based on multiple specimens. These quantitative data support the presence of reversed sexual size dimorphism (SSD), with females larger than males.
Adult specimens of Hemicyclops tanakai from the Vellar estuary, southeast coast of India. (A) Adult female with paired egg sacs, dorsal view. (B) Adult male, lateral view. Scale bars = 300 μm.
Figure 2 Long description
Image A shows a dorsoventrally compressed copepod with a robust body and visible appendages. The specimen is oriented with the head at the top and the tail at the bottom, measuring 300 micrometers. Image B shows a lateral view of a copepod, displaying its segmented body and appendages. The specimen is positioned diagonally, also measuring 300 micrometers. Both images highlight the structural features of the copepods, including their setae and body segmentation.
Line drawings of Hemicyclops tanakai: (A) male, dorsal view; (B) female, dorsal view; (C) antennule, male; (D) antennule, female; (E) mandible; (F) maxillule; (G) maxilla; (H) maxilliped; (I) antenna; (J) P1; (K) P2; (L) P3; (M) P4; (N) P5. Scale bars: A, B = 300 µm; C, D, I, J–N = 100 µm; E–H = 50 µm.
Figure 3 Long description
The image shows detailed illustrations of copepod anatomy. A and B depict the dorsal view of the copepod body with segmented structures and caudal setae. C and D illustrate appendages with multiple segmented branches. E to H show close-ups of smaller appendages with fine details. I to N present various views of appendages with intricate branching patterns and setae. Each illustration is labeled with scale bars indicating measurements in micrometers.
Key body measurements (µm) of Hemicyclops tanakai (mean ± SD, with observed range in parentheses)
Table 1 Long description
The table reports mean body measurements in micrometers for female and male Hemicyclops tanakai, with standard deviations and observed ranges. Females have greater total length including caudal setae (2050±50; 2000–2100) than males (1950±50; 1900–2000). Prosome length is longer in females (1250±20; 1230–1270) than males (1180±25; 1150–1200), and urosome length is also longer in females (800±15; 780–820) than males (770±20; 750–790). The genital double-somite is larger in females (68±3 by 115±4; range 65–70 by 110–120) than in males (52±2 by 95±3; range 50–55 by 90–100). Caudal ramus length is greater in females (88±2; 85–90) than males (72±2; 70–75). Despite size differences, the caudal ramus length-to-width ratio is the same for both sexes (2.1–2.2). Values are summarized as mean±SD with ranges, so individual specimens may vary within the reported intervals.
Differential diagnosis
The diagnostic features of the Vellar estuary specimens conform to the original description of H. tanakai (Itoh and Nishida, Reference Itoh and Nishida2002), particularly the enlarged, heart-shaped genital somite in males and the distinctly swollen genital double-somite in females. These characters separate H. tanakai from morphologically close congeners. Hemicyclops gomsoensis (Itoh and Nishida, Reference Itoh and Nishida2007) differs by having a narrower male genital somite and a less swollen female genital double-somite. Hemicyclops spinulosus (Itoh and Nishida, Reference Itoh and Nishida2008) shows strong male polymorphism and long spinules on the second antennal segment, which are absent in H. tanakai. Hemicyclops xiamenensis (Ohtsuka et al., Reference Ohtsuka, Tomikawa and Shang2010) can be distinguished by differences in caudal ramus proportions and antennal ornamentation. Hemicyclops thalassius (Vervoort and Ramírez, Reference Vervoort and Ramírez1966) shares general leg morphology with H. tanakai but differs in the shape of the female genital double-somite and reduction of the male sixth leg. The combination of these morphological characters confirms the identity of the Indian material as H. tanakai.
BLAST
The 18S rDNA sequence of H. tanakai obtained from the Vellar estuary population is 644 base pairs long (GenBank Accession number: PQ845087.1). BLAST analysis against the GenBank nucleotide database confirmed a high degree of similarity with other H. tanakai sequences, showing 99.53% similarity to H. tanakai voucher LEGO-POE050 (KR048769.1). Additional close matches include Hemicyclops sp. SYB-2016 (KT030266.1; 98.29%), Hemicyclops ctenidis (KR048744.1; 97.98%), and H. thalassius (JF781552.1; 97.98%). Beyond the genus Hemicyclops, the sequence also displayed moderate similarity to other clausidiid copepods, such as Clausidium vancouverense (JF781553.1; 97.05%), Clausia sp. SYB-2016 (KR048749.1; 95.04%), and members of related families like Catiniidae sp. (JF781554.1; 93.82%), Anthessius cucullatus (ON905677.1; 93.81%), and Giselinidae sp. (MK370214.1; 93.65%).
Phylogenetic analysis
The maximum likelihood (ML) tree constructed using partial 18S rRNA sequences grouped the Vellar estuary specimen within the H. tanakai clade (Fig. 4). The present sequence (PQ845087.1) clustered closely with H. tanakai reference sequences KR048769, supported by a high bootstrap value of 99%. Other Hemicyclops species (H. ctenidis, H. thalassius, Hemicyclops sp. SYB-2016) formed a sister group with moderate support (53–72%). Non-Hemicyclops clausidiid taxa (C. vancouverense, Clausia sp.) were placed in a separate lineage. To reduce topological error and improve tree stability, several outgroup taxa were added: Synaptiphilus longicaudus, Catiniidae sp., A. cucullatus, Anthessiidae gen. n. sp. n., and Giselinidae sp. (two sequences). Inclusion of multiple non-Hemicyclops representatives clarified higher-level relationships, showing Hemicyclops as a distinct monophyletic group within Clausidiidae. These molecular results corroborate the morphological identification of H. tanakai from the Vellar estuary, representing the first record of this species in Indian waters.
Maximum-likelihood phylogenetic tree based on partial 18S rRNA gene sequences of Hemicyclops tanakai and related clausidiid copepods. Numbers at nodes indicate bootstrap support values (1000 replicates). The present sequence (PQ845087.1) from the Vellar estuary is highlighted in red. Outgroups include Synaptiphilus longicaudus, Catiniidae sp., Anthessius cucullatus, Anthessiidae gen. n. sp. n., and Giselinidae sp. Sequences are labelled with GenBank accession numbers and taxon names.
Figure 4 Long description
The diagram is a maximum likelihood tree oriented vertically, illustrating the phylogenetic relationships among various species based on partial 18S ribosomal RNA gene sequences. At the top, the sequence labeled PQ845087.1, identified as Hemicyclops tanakai voucher TN-921, is highlighted in red. This sequence is closely related to KR048769.1, another Hemicyclops tanakai voucher, with a bootstrap value of 99 percent. Below, JF731961.1, identified as Hemicyclops thalassius and JF731962.1, identified as Hemicyclops ctenidis, form a sister group with moderate support values ranging from 53 percent to 72 percent. Further down, Hemicyclops sp. SYB-2016 is grouped with a bootstrap value of 53 percent. Non-Hemicyclops species, such as Clausidium vancouverense and Clausia sp., are placed in a separate lineage. Outgroup taxa, including Synaptiphilus longicaudus, Catiniidae sp., Anthessius cucullatus, Anthessiidae gen. n. sp. n. and Giselinidae sp., are included to clarify higher-level relationships. The tree shows Hemicyclops as a distinct monophyletic group within Clausidiidae, supported by various bootstrap values indicated at each node.
Discussion
This record of H. tanakai from the Vellar estuary is an important contribution to the geographic distribution of the species. Originally reported by Itoh and Nishida (Reference Itoh and Nishida2002) from the Pacific coast of Japan, H. tanakai has since been reported from Korean coastal waters (Karanovic and Lee, Reference Karanovic and Lee2016), but not from Indian ecosystems until the present report. The present record, based on the combination of morphological and molecular evidence, is the first definite report of H. tanakai from the Indian subcontinent, with a specific record from a tropical estuarine system along the southeast coast. Morphological study of the Indian specimens showed close agreement with these previous descriptions, especially in prosome-urosome ratio, genital somite structure, and sexually dimorphic features like the maxillipeds and antennal segmentation. A key difference was the reversal of SSD. Unlike earlier records, where males were slightly larger, the Vellar estuary population showed consistently larger females. This reversal may reflect ecological or evolutionary pressures linked to mating strategies, fecundity optimization, or male competition. Parallel SSD reversals have also been observed in copepods such as Centropages hamatus, where environmental stress caused smaller females (Gilbert and Williamson, Reference Gilbert and Williamson1983), and Acartia grani, where seasonal ecological fluctuation caused changes in SSD direction (Rodriguez and Jiménez, Reference Rodriguez and Jiménez1990). Rohner et al. (Reference Rohner, Blanckenhorn and Puniamoorthy2016) further pointed out that SSD can evolve or reverse quickly under changing selection regimes. The reversed SSD in H. tanakai might be a reflection of localized ecological pressures like host-specificity, sediment assemblages, and resource partitioning influencing sex-specific growth strategy or fecundity trade-offs.
Taxonomically, the genus Hemicyclops is characterized by remarkable morphological diversity, frequently making species demarcation difficult. H. tanakai is uniquely characterized among its congeners by the presence of an enlarged, heart-shaped genital somite in males (Fig. 2). Research by Vervoort and Ramírez (Reference Vervoort and Ramírez1966) and later Kim (Reference Kim2009), who reclassified and revised some of the species in the genus, has presented finer diagnostic guidelines. The Indian specimens fit into these schemata, especially in appendage segmentation and genitalia, but also with differences from morphologically close species like H. gomsoensis (Itoh and Nishida, Reference Itoh and Nishida2007), H. spinulosus (Itoh and Nishida, Reference Itoh and Nishida2008), and H. xiamenensis (Ohtsuka et al., Reference Ohtsuka, Tomikawa and Shang2010). Although these taxa share overall morphology, they differ in caudal-ramus proportions, antennal ornamentation, and maxilliped structure. The pronounced male polymorphism in H. spinulosus further supports its distinction from H. tanakai. Revision of H. thalassius (Vervoort and Ramírez, Reference Vervoort and Ramírez1966) uncovered a number of characters shared with H. tanakai, particularly in caudal rami shape and biramous leg morphology, but stable differences in the female genital double-somite and male sixth leg also justify H. tanakai's species status. Although detailed morphometric statistical analyses were not conducted, the inclusion of descriptive measurements (Table 1) provides quantitative support for reversed SSD and strengthens morphological identification when combined with molecular evidence. Future morphometric comparisons with East Asian populations could further clarify intraspecific variability.
Molecular analysis concurred with morphological identification: 18S rDNA from the Vellar specimens was 99.53% similar to the H. tanakai reference sequence (KR048769.1) and showed close affinity with H. ctenidis and H. thalassius, echoing morphological patterns. While 18S rDNA confirmed species identity with strong support, this marker is relatively conserved and may not resolve fine-scale variation. Future multi-locus studies (COI, ITS, 28S) will provide higher confidence in delineating Hemicyclops lineages (Bucklin et al., Reference Bucklin, Ortman, Jennings, Nigro, Sweetman, Copley and Wiebe2010; Tang et al., Reference Tang, Leasi, Obertegger, Kieneke, Barraclough and Fontaneto2012). Homology of more than 93% sequence with other clausidiid copepods, that is, C. vancouverense and Clausia sp., which is consistent with the conserved status of the 18S locus, supporting the usefulness of other markers such as COI and ITS in resolving fine-scale phylogenies (Suzuki et al., Reference Suzuki, Nakayama and Tanaka2013; Korzhavina et al., Reference Korzhavina, Nikitin, Hoeksema, Armenteros, Reimer and Ivanenko2024; Nitta, Reference Nitta2024).
Ecologically, H. tanakai reflects the genus’s close association with burrow-dwelling decapods within sediment-rich estuarine environments. Ecologically, Hemicyclops copepods represent a functionally significant group in estuarine environments due to their dual life strategy, with nauplii occurring in the plankton and later stages occupying benthic habitats such as decapod burrows (Itoh and Nishida, Reference Itoh and Nishida2007, Reference Itoh and Nishida2008; Ohtsuka et al., Reference Ohtsuka, Tomikawa and Shang2010). This inhabitation chain facilitates energy transfer between pelagic and benthic compartments and stabilizes host-associated micro-communities (Boxshall and Defaye, Reference Boxshall and Defaye2008). Their close associations with crustaceans and other invertebrates suggest potential influences on host fitness and burrow dynamics, indirectly affecting sediment turnover and nutrient cycling (Itoh and Nishida, Reference Itoh and Nishida2002; Karanovic and Lee, Reference Karanovic and Lee2016). The discovery of H. tanakai in Indian estuarine waters thus adds not only to the biogeographic records of the genus but also emphasizes the ecological role of symbiotic copepods in modulating estuarine community structure and functioning (Forrester et al., Reference Forrester, Mccaffrey, Terpis and Lane2021; Korzhavina et al., Reference Korzhavina, Nikitin, Hoeksema, Armenteros, Reimer and Ivanenko2024). Species of Hemicyclops exhibit associations with a diverse array of hosts, frequently occupying their burrows, including sponges, scleractinian corals, gastropods, bivalves, polychaetes, crustaceans, and echiurans (Karanovic and Lee, Reference Karanovic and Lee2016). First identified in relation to Upogebia major burrows (Itoh and Nishida, Reference Itoh and Nishida2002), and later recorded from other muddy habitats (Karanovic and Lee, Reference Karanovic and Lee2016), the occurrence of this species in the Vellar estuary implies wider Indo-Pacific dispersal potential. suggested that Hemicyclops spp. have partial planktonic life cycles, which enable coastal dispersal, and probably add to the patchy but increasing biogeographic range of the species. Estuarine environments like the Vellar estuary, with varying salinity, turbidity, and richness in organic matter, present the best possible niches for symbiotic copepods by both protecting and provisioning them. The findings of Forrester et al. (Reference Forrester, Mccaffrey, Terpis and Lane2021) and Korzhavina et al. (Reference Korzhavina, Nikitin, Hoeksema, Armenteros, Reimer and Ivanenko2024) also concur that the local availability of hosts, filtering by the environment, and the properties of sediment may influence the composition of assemblages and guide evolutionary directions in copepod populations. The simultaneous occurrence of reversed SSD in this group contributes to findings of ecological plasticity and can indicate nascent phases of local adaptation or population divergence.
The occurrence of H. tanakai in the Indian subcontinent, well beyond its earlier documented range in East Asia, does pose significant questions regarding its likely routes of introduction. With its close ecological connection to burrow-dwelling decapods and benthic environments, natural range expansion via larval dispersal becomes a likely scenario. Most poecilostomatoid copepods, such as Hemicyclops, have planktonic naupliar and copepodid stages that can disperse passively by coastal currents and estuarine flushing. Monsoonal seasonal circulation and coastal current systems on the eastern coast of India might enable southward or westward dispersal from Southeast Asia or East Asia. Alternatively, human vectors like ballast water discharge from overseas shipping might have been involved. Ballast-mediated translocation is a widely documented vector for the introduction of non-native copepods, as evidenced in the worldwide distribution of A. tonsa and O. davisae (Carlton and Geller, Reference Carlton and Geller1993). Indian ports like Chennai and Tuticorin have commercial traffic from East and Southeast Asia, including Japan and Korea, where H. tanakai has been reported to exist. Also, aquaculture-mediated transfer of host species, decapods deployed in shrimp hatcheries or employed as biofilter agents, can be a likely indirect vector. Symbiotic copepods and their crustacean host have previously been demonstrated to be inadvertently introduced through aquaculture, which has been recorded for copepod-crab symbiosis in Asia and the Mediterranean (Carlton et al., Reference Carlton and Arlington2003; Boxshall and Defaye, Reference Boxshall and Defaye2008).
The female-biased sex ratio observed in the cultured population (83♀: 44♂) aligns with reports of environmental influences on copepod sex determination (Souissi et al., Reference Souissi, Souissi, Devreker and Hwang2010). Temperature-driven variation in sex ratios, as noted in other copepods (Eurytemora affinis, A. tonsa), may also occur in H. tanakai populations and deserves experimental investigation. The size dimorphism reversal found in H. tanakai can also be an expression of developmental plasticity, a process by which organisms adjust growth patterns based on extrinsic environmental cues. In copepods, abiotic factors such as temperature, salinity, turbidity, and nutrient level are reported to influence developmental and morphometric features, including sexual dimorphism (Svensen and Tande, Reference Svensen and Tande1999; Glazier, Reference Glazier2015). The estuarine habitat of the Vellar, having a dynamic and strong seasonality in salinity, high loading of organics, and input of suspended sediment (Rajasegar, Reference Rajasegar2003; Ajmal; Khan et al., Reference Khan, Manokaran and Lyla2014), is presumed to exert the selective pressures facilitating plastic growth adaptation. This plasticity has been reported in the likes of E. affinis and A. tonsa, whose developmental timing and adult morphology were affected by variation in rearing conditions (Holste and Peck, Reference Holste and Peck2006; Lee et al., Reference Lee, Kiergaard, Gelembiuk, Eads and Posavi2011). The universal larger size of females in the Vellar estuary population could be an adaptation in a context-dependent manner that increases fecundity in a nutrient-replete but variable environment. Therefore, morphological diversity between populations must not be viewed as fixed, but instead as an aspect of a species’ ability for plastic phenotypic expression within dynamic estuarine environments. In combination, the morphological, molecular, and ecological data revealed herein not only expand the geographic distribution of H. tanakai to the Indian subcontinent but also shed new light on its phenotypic plasticity, evolutionary versatility, and ecological specialization.
Acknowledgements
The authors are grateful to the Centre of Advanced Study in Marine Biology, Annamalai University, for providing the necessary facilities and support to carry out this study. The authors sincerely thank the Rajiv Gandhi Centre for Aquaculture (RGCA) for assisting with molecular analysis and DNA sequencing.
Author contributions
All authors, M.B., A.G., and T.M., contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by M.B. under the supervision and guidance of A.G. and T.M. The first draft of the manuscript was written by M.B., and all authors commented on and revised previous versions. All authors read and approved the final manuscript.
Funding
This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors acknowledge the Centre of Advanced Study in Marine Biology, Annamalai University, for providing laboratory and research facilities.
Competing interests
The authors declare that they have no conflicts of interest or competing interests associated with this publication. There has been no financial or personal relationship that could have influenced the outcome of this study.
Availability of data and materials
The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. The 18S rDNA sequence obtained in this study has been deposited in GenBank under the accession number PQ845087.1.
