Research Article
Research Article
Supplemental re-description of a deep-sea ascidian, Fimbrora calsubia (Ascidiacea, Enterogona), with an inference of its phylogenetic position
expand article infoNaohiro Hasegawa, Natsumi Hookabe§, Yoshihiro Fujiwara§, Naoto Jimi|, Hiroshi Kajihara
‡ Hokkaido University, Sapporo, Japan
§ Research Institute for Global Change (RIGC), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan
| Nagoya University, Toba, Japan
¶ Universiti Sains Malaysia, Penang, Malaysia
Open Access


Fimbrora Monniot & Monniot, 1991, a macrophagous ascidian genus within the family Ascidiidae Adams & Adams, 1858, is currently monotypic, represented by F. calsubia Monniot & Monniot, 1991, a species previously recorded from the bottom of the South Pacific at depths of 1000–1860 m. The taxonomic status of Fimbrora has remained ambiguous because characteristics in its branchial papillae and neural-gland opening are incompletely known in previous studies, while these traits are essential for distinguishing other ascidiid genera. So far, no nucleotide sequence representing F. calsubia is available. In this study, we collected a single specimen of F. calsubia at a depth of 2027 m, about 400 km off the Pacific coast of Honshu, Japan. This is the deepest record, as well as the first report from the North Pacific, for the species. Our examination indicates that Fimbrora is morphologically similar to another ascidiid genus, Psammascidia Monniot, 1962, by having only secondary branchial papillae in the pharynx. Our phylogenetic analysis, based on the 18S ribosomal RNA and cytochrome c oxidase subunit I genes, along with those of 27 ascidian species available in public databases, showed that F. calsubia was more closely related to Ascidia zara Oka, 1935, Phallusia fumigata (Grube, 1864) and Phallusia mammilata (Cuvier, 1815) than to Ascidia ceratodes (Huntsman, 1912), Ascidiella aspersa (Müller, 1776) and Ascidiella scabra (Müller, 1776). Our results also indicated that acquisitions of macrophagous feeding by deep-sea members happened independently at least three times in the evolutionary history of the entire Ascidiacea.

Key Words

bathyal zone, biogeography, Chordata, phylogeny, taxonomy, Tunicata, Urochordata


The ascidiid genus Fimbrora Monniot & Monniot, 1991a is currently monotypic, consisting of the deep-sea ascidian Fimbrora calsubia Monniot & Monniot, 1991a. The taxonomic identity of Fimbrora is not fully established because states of some characters used for distinguishing other ascidiid genera are not known for this taxon. Apart from Fimbrora, the family Ascidiidae Adams & Adams, 1858 also contains four genera: Ascidia Linnaeus, 1767; Ascidiella Roule, 1884; Phallusia Savigny, 1816; and Psammascidia Monniot, 1962. Fimbrora is supposed to be distinguished from the other ascidiid genera by having a combination of three characteristics: i) the large, cup-shaped oral siphon with thin, uniformly long, and soft lobes, ii) two large blood vessels running on the oral-siphon wall and iii) macrophagous feeding behaviour (cf. Monniot and Monniot (1991a)). The remaining four genera are distinguished from each other, based on: i) whether primary and/or secondary branchial papillae in the pharynx are present and ii) whether accessory openings of the neural gland are present (e.g. Kott (1985); Monniot et al. (1991); Rocha et al. (2012)). However, while the branchial papillae have been reported to be present in Fimbrora (Monniot & Monniot, 1991a), whether they are primary and/or secondary was not mentioned in any of the previous literature (Monniot and Monniot 1991a; Monniot 1993; Monniot and López-Legentil 2017); also, the nature of the neural-gland opening (or, whether accessory openings are present) has not been stated in any of these works.

