Introduction
The phylum Nemertea, commonly known as ribbon worms, includes unsegmented vermiform invertebrates widely distributed in marine environments (Hookabe Reference Hookabe2022). Although the phylum currently comprises more than 1,300 valid species worldwide (Norenburg et al. Reference Norenburg, Chernyshev, Kajihara and Maslakova2026), recent molecular phylogenetic studies have continued to reveal numerous undescribed and cryptic taxa (e.g., Abato and Kajihara Reference Abato and Kajihara2025; Chernyshev et al. Reference Chernyshev, Abukawa and Kajihara2015; Chernyshev and Polyakova Reference Chernyshev and Polyakova2018a, Reference Chernyshev and Polyakova2018b, Reference Chernyshev and Polyakova2019).
Most marine nemerteans inhabit shallow environments, whereas a few species have been known from the deep sea (deeper than 200 m) (Chernyshev Reference Chernyshev2013). Before 2013, only 14 described species of benthic nemerteans had been recorded from depths greater than 1,000 m (Chernyshev Reference Chernyshev2013). Over the past two decades, however, several expeditions focused on deep-sea biodiversity, including SoJaBio, KuramBio, SokhoBio, Vema-TRANSIT, and the research vessel Hakuho-maru cruises (KH-22-8, KH-23-5), have greatly expanded our understanding of the nemertean diversity in the deep sea (e.g., Abato and Kajihara Reference Abato and Kajihara2025; Chernyshev and Polyakova Reference Chernyshev and Polyakova2018a, Reference Chernyshev and Polyakova2018b, Reference Chernyshev and Polyakova2019). These surveys revealed that nemerteans are distributed across a much broader bathymetric range than previously recognised, reaching hadal depths (deeper than 6,000 m) with five new species (Abato and Kajihara Reference Abato and Kajihara2025; Chernyshev and Polyakova Reference Chernyshev and Polyakova2018a, Reference Chernyshev and Polyakova2019).
The majority of nemerteans are free-living, whereas more than 50 species are known to be associated with other invertebrates including bivalves, crustaceans, and echinoderms (reviewed by Majhi et al. Reference Majhi, Dhara, Madhu, Hansell, Ghorai, Madhu, Sanyal, Sen, Ganguly and Hansell2024). Such associations are also reported from the deep sea, but their true diversity remains poorly understood because nemerteans are frequently detached from their hosts during collection (Chernyshev and Kuznetsov Reference Chernyshev and Kuznetsov2025). Associations with ascidians (class Ascidiacea in the phylum Chordata) are no exception. To date, several nemertean species have been reported as commensals of ascidians, most of which belong to the hoplonemertean family Oerstediidae Chernyshev, 1993 (Chernyshev Reference Chernyshev, Saeedi and Brandt2020; Junoy et al. Reference Junoy, Andrade and Giribet2011; Kvist et al. Reference Kvist, Chernyshev and Giribet2015; Monniot Reference Monniot and Kinne1990). Except for shallow-water species such as Gononemertes australiensis Gibson, 1974, Gononemertes parasita Bergendal, 1900, and Vieitezia luzmurubeae Junoy, Andrade and Giribet, 2010, all deep-sea nemerteans are associated with ascidians of the genus Culeolus Herdman, 1881 (Chernyshev Reference Chernyshev, Saeedi and Brandt2020; Kvist et al. Reference Kvist, Chernyshev and Giribet2015).
During a deep-sea faunal survey conducted by the R/V Shinsei-maru (Japan Agency for Marine-Earth Science and Technology) along the continental slope of the Japan Trench in the northwestern Pacific Ocean, we collected styelid ascidians attached to small rocks and recovered nemerteans from the peribranchial cavities of these ascidians. In this study, we inferred the phylogenetic positions of these nemerteans using sequences of mitochondrial cytochrome c oxidase subunit I (COI), 16S rRNA (16S), nuclear histone H3 (H3), 18S rRNA (18S), and 28S rRNA (28S) genes, providing new insights into host utilisation and diversity of ascidian-associated oerstediid nemerteans.
Materials and methods
The host ascidians, attached to small rocks, were collected in a substrate sample obtained using an ORI-type 3-m beam trawl on 23 March 2026 during the KS-26-3 cruise of the R/V Shinsei-maru, at depths of 1,400–1,409 m at Station FS1500 BT (37°46.72′ N, 142°41.75′ E to 37°46.30′ N, 142°41.04′ E). The ascidian tunics were visually examined for symbiotic organisms. Nemerteans thus obtained were preserved in 99% ethanol, and some individuals were photographed before preservation. Two individuals, each recovered from a different ascidian host and used for molecular analyses, were deposited in the Invertebrate Collection of the Hokkaido University Museum (ICHUM), Sapporo, Japan, under catalogue numbers ICHUM 9472 and 9473. The remaining specimens are retained in the collection of Dr. Hiroshi Kajihara at Hokkaido University, Sapporo, Japan.
Genomic DNA was extracted from the clipped tissue of two nemertean individuals using the DNeasy Blood and Tissue Kit (Qiagen, Netherlands). Partial fragments of COI, 16S, H3, 18S, and 28S genes were PCR-amplified using the primers listed in Supplementary Table S1. Each PCR reaction was conducted in a total volume of 25 μL containing 17.3 μL of double-distilled water, 2.5 μL of genomic DNA, 2.5 μL of Ex Taq Buffer (×10), 2.0 μL of dNTP mixture, 0.3 μL of forward and reverse primers (20 μM each), and 0.1 μL of TaKaRa Ex Taq Hot Start Version (TaKaRa Bio, Japan). The thermal cycling consisted of an initial denaturation at 94.5°C for 2 min; 35 cycles of denaturation at 94.5°C for 30 s, annealing at 42.0°C (COI) or 50.0°C (16S, H3, 18S, 28S) for 40 s, and extension at 72.0°C for 1 min (COI, 16S, H3), 1.5 min (28S), or 2 min (18S); followed by a final extension at 72.0°C for 5 min. Successful amplicons were purified with ExoSAP-IT Express (Applied Biosystems, USA) and sequencing reactions were performed with a BigDye Terminator Cycle Sequence Kit 3.1 (Applied Biosystems) and the primers shown in Supplementary Table S1. The reaction mixtures were purified with a BigDye XTerminator Purification Kit (Applied Biosystems) and sequenced on an ABI PRISM 3500xL sequencer (Applied Biosystems). In addition to the nemerteans, the COI and 18S sequences of the ascidian hosts of the two nemertean specimens were also generated using the same methods described above for their species identification. The newly generated sequences were deposited in DDBJ under the accession numbers LC942292–LC942303 (see Supplementary Tables S2 and S3 for details).
A BLAST search (Johnson et al. Reference Johnson, Zaretskaya, Raytselis, Merezhuk, McGinnis and Madden2008) of the newly obtained 18S and 28S sequences suggested that the nemerteans should be assigned to the hoplonemertean family Oerstediidae. To infer their phylogenetic position within this family, we conducted a maximum-likelihood (ML) analysis based on a concatenated COI+16S+H3+18S+28S dataset comprising our oerstediid specimens, complemented with 36 additional species from Oerstediidae and three outgroup taxa (Acteonemertes orientalis Hookabe, Fujino, Jimi and Ueshima, 2023, Argonemertes australiensis (Dendy, 1892), and Potamonemertes percivali Moore and Gibson, 1973) retrieved from GenBank (Supplementary Table S2). The ribosomal rDNA sequences (16S, 18S, and 28S) were aligned separately using the online version of MAFFT 7.520 (Katoh et al. Reference Katoh, Rozewicki and Yamada2019) with the Q-INS-i strategy (Katoh and Toh Reference Katoh and Toh2008). The COI (658 bp) and H3 (328 bp) fragments were aligned by eye as deduced amino acid sequences (no indels were observed). Ambiguously aligned regions were removed with Gblocks version 0.91b (Castresana Reference Castresana2000) under less stringent settings, leaving final alignments of 608 bp (COI), 414 bp (16S), 323 bp (H3), 1,753 bp (18S), and 1,123 bp (28S). The best-fit substitution models selected in jModelTest 2.1.10 (Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012) under the Akaike information criterion (AIC; Akaike Reference Akaike1974) were TVM+Γ+I for COI, TIM3+Γ+I for 16S, TIM1+Γ+I for H3, TIM2+Γ+I for 18S, and GTR+Γ+I for 28S. ML analyses of the five genes concatenated were carried out in RAxML-NG (Kozlov et al. Reference Kozlov, Darriba, Flouri, Morel and Stamatakis2019), with nodal support assessed using 1,000 bootstrap replicates (BS, %). Trees were visualized in FigTree 1.4.4 (Rambaut Reference Rambaut2018).
Results and discussion
A total of six nemertean individuals were found in the peribranchial cavities of three ascidians attached to small rock fragments (Figure 1a). The host ascidians had an oval and dorsoventrally flattened body, reaching ca. 50 mm in maximum length. The tunic was thick and covered with sand grains and small pebbles, giving the animals an overall muddy appearance. The live nemerteans were ca. 10–20 mm in body length and pale orange (Figure 1b). Ethanol-preserved specimens were strongly shrunken and faded in colouration. The BLAST search results for COI and 18S revealed that the host ascidians belonged to the family Styelidae Sluiter, 1895 (Supplementary Table S3). Because only external morphology was examined and species- or genus-level identification in styelid ascidians generally requires internal morphological characters such as the gonads, the host ascidian was hereafter treated as Styelidae gen. et sp. based on the molecular data.
Oerstediid nemerteans collected from deep-sea styelid ascidians off northeastern Japan in the northwestern Pacific Ocean. (a) A host ascidian attached to a rock. (b) A nemertean specimen found in the peribranchial cavity of the ascidian. (c) Maximum-likelihood phylogeny for Oerstediidae reconstructed from a concatenated COI+16S+H3+18S+28S dataset (4,221 sites in total). Numbers near nodes are bootstrap values (BS, %); values below 50% are not shown. Several genera are collapsed for simplicity and readability (see Supplementary Figure S1 for full tree).
Figure 1. Long description
Panel a is a photo of a host ascidian with a rough, sediment-covered exterior attached to a rock. A pale pink, worm-like nemertean is visible on the surface. Panel b is a close-up photo of a long, slender, pale pink nemertean specimen against a black background, with a 5 mm scale bar. Panel c is a phylogenetic tree. The root on the left branches into an outgroup containing Argonemertes, Potamonemertes, and Acteonemertes. The main Oerstediidae clade is highlighted in a light gray box. Within this, a blue-shaded region labeled Clade Diplomma includes Diplomma serpentinum, Diplomma bothwellae, and several Gononemertes species. Below this, a green-shaded region labeled Clade Gononemertes contains various genera including Galathenemertes, Vieitezia, and Alvinonemertes. Two specific specimens, Oerstediidae gen. et sp. I C H U M 9473 and 9472, are highlighted in bold within the green clade, both noted as having Styelidae hosts. Bootstrap values are indicated at the nodes, such as 100 at the base of the Oerstediidae clade and 93 at the base of Clade Gononemertes. A scale bar for genetic distance of 0.06 is located at the bottom left.
In the ML tree on the concatenated COI+16S+H3+18S+28S dataset ( Figure 1c ), the monophyly of the family Oerstediidae was strongly supported (BS = 100%). The two newly obtained nemertean sequences were nested within the well-supported ‘Clade Gononemertes’ sensu Chernyshev and Kuznetsov (Reference Chernyshev and Kuznetsov2025) (93%), together with representatives of the genera Alvinonemertes Sagorny, von Döhren, Rouse and Tilic, 2022, Chernyshevia Sagorny, von Döhren, Rouse and Tilic, 2022, Galathenemertes Chernyshev and Polyakova, Reference Chernyshev and Polyakova2019, Gononemertes Bergendal, 1900, Sagornya Chernyshev and Kuznetsov, Reference Chernyshev and Kuznetsov2025, and Vieitezia Junoy, Andrade and Giribet, 2010. However, phylogenetic relationships within this clade remained largely unresolved, probably due to the limited resolution of the DNA markers used in the analyses; for example, the two genera Alvinonemertes and Sagornya were recovered as polyphyletic, consistent with the results of Hookabe et al. (Reference Hookabe, Manzano, Jimi, Kise, Reimer, Tsuchida and Fujiwara2025, fig. 3). Additionally, the ‘Clade Diplomma’ sensu Chernyshev and Kuznetsov (Reference Chernyshev and Kuznetsov2025), including two species of Diplomma Stimpson, 1857 and two species of ‘Gononemertes’, was recovered with moderate support (87%).
The family Oerstediidae is distributed worldwide across the full bathymetric range, from intertidal to hadal depths (Chernyshev and Polyakova Reference Chernyshev and Polyakova2019). This family is also notable for comprising numerous commensal species associated with a wide variety of invertebrate hosts, including sponges, corals, sea anemones, sea stars, and ascidians (e.g., Majhi et al. Reference Majhi, Dhara, Madhu, Hansell, Ghorai, Madhu, Sanyal, Sen, Ganguly and Hansell2024; Chernyshev and Kuznetsov Reference Chernyshev and Kuznetsov2025; Hookabe et al. Reference Hookabe, Manzano, Jimi, Kise, Reimer, Tsuchida and Fujiwara2025). Several ascidian-associated nemerteans belonging to Oerstediidae have been reported (Monniot Reference Monniot and Kinne1990; Chernyshev Reference Chernyshev, Saeedi and Brandt2020; Majhi et al. Reference Majhi, Dhara, Madhu, Hansell, Ghorai, Madhu, Sanyal, Sen, Ganguly and Hansell2024), and G. parasita and four Gononemertes-like species have been recorded from the deep sea (Table 1). Among these, ‘Gononemertes’ sp. 2 (= Gononemertes sp. in Chernyshev et al. Reference Chernyshev, Abukawa and Kajihara2015; Gononemertes sp. IZ 45558 in Kvist et al. Reference Kvist, Chernyshev and Giribet2015) and ‘Gononemertes’ sp. 3 (= Gononemertes sp. 6DS in Chernyshev and Polyakova Reference Chernyshev and Polyakova2018b) are placed in the ‘Clade Diplomma’, whereas the nemertean obtained in the present study and two ascidian-associated species, G. parasita and V. luzmurubeae, are included in the ‘Clade Gononemertes’. This suggests that commensalism between nemerteans and ascidians evolved independently at least twice within Oerstediidae. However, molecular data are still unavailable for two of the four ‘Gononemertes’ spp. and the evolutionary history of this association remains poorly understood.
List of commensal nemertean species associated with deep-sea ascidians
Table 1. Long description
The table contains five columns and six data rows.
* Row 1: Species is Gononemertes parasita. Host is Ascidia obliqua. Locality is Northeastern Atlantic. Depth is 15 to 886 meters. References are Brinkmann 1927 and Chernyshev and Polyakova 2022.
* Row 2: Species is Gononemertes sp. 1. Host is Culeolus nadejdi. Locality is Sea of Okhotsk. Depth is 1,040 to 1,050 meters. Reference is Sanamyan 1992.
* Row 3: Species is Gononemertes sp. 2. Host is Culeolus cf. murrayi. Locality is Abyssal plain adjacent to the Kuril-Kamchatka Trench. Depth is 5,124 to 5,125 meters. References are Chernyshev et al 2015 and Kvist et al. 2015.
* Row 4: Species is Gononemertes sp. 3. Host is Culeolus sp. Locality is Kuril Basin of the Sea of Okhotsk. Depth is 3,300 to 3,303 meters. Reference is Chernyshev and Polyakova 2018b.
* Row 5: Species is Gononemertes sp. 4. Host is Culeolus barryi. Locality is Off the coast of California, U S A. Depth is 1,200 meters. Reference is Sanamyan et al. 2018.
* Row 6: Species is Oerstediidae gen. et sp. Host is Styelidae gen. et sp. Locality is Off the Pacific coast of Honshu Island, Japan. Depth is 1,400 to 1,409 meters. Reference is This study.
A footnote indicates that for the deep-sea record of Gononemertes parasita at 886 meters, the host is unknown.
a Host unknown for the deep-sea record (886 m depth; Chernyshev and Polyakova Reference Chernyshev and Polyakova2022).
Associations between nemerteans and ascidian hosts have been reported from intertidal to abyssal depths (Chernyshev Reference Chernyshev, Saeedi and Brandt2020; Majhi et al. Reference Majhi, Dhara, Madhu, Hansell, Ghorai, Madhu, Sanyal, Sen, Ganguly and Hansell2024; Monniot Reference Monniot and Kinne1990). Among shallow-water species, G. parasita is one of the best-studied examples (e.g., Brinkmann Reference Brinkmann1927; Franzén and Sensenbaugh Reference Franzén and Sensenbaugh1988) and is known to inhabit the peribranchial cavity of the ascidian Ascidia obliqua Alder, 1863 (Brinkmann Reference Brinkmann1927). Although a specimen identified as G. parasita was reported from the Iceland Basin at a depth of 886 m, it was collected without a host, and its host in deep-sea environments remains unknown (Chernyshev and Polyakova Reference Chernyshev and Polyakova2022). It is noteworthy that all confirmed records of deep-sea ascidian-associated nemerteans utilise the ascidian genus Culeolus in the family Pyuridae Hartmeyer, 1908 (Chernyshev et al. Reference Chernyshev, Abukawa and Kajihara2015; Chernyshev and Polyakova Reference Chernyshev and Polyakova2018b; Kvist et al. Reference Kvist, Chernyshev and Giribet2015; Sanamyan Reference Sanamyan1992; Sanamyan et al. Reference Sanamyan, Sanamyan and Kuhnz2018; Table 1). In contrast, we found oerstediid nemerteans from ascidians of the family Styelidae, indicating that deep-sea commensal nemerteans may utilise a broader range of ascidian hosts than previously recognised. The apparent predominance of Culeolus among previous records may reflect not only biological host preference but also a sampling bias. The styelid ascidians examined here were entirely covered with an opaque brown tunic (Figure 1a), whereas Culeolus species possess a translucent one (e.g., Chernyshev and Polyakova Reference Chernyshev and Polyakova2018b, fig. 6F; Sanamyan et al. Reference Sanamyan, Sanamyan and Kuhnz2018, fig. 1E), potentially making commensal nemerteans easier to detect in the latter hosts. Further anatomical examinations of taxonomically diverse deep-sea ascidians will likely reveal additional nemertean–ascidian associations that have thus far escaped detection.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X26102223.
Acknowledgements
We are grateful to the officers and crew of the R/V Shinsei-maru and all scientists and technical staff on board the KS-26-3 expedition, especially Dr. Mizuki Ohta (Hokkaido University), Dr. Genki Ishiyama (Marine Ecology Research Institute Foundation), Dr. Takuya Yahagi (The University of Tokyo), and Mr. Hiroki Ushiromura (Marine Works Japan Ltd.), for their invaluable assistance during the research cruise. Our thanks also go to Dr. Samuel Abalde Lago (The University of Tokyo) for valuable comments and linguistic corrections to the manuscript. We express our gratitude to Dr. Hiroshi Kajihara (Hokkaido University) for facilitating the deposition of the nemertean specimens. We also thank the editor, Dr. Alexei Chernyshev (Russian Academy of Sciences), and an anonymous reviewer for their constructive comments and suggestions, which substantially improved our manuscript.
Financial support
This study was supported by the Cooperative Research Program of Atmosphere and Ocean Research Institute, The University of Tokyo (R/V Shinsei-maru, Number JURCAOSS25-31).
Competing interests
The authors declare none.
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