Introduction
The phylum Nematoda encompasses a diverse range of species, including both parasitic and free-living forms (Heip et al., Reference Heip, Vincx and Vranken1985). Free-living nematodes inhabit a wide range of ecosystems and are distributed across terrestrial and aquatic environments, with marine substrata representing particularly important habitats (Ptatscheck and Traunspurger, Reference Ptatscheck and Traunspurger2020). To date, approximately 6,900 valid species of free-living marine nematodes have been described worldwide (Appeltans et al., Reference Appeltans, Ahyong, Anderson, Angel, Artois, Bailly and Costello2012). Within this diversity, the family Chromadoridae Filipjev, Reference Filipjev1917 (class Chromadorea) encompasses numerous genera of free-living marine nematodes (Decraemer and Smol, Reference Decraemer and Smol2006). Euchromadora de Man, Reference de Man1886 is one such genus within Chromadoridae and currently includes 11 valid species (Venekey et al., Reference Venekey, Gheller, Kandratavicius, Cunha, Vilas-Boas, Fonseca and Maria2019; Leduc, Reference Leduc2025; Nemys eds, 2025). Species of Euchromadora are predominantly associated with hard and coarse substrates, often in the presence of vegetative growth, as documented in several studies (Bastian, Reference Bastian1865; Inglis, Reference Inglis1969; Kito, Reference Kito1977; Kulikov et al., Reference Kulikov, Dashchenko, Koloss and Yushin1998; Leduc, Reference Leduc2025).
The type species, Euchromadora vulgaris (Bastian, Reference Bastian1865) de Man, Reference de Man1886, was originally described as Chromadora vulgaris by Bastian (Reference Bastian1865) from intertidal habitats of the North Sea characterized by green filamentous algae. Among the currently recognized species of the genus, E. vulgaris has been the only species previously reported from Indian waters (Annapurna et al., Reference Annapurna, Rao, Bhanu, Ambedkar, Satyanarayana and Rao2017). In contrast, Euchromadora atypica Blome, 1985 was originally described from the Galápagos Islands (Blome, Reference Blome1985) and subsequently reported from Cuba (Armenteros et al., Reference Armenteros, del Pino-Machado, González-González, Ruiz-Abierno and Pérez-García2014), with no additional records documented thereafter. The present study expands the known distribution of E. atypica by documenting its first record from the Indian Ocean and provides the first molecular evidence for the species based on two ribosomal gene loci. Additionally, phylogenetic relationships within the family Chromadoridae are examined, and a global distributional map of all valid Euchromadora species is presented.
Materials and methods
Sediment samples were collected in October 2024 (post-monsoon season) from Arjyapalli Beach (19°18.26764′N, 84°57.92365′E) along the eastern coast of India, Odisha. The intertidal zone at Arjyapalli is characterized by numerous rocky formations, predominantly of anthropogenic origin. Small rock fragments bearing visible green algal growth were collected to investigate the benthic marine nematode assemblages associated with these substrates. In the laboratory, the rock fragments were immersed in 10% formalin for 30 minutes to detach organisms associated with the vegetation, after which the surfaces were carefully scraped with a scalpel to recover additional fauna. An identical procedure was followed for a parallel replicate preserved in DESS solution. The material from both replicates was subsequently passed through a sieve stack comprising a 500 µm mesh over a 63 µm mesh. The fraction retained on the 63 µm mesh was preserved in 4% buffered formalin and DESS solution, respectively, for further analyses. Nematodes were sorted under a stereo zoom microscope (Magnus TZM 6), mounted in glycerin, and sealed with paraffin wax. Morphological observations, photomicrography, and morphometric measurements were conducted using an Olympus BX53 compound microscope equipped with a DP23 digital camera and cellSens Standard software. Illustrations were generated using the same imaging system. Genus-level identification followed the diagnostic criteria outlined by Decraemer and Smol (Reference Decraemer and Smol2006), Tchesunov (Reference Tchesunov2014), and Venekey et al. (Reference Venekey, Gheller, Kandratavicius, Cunha, Vilas-Boas, Fonseca and Maria2019), while species-level identification was based on the identification key of Leduc (Reference Leduc2025) and original species description. Voucher specimens were deposited in the national repository at the Estuarine Biological Research Centre, Zoological Survey of India, Gopalpur-on-Sea.
For molecular analyses, a single specimen from the DESS-preserved replicate was used for DNA extraction using the HiPurA® Insect DNA Purification Kit, following the manufacturer’s instructions with a minor modification in the duration of thermolysis to overnight, instead of three hours. The LSU region and two SSU rRNA gene fragments were amplified under standardized Polymerase Chain Reaction (PCR) conditions using the same primer pairs as described by Leduc and Zhao (Reference Leduc and Zhao2025). The PCR products were subsequently sequenced using Sanger sequencing. Sequence data for both gene loci were edited and analysed in BioEdit v7.2. The SSU fragments were manually assembled after identifying overlapping regions. The assembled SSU and the LSU sequences were submitted to NCBI (GenBank accession numbers PX468573 and PX585119, respectively). For phylogenetic analyses, sequences of both gene loci representing all genera within the family Chromadoridae, available in NCBI, were included. Multiple sequence alignment was performed using MAFFT v7 (Katoh et al., Reference Katoh, Rozewicki and Yamada2019). Bayesian inference (BI) phylogenetic analyses were conducted in MrBayes v3.2.7, with analyses run for 1,000,000 generations and sampling every 1,000 generations under the best-fit GTR + I + G substitution model, as determined by MrModeltest2 v2.4. The first 25% of sampled trees were discarded as burn-in. Convergence and stability of the analyses were evaluated by examining effective sample size and potential scale reduction factor values in Tracer v1.6. The resulting phylogenetic trees were visualized in FigTree, with branch support indicated by posterior probability (PP) values to assess the robustness of inferred lineages.
Results
Systematics:
Phylum Nematoda Cobb, Reference Cobb1932
Class Chromadorea Inglis, Reference Inglis1983
Order Chromadorida Chitwood, Reference Chitwood1933
Family Chromadoridae Filipjev, Reference Filipjev1917
Genus Euchromadora de Man, Reference de Man1886
List of valid species:
E. atypica Blome, Reference Heip, Vincx and Vranken1985
E. eileenae Inglis, Reference Inglis1969
E. ezoensis Kito, Reference Kito1977
E. gaulica Inglis, Reference Inglis1962
=E. chitwoodi Coles, Reference Coles1965
=E. tridentata sensu Wieser, Reference Wieser1951
E. meadi Wieser & Hopper, Reference Wieser and Hopper1967
E. permutabilis Wieser, Reference Wieser1954
E. rebeccae Leduc, Reference Leduc2025
E. robusta Kulikov, Dashchenko, Koloss & Yushin, Reference Kulikov, Dashchenko, Koloss and Yushin1998
E. striata (Eberth, Reference Eberth1863) de Man, Reference de Man1886
=Odontobius striatus Eberth, Reference Eberth1863
E. tokiokai Wieser, Reference Wieser1955
E. vulgaris (Bastian, Reference Bastian1865) de Man, Reference de Man1886
=Chromadora vulgaris Bastian, Reference Bastian1865
Euchromadora atypica Blome, 1985 (Figures 1–2, Table 1)
Euchromadora atypica: (A) Cephalic region of male; (B) tail region of male (RS: right spicule; LS: left spicule, GB: gubernaculum, TM: telamon); (C) punctuation pattern and pharyngeal region of female; (D) vulva opening and reproductive parts of female; (E) posterior portion of female.
Micrographs of Euchromadora atypica: (A) Entire male; (B) cephalic region of male, arrows indicate excretory pore opening and end of buccal cavity; (C) posterior body region of male with copulatory apparatus, arrow indicates ovoid structure; (D) left spicule, telamon, gubernaculum, arrow indicates the pre-cloacal setae; (E) right spicule; (F) entire female; (G) pharyngeal region of female; (H) reproductive parts of female, arrow indicates vulva opening; (I) posterior portion of female.
Morphometric measurements (in μm) of Euchromadora atypica
Table 1 Long description
Morphometric measurements in micrometers are listed for four specimens labeled M1, M2, F1, and F2 under registration numbers ZSI/EBRC/N-18943 through ZSI/EBRC/N-18946. Total length values are 1533 for M1, 1537 for M2, 1884 for F1, and 1702 for F2. Ratios a, b, c, and c prime are given as 22, 5.9, 8.9, and 3.8 for M1; 22, 5.9, 8.6, and 4.6 for M2; 17, 6, 9.3, and 5.1 for F1; and 17, 5.7, 8.6, and 4.9 for F2. Buccal cavity length is 30, 23, 31, and 27; body diameter at cephalic setae is 24, 24, 32, and 28; and cephalic setae length is 8, 8, 8.9, and 8.5. Nerve ring from anterior end is 98, 106, 116, and 110, and nerve ring cbd is 43, 40, 51, and 45; pharynx length is 260, 262, 316, and 298, with cbd at pharynx base 51, 52, 67, and 61. Maximum body diameter is 70, 71, 113, and 99; cloacal or anal body diameter is 46, 39, 40, and 41; tail length is 172, 179, 203, and 197; and tail length in cbd is 3.8, 4.6, 5.1, and 4.9. Right spicule length, left spicule length, gubernaculum length, and telamon length are listed for M1 and M2 only, with values 56 and 56, 56 and 55, 36 and 37, and 37 and 37, while F1 and F2 show a dash in those rows. Female only rows list body diameter at vulva as a dash for M1 and M2 and as 113 for F1 and 98 for F2, V as a dash for M1 and M2 and as 925 for F1 and 837 for F2, and percent V as a dash for M1 and M2 and as 49 for both F1 and F2.
Materials examined: Two males (slide ZSI/EBRC/N-18943 and slide ZSI/EBRC/N-18944), two females (slide ZSI/EBRC/N-18945 and slide ZSI/EBRC/N-18946).
Diagnostic characters: Excretory pore opens at the level of the buccal cavity end. Both spicule size is similar, but the shape of the left spicule is relatively much wider than the uniformly tapered right spicule. Gubernaculum rod-like. Telamon without denticle. Ovoid whitish structure at base of spicule.
Description: Males. Body cylindrical, tapering slightly towards anterior extremities in comparison to the body, but the labial region little wider, forming a posterior neck. Six outer labial and four cephalic sensilla setose. Lips high. Heterogeneous cuticular punctuation. Punctuation most prominent and nearly hexagonal between the cephalic end and the nerve ring. Punctuation on rest of the body is tubular and narrower. Buccal cavity conical with one distinct and large dorsal tooth inserted in pharyngeal lining, two small ventral teeth. Rows of noticeable denticles anterior to teeth. Indistinct amphid. Ocelli absent. Pharynx swollen anteriorly surrounding the buccal cavity, uniformly tubular in the middle, gradually becomes wider posteriorly without accurate bulb. Excretory pore opens at the level of end of buccal cavity. Testis monorchic, situated to right of intestine. Paired curved spicules with almost of similar size but of different shape. Right spicule evenly tapered throughout its length. Left spicule uneven in shape, proximally narrower, becomes much wider including calomus region, again distally becomes narrower near to tip. Thin rod-shaped gubernaculum close to the spicule. Paired telamon, well cuticularized, L-shaped, without denticles. At base of reproductive aperture, ovoid whitish structure observed. A pair of pre-cloacal setae present. Tail conical. Three overlapping thin and elongated caudal glands present. Spinneret distinct with terminal opening.
Females: Similar to males. Slightly longer and wider body than male. Reproductive system didelphic, with two opposed and reflexed ovaries, located to the right of intestine. Vulva well cutinized, located near mid-body. Vagina with muscular wall.
Phylogenetic relationship
The BI phylogenetic tree based on the SSU gene (Figure 3) recovered two major clades within the family Chromadoridae, each comprising well-supported generic subclades. The genus Euchromadora formed a monophyletic subclade that included the newly generated sequence of E. atypica, with strong support (PP = 1.0). The E. atypica sequence (PX468573) showed a close phylogenetic affinity to Euchromadora sp. (MH302926) from Panama, sharing 97.9% sequence similarity. The BI phylogenetic tree inferred from the LSU gene (Figure 4) resolved two major and two minor clades within Chromadoridae. The E. atypica sequence from India (PX585119) clustered closely with E. ezoensis (OQ417519), forming a well-supported subclade. Overall, the branching patterns indicate clear genetic differentiation among genera, whereas species-level relationships are reflected by shorter branch lengths and nested subclades in both gene-based phylogenies.
BI phylogeny tree with PP value, inferred from SSU data for Chromadoridae based on GTR + I + G substitution model.
Figure 3 Long description
The circular phylogenetic tree displays evolutionary relationships among various species within the Chromadoridae family. The tree is organized radially, with branches extending outward from the center. The tree is color-coded, with red, yellow and blue branches indicating different clades or subclades. The red branches highlight a specific clade, including Euchromadora atypica India, Euchromadora ezoensis and Euchromadora sp. Panama, showing close phylogenetic affinity. The blue section emphasizes the Euchromadora genus, forming a monophyletic subclade. The layout visually represents genetic differentiation among genera and species-level relationships, with shorter branch lengths indicating closer genetic relationships.
BI phylogeny tree with PP value, inferred from LSU data for Chromadoridae based on GTR + I + G substitution model.
Figure 4 Long description
The circular phylogenetic tree illustrates the evolutionary relationships within the Chromadoridae family. The tree is centered with branches radiating outward, each labeled with species names and accession numbers. The branches are color-coded, with yellow, black, blue and red lines indicating different clades or subclades. Notable sequences include PX585119, Euchromadora atypica, highlighted in red and OQ417519, Euchromadora ezoensis, highlighted in blue. The tree structure shows branching patterns that reflect genetic differentiation among genera, with shorter branch lengths indicating species-level relationships. The layout is radial, emphasizing the connections and divergence among the various species within the family.
Distribution of genus
Species of the genus Euchromadora are distributed across distinct pockets within different climatic zones. The type species, E. vulgaris, exhibits a broad global distribution compared to other species in the genus and is represented by numerous distributional records. Similarly, E. striata shows a comparatively wide geographic range, although it is supported by fewer occurrence records. In contrast, the remaining species of the genus appear to have more restricted distributions, being largely confined to specific geographic regions. Notably, much of the African coastline and extreme South Asian countries currently lack documented records of Euchromadora. The global distribution of all 11 valid species is illustrated in Figure 5.
Map presents the distributional locations of 11 currently valid species of the genus Euchromadora.
Figure 5 Long description
A thematic world map displays the distribution of eleven Euchromadora species using distinct point symbols. North is oriented at the top, indicated by a north arrow. The map includes a scale bar, though its relevance on a global scale is limited. Each species is represented by a unique symbol: E. atypica (orange star), E. robusta (red star), E. eileenae (green circle), E. striata (purple circle), E. exoensis (orange circle), E. tokiokai (purple square), E. gaulica (yellow circle), E. vulgaris (purple diamond), E. meadi (black triangle), E. permatabilis (pink circle) and E. rebecca (blue diamond). Distribution points are concentrated in specific regions: North America, South America, Europe, Asia and Australia, with notable clusters in coastal areas and some inland regions. The African coastline and parts of South Asia show a lack of documented records. The map effectively illustrates the global spread and regional concentrations of these species.
Discussion
Euchromadora atypica exhibits several distinct morphological characters that clearly differentiate it from the only previously recorded Euchromadora species from India, E. vulgaris. These differences include total body length (1.2–1.9 mm in E. atypica versus 2.0–2.4 mm in E. vulgaris), absence of lateral pigment spots or ocelli (present in E. vulgaris), shorter spicules (55–60 µm versus 73–91 µm in E. vulgaris), dimorphic spicules (symmetrical in E. vulgaris), and reduced lengths of the gubernaculum (36–37 µm versus 47–48 µm in E. vulgaris) and telamon (37–39 µm versus 44–48 µm in E. vulgaris). Notably, specimens examined in the present study (1,533–1,884 µm) are slightly larger than those originally described by Blome (Reference Blome1985) from the Galápagos Islands (1,220–1,511 µm), a variation that may reflect geographic or environmental influences.
The known distribution pattern of the genus Euchromadora indicates a predominantly patchy distribution for most valid species, with the exceptions of E. vulgaris and E. striata, which exhibit comparatively wide geographic ranges. Furthermore, many regions with suitable habitats lack records of Euchromadora species, likely reflecting limited or inadequate taxonomic investigation rather than true absence. Euchromadora atypica was originally described from the Galapagos Islands in the eastern Pacific Ocean (Blome, Reference Blome1985), with the only subsequent record reported from Cuba in the Atlantic Ocean (Armenteros et al., Reference Armenteros, del Pino-Machado, González-González, Ruiz-Abierno and Pérez-García2014). The present record from the Indian Ocean substantially extends the known distribution of this species and confirms its occurrence across tropical marine regions.
Molecular characterization of E. atypica is reported here for the first time. Of the 11 valid species currently recognized within the genus, SSU gene sequences are now available for only four species, while LSU gene sequences are available for only two, including E. atypica. The recovery of a well-supported monophyletic clade for Euchromadora corroborates the current morphology-based taxonomy of the genus. However, the limited molecular representation highlights the need for additional sequence data to better resolve phylogenetic relationships within the genus. Phylogenetic analysis based on the SSU rRNA gene revealed a close affinity between E. atypica reported in the present study and Euchromadora sp. from Panama, a locality geographically proximate to the type region of E. atypica (Galapagos Islands). This molecular evidence further supports the presence of eastern Pacific Euchromadora lineages in the Indian Ocean. In contrast, the LSU-based phylogeny primarily corroborates the generic placement of E. atypica, as the limited availability of comparative sequence data restricts phylogenetic resolution largely to the family level.
Ecologically, E. atypica was found to be abundant on rocky substrates with associated vegetative growth at Arjyapalli Beach, Odisha. This habitat preference is consistent with previous reports of Euchromadora species occurring on hard substrata with algal cover (Bastian, Reference Bastian1865; Inglis, Reference Inglis1969; Kito, Reference Kito1977; Kulikov et al., Reference Kulikov, Dashchenko, Koloss and Yushin1998; Armenteros et al., Reference Armenteros, del Pino-Machado, González-González, Ruiz-Abierno and Pérez-García2014; Leduc, Reference Leduc2025). With the addition of E. atypica from the Odisha coast, the number of Euchromadora species recorded from Indian waters now increases to two.
Acknowledgements
The authors express their sincere gratitude to the Vice Chancellor of Berhampur University for providing the essential facilities required to conduct this research. The first author also gratefully acknowledges the financial support received from the Odisha Government under the MRIP-2023 programme through the MRF Fellowship.
Author contributions
S.P.: Data collection, species identification, molecular analyses, preparation of illustrations, and manuscript drafting. S.P.: Data verification, critical evaluation of results, and revision and improvement of the manuscript.
Declaration of interest
The authors declare no conflict of interest.
Data availability statement
All essential data are provided in this article.
Ethics and approach
Not applicable.
Funding statement
No funding.