While ascidians are generally suspension feeders that filter food particles, such as phytoplankton, from the surrounding seawater (Millar 1971), 40 species have hitherto been identified as macrophagous, based on their large oral siphons and unciliated pharynges; direct confirmation of this feeding behaviour was made in 10 species by the presence of small crustaceans in their gut contents (Table 1). These macrophagous ascidians exclusively inhabit deep waters below 200 m with one exception, Oligotrema psammites Bourne, 1903, which is also distributed up to 90 m (Table 1). In addition to Fimbrora, another three ascidian taxa—the family Octacnemidae, Herdman 1888 (with 26 species in 10 genera), as well as the two molgulid genera Asajirus Kott, 1989 (with eight species) and Oligotrema Bourne, 1903 (with five species)—are known to consist of macrophagous members (Table 1). Previously, certain morphological data suggested that macrophagous feeding amongst ascidians evolved convergently, probably due to difficulty in filter-feeding in the deep sea (Millar 1959). This view was confirmed by the phylogenetic study of Tatián et al. (2011), including two macrophagous taxa, the octacnemid Megalodicopia Oka, 1918 and the molgulid Oligotrema, but F. calsubia has not been represented with any molecular sequence data.

Table 1.

List of macrophagous species in Ascidiacea with information about family, species, depth, evidence for macrophagous feeding and references.

Family Species Depth (m) Evidence for macrophagous feeding* References
Ascidiidae Fimbrora calsubia Monniot & Monniot, 1991 1000–2027 m/c Monniot and Monniot (1991a), Monniot (1993), Monniot and López-Legentil (2017), present study
Octacnemidae Benthascidia michaelseni Ritter, 1907 399 m Ritter (1907), Monniot (1998)
Cibacapsa gulosa Monniot & Monniot, 1983 567 m/c Monniot and Monniot (1983)
Cryptia planum Monniot & Monniot, 1985 4930 m/c Monniot and Monniot (1985a)
Dicopia antirrhinum Monniot, 1972 600–4300 m/c Monniot (1972), Monniot and Monniot (1974, 1985a), Sanamyan (2014)
Dicopia fimbriata Sluiter, 1905 1210 m Sluiter (1905a), Monniot and Monniot (1991b), Monniot and López-Legentil (2017), Sanamyan and Sanamyan (1999)
Dicopia japonica Oka, 1913 4526–4609 m Oka (1913), Millar (1988)
Kaikoja globosa Monniot, 1998 1978 m Monniot (1998)
Kaikoja multitentaculata (Vinogradova, 1975) 4485–4520 m Vinogradova (1975), Sanamyan and Sanamyan (2002)
Megalodicopia hians Oka, 1918 200–5325 m/c Oka (1918), Tokioka (1953), Kott (1969), Nishikawa (1991), Sanamyan (1998), Okuyama et al. (2002), Havenhand et al. (2006)
Megalodicopia rineharti (Monniot & Monniot, 1989) 695–3970 m Monniot and Monniot (1989), Sanamyan and Sanamyan (2002)
Myopegma melanesium Monniot & Monniot, 2003 445–472 m/c Monniot and Monniot (2003)
Myopegma midatlantica Monniot, 2011 2087 m Monniot (2011)
Octacnemus alatus Monniot & Monniot, 1985 3344 m Monniot and Monniot (1985b)
Octacnemus bythius Moseley, 1876 1957–4087 m/c Moseley (1876), Ritter (1906), Ihle (1935), Millar (1959), Monniot and López-Legentil (2017)
Octacnemus ingolfi Madsen, 1947 640–4655 m Madsen (1947), Monniot and Monniot (1973, 1976, 1985a, 1985b, 1985c, 1991b, 2003), Sanamyan (2014)
Octacnemus kottae Sanamyan & Sanamyan, 2002 3700–3910 m Sanamyan and Sanamyan (2002)
Octacnemus vinogradovae Sanamyan & Sanamyan, 1999 5400 m Sanamyan and Sanamyan (1999)
Octacnemus zarcoi Monniot & Monniot, 1984 4260–4270 m/c Monniot and Monniot (1984a), Sanamyan (2014)
Polyoctacnemus patagoniensis (Metcalf, 1893) 1920 m Metcalf (1893), Ihle (1935)
Situla cuculli Monniot & Monniot, 1991 2040 m Monniot and Monniot (1991b)
Situla galeata Monniot & Monniot, 1991 1395–4891 m Monniot and Monniot (1991b), Sanamyan and Sanamyan (1998)
Situla lanosa Monniot & Monniot, 1973 1800–4990 m Monniot and Monniot (1973, 1974, 1985a), Sanamyan (2014)
Situla macdonaldi Monniot & Monniot, 1977 790 m Monniot and Monniot (1977)
Situla pelliculosa Vinogoradova, 1969 5035–8400 m Vinogradova (1969)
Situla rebainsi Vinogradova, 1975 3700–5651 m Vinogradova (1975), Sanamyan and Sanamyan (2002)
Situla rineharti Monniot & Monniot, 1989 695–3680 m Monniot and Monniot (1989, 1991b)
Molgulidae Asajirus arcticus (Hartmeyer, 1923) 905–1283 m Hartmeyer (1923)
Asajirus dichotomus (Monniot & Monniot, 1984) 3550 m Monniot and Monniot (1984a, 1985a), Kott (1989)
Asajirus eunuchus (Monniot & Monniot, 1976) 2000–5000 m Monniot and Monniot (1976)
Asajirus gulosus (Monniot & Monniot, 1984) 1800–2500 m Monniot and Monniot (1984a), Kott (1989)
Asajirus hemisphericus (Monniot & Monniot, 1990) 3680–3740 m Monniot and Monniot (1990)
Asajirus indicus (Oka, 1913) 800–5000 m/c Oka (1913), Hartmeyer (1923), Van Name (1945), Millar (1959, 1970), Kott (1957, 1969, 1989), Monniot (1969, 1971), Monniot and Monniot (1968, 1970, 1973, 1974, 1976, 1982, 1984a, 1984b, 1985a, 1985b, 1990), Sanamyan and Sanamyan (2006), Maggioni et al. (2018, 2022)
Asajirus ledanoisi (Monniot & Monniot, 1990) 720–4829 m Monniot and Monniot 1973; 1974; 1977; 1985b; 1990; Sanamyan 2014
Asajirus ovirarus (Monniot & Monniot, 1990) 820–1900 m Monniot and Monniot 1990; 2003
Oligotrema lyra (Monniot & Monniot, 1973) 3360–4680 m/c Monniot C. and Monniot F. (1973, 1974, 1984b, 1985a, 1990), Kott (1989), Sanamyan and Sanamyan (1999), Sanamyan (2014)
Oligotrema psammatodes (Sluiter, 1905) 1158 m Millar (1969), Sluiter (1905a, 1905b), Monniot and Monniot (1990)
Oligotrema psammites Bourne, 1903 90–4000 m Bourne (1903), Monniot and Monniot (1990), Monniot (2022), Kott (1992, 2009)
Oligotrema sandersi (Monniot & Monniot, 1968) 2200–5020 m Monniot and Monniot (1968, 1970, 1974, 1985a, 1990), Millar (1970), Kott (1989), Sanamyan (2014)
Oligotrema unigonas (Monniot Monniot, 1974) 2300–5500 m Monniot and Monniot (1974, 1984b, 1985a, 1985b, 1990), Kott (1989), Sanamyan (2014)

The taxonomy of macrophagous molgulids has experienced twists and turns. Historically, Asajirus and Oligotrema were once considered by Kott (1989) to comprise the now-abandoned family Hexacrobylidae Seeliger, 1906, for which the monofamilial order Aspiraculata and the mono-order class Sorberacea had been established by Seeliger (1906) and Monniot et al. (1975), respectively. These suprafamilial higher taxa were rejected by Kott (1989), who also noted morphological similarities between Hexacrobylidae and Molgulidae Lacaze-Duthiers, 1877. At another time, Hexacrobylidae was regarded by Monniot and Monniot (1990) to consist of the four genera Hexacrobylus Sluiter, 1905a, Gasterascidia Monniot & Monniot, 1968, Sorbera Monniot & Monniot, 1974 and Hexadactylus Monniot & Monniot, 1990, the first three of which were synonymised with Oligotrema by Kott (1989) and the last was synonymised with Asajirus by Kott (1992). Later, Hexacrobylidae was demonstrated to be a junior synonym of Molgulidae by a molecular phylogenetic analysis supporting the inclusion of O. lyra (Monniot & Monniot, 1973) in the latter family (Tatián et al. 2011). Until then, Hexacrobylidae/Aspiraculata/Sorberacea had been occasionally considered valid in certain publications (e.g. Monniot (2001)).

So far, F. calsubia has been known from the South Pacific bathyal zone in three publications, based on a total of 13 specimens: three specimens at a depth of 1865 m in New Caledonian waters (Monniot and Monniot 1991a), two specimens at about 1000 m depth in Indonesia (Monniot 1993) and eight specimens at 1000–1200 m depth in Papua New Guinea (Monniot and López-Legentil 2017). Meanwhile, during a biodiversity survey in an off-shore submarine nature conservation area around Nishi-Shichito Ridge in the western North Pacific, a 14th individual of F. calsubia was obtained. Here, we provide a morphological re-description and an inference of its molecular phylogenetic position within the class Ascidiacea.

Materials and methods

A single specimen of F. calsubia was collected near the south of Hoei Seamount, about 400 km off the Pacific coast of Honshu, Japan (Fig. 1), with a manipulator of the human-occupied vehicle Shinkai 6500 (Dive No. 1651) during the cruise YK22-17C of the R/V Yokosuka (Suppl. material 1, 2). The live animal was photographed with an OM-D E-M1X digital still camera (Olympus, Tokyo, Japan) attached to an M.Zuiko Digital ED 30 mm F3.5 Macro lens (Olympus). Two of the thread-like lobes of the specimen were dissected from the oral siphon; one was preserved in 99% ethanol for DNA extraction, the other in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) for future analysis; the remaining body was fixed in 10% formalin seawater for morphological observation. For detailed examination, the pharynx was stained with haematoxylin. The voucher specimens have been deposited in the Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, with the catalogue number JAMSTEC No. 111618 for the formalin-fixed specimen, JAMSTEC No. 111619 for the lobe in 99% ethanol and JAMSTEC No. 111620 for the lobe in RNAlater.

Figure 1. 

Maps showing the sampling site (red circle), south of Houei Seamount (of which the top is indicated with a red triangle). The images were generated by using GMT 6 (Wessel et al. 2019), based on grid data provided by the General Bathymetric Chart of the Oceans.

Total DNA was extracted using a DNeasy Tissue Kit (Qiagen, Hilden, Germany). For amplification, KOD One PCR Master Mix (TOYOBO, Osaka, Japan) was used. Partial sequences of the 18S rRNA (18S) gene and the mitochondrial cytochrome c oxidase subunit I (COI) gene were PCR amplified from the total DNA; the primer pairs 1F/9R (Giribet et al. 1996) and dinF/Nux1R (Brunetti et al. 2017) were used for 18S and COI, respectively. PCRs were performed under the following conditions. For 18S: 94 °C for 2 min; 35 cycles of 94 °C for 45 sec, 52 °C for 50 sec and 72 °C for 90 sec; then 72 °C for 5 min. For COI: 94 °C for 2 min; 35 cycles of 94 °C for 40 sec, 50 °C for 60 sec and 72 °C for 60 sec; then 72 °C for 7 min. Purification of PCR products was conducted by enzymatic reaction with ExoSAP-IT (Applied Biosystem, Waltham, MA, USA). The purified products were sequenced with an ABI BigDye Terminator ver. 3.1 Cycle Sequencing Kit and an ABI 3100 Avant Genetic Analyzer (Applied Biosystem), using the same primer pairs for amplification; for 18S, the internal primers 3F and 5R (Giribet et al. 1996), as well as a2.0 and bi (Whiting et al. 1997), were also used.

For phylogenetic analysis, 18S and COI sequences of 27 ascidian species and those of the lancelet Branchiostoma floridae Hubbs, 1922 were downloaded from GenBank (Table 2). The dataset of 18S was aligned using MAFFT ver. 7.310 with E-INS-I strategy (Katoh and Standley 2013); the aligned 18S dataset was trimmed by using trimAl ver. 1.4. rev15 with gappyout command (Capella-Gutiérrez et al. 2009). An alignment of COI was obtained by using MEGA X (Kumar et al. 2018) following Hasegawa and Kajihara (2019). Then, the 18S and COI sequences were concatenated on MEGA X (Kumar et al. 2018).

Table 2.

The GenBank accession numbers of 18S and COI sequences of Fimbrora calsubia Monniot & Monniot, 1991a, as well as 27 ascidian species and the lancelet Branchiostoma floridae Hubbs, 1922, used for phylogenetic analysis in this study.

Species 18S COI
Ascidia ceratodes L12378 MW872268
Ascidia zara LC547325 KY235397
Ascidiella aspersa LC547321 KF886702
Ascidiella scabra AB811928 MN064599
Botrylloides violaceus LC432326 LC432331
Chelyosoma siboja AF165821 AB104867
Ciona robusta AB013017 MF479417
Ciona savignyi LC547329 MK512499
Clavelina lepadiformis JN573225 AY603104
Clavelina meridionalis FM244840 AM706470
Corella eumyota FM244846 KU299765
Ecteinascidia herdmanni FM244847 AY600968
Ecteinascidia turbinata FM244848 MT873564
Fimbrora calsubia LC777587 LC777585
Halocynthia roretzi AB013016 HM151268
Herdmania momus AF165827 KM411616
Megalodicopia hians AB075543 AB104866
Molgula manhattensis L12426 MT873565
Oligotrema lyra JN565043
Perophora japonica AB499607 MN064600
Perophora viridis FM244849 OM912740
Phallusia fumigata FM244844 KF309548
Phallusia mammillata AF236803 MN064634
Pycnoclavella diminuta KJ632948 KC017435
Pyura mirabilis LC432327 LC432332
Styela clava LC432329 LC432334
Symplegma reptans AF165826 LS992553
Syncarpa composita LC432325 LC432330
Branchiostoma floridae M97571 AB478593

For constructing phylogenetic trees, Bayesian Inference (BI) and Maximum Likelihood (ML) analyses were performed; MrBayes ver. 3.2.6 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003: Ronquist et al. 2012) for BI and the ultrafast bootstrap method (Hoang et al. 2018) implemented in IQtree (Nguyen et al. 2015) for ML. PartitionFinder ver. 2.1.1 (Lanfear et al. 2016) was used for selecting the best-fit substitution models, which suggested GTR + I + G for 18S and COI first codon position and GTR + G for COI second and third codon positions. For BI, Markov chains were started from a random tree and run for 107 generations; trees were picked up every 100 generations from the chain. Burn-in was set at 25%. The “sumt” command was used for calculating a consensus of trees; the posterior probability (PP) for each node was collected to assess the certainty of the inference. Run convergence was assumed, based on the following values of variables: average standard deviation of split frequencies = 0.002002; average estimated sample size of all parameters > 200; and potential scale reduction factor for all parameters ≤ 1.008. For ML analysis, branch support was calculated with 1000 ultrafast bootstraps (Minh et al. 2013).


Taxonomy and morphology

Order Enterogona

Suborder Phlebobranchia

Family Ascidiidae Adams & Adams, 1858

Fimbrora calsubia Monniot & Monniot, 1991a

Figs 2, 3, 4

Fimbrora calsubia Monniot & Monniot, 1991a, p. 384, figs 1–6; Monniot (1993), p. 356; Monniot and López-Legentil (2017), p. 531, figs 1, 2.

New Japanese name

Yorifusa-boya, from yorifusa, an ornament for kimonos and Japanese accessories and boya, a phonological variant of hoya, meaning a sea squirt.

Material examined

One individual, JAMSTEC No. 111618, collected by N. Hookabe on 26 September 2022, about 400 km off the Pacific coast of middle Honshu, Japan, 30°47.05'N, 138°44.72'E, at a depth of 2027 m (Fig. 1).


Individual ca. 20 cm in length including oral siphon (Fig. 2A, B). Tunic opaque and gelatinous; blood vessels running on surface of tunic (Fig. 2B); fine warts, each about 0.5 mm in diameter, scattered evenly over entire tunic. Body attached to substrate with its posterior end (Fig. 2A, B). Oral siphon enlarged, ca. 10 cm in diameter; single annular muscle strand running on outer edge of oral siphon; thread-like lobes, 52 in number, tightly arranged to each other on oral-siphon edge; single groove radially arranged on edge of oral siphon between base of each lobe; muscle strand associated to each lobe, running on inner wall of oral-siphon edge from lobe base for ca. 1 cm; beneath inner surface of oral siphon, neural cords radially running from neural ganglion (Fig. 2C). Oral aperture situated 2.5 cm anterior to neural ganglion. Atrial siphon 1.5 cm in diameter; 37 blood vessels longitudinally running on surface of atrial siphon (Fig. 2D).

Figure 2. 

Fimbrora calsubia Monniot & Monniot, 1991a, photographs showing external appearance of JAMSTEC No. 111618. A. The individual in situ (white arrow), attaching to a dead sponge (yellow arrowhead) along with a euplectellid glass sponge (yellow arrow); B. Left view in life; C. Inner surface of the oral siphon in fixed state; D. Enlarged view of atrial siphon in life.

Body wall attached to tunic on oral siphon, heart and renal vesicles; irregular cavity existing between tunic and body wall; inner surface of tunic covered with epithelial tissue. Neural ganglion situated between oral siphon and atrial siphon. On base of oral siphon, 105 oral tentacles present, each being ca. 8 mm in length. Peripharyngeal band made of single lamina running in a short distance posterior to oral tentacles, forming V-shape posterior to neural gland aperture (Fig. 3A); latter being single in number, almost straight in shape (Fig. 3A) and opening at dorsal tubercle. Pharynx connected by mesenteries to peripharyngeal epithelium; mesenteries 0.5–3.0 mm in diameter (Fig. 3B). Smooth dorsal lamina running along mid-line on ventral side of pharynx (Fig. 3A, B). Longitudinal and transverse vessels running on inner surface of pharynx (Fig. 3C); 6–10 stigmata without lateral cilia per mesh (Fig. 3C). Secondary branchial papillae present on intersections of longitudinal and transverse vessels (Fig. 3C).

Figure 3. 

Fimbrora calsubia Monniot & Monniot, 1991a (JAMSTEC No. 111618). A. Drawing of dissected specimen, showing the shape of neural-gland aperture, peripharyngeal band and dorsal lamina; B. Photograph of dissected pharynx cut open from ventral side; C. Magnification of the rectangle on B, showing the arrangement of longitudinal vessels, transverse vessels, stigmata and secondary branchial papillae (indicated with arrows).

Digestive tract positioned on left side of body (Fig. 4A). Oesophagus opening to left side of dorso-posterior part of pharynx. Stomach about 1.5 cm in length, having 10 folds, surrounded with renal vesicles (Fig. 4A); multiple crustaceans (probably copepods) found in stomach lumen (Fig. 4B). Intestinal loop S-shaped, having primary loop and secondary loop; intestine ca. 7 cm in length, ca. 5 mm in diameter (Fig. 4A). Anus smoothly edged, opening close to atrial siphon (Fig. 4A).

Figure 4. 

Fimbrora calsubia Monniot & Monniot, 1991a (JAMSTEC No. 111618), photographs of fixed specimen. A. Sinistero-posterior portion of body, viewed from outside, showing alimentary canal and reproductive system; B. Cross section of stomach, showing the prey crustacean (probably a copepod); arrows indicating stomach folds; C. Gonads; D. Magnification of the rectangle on C, showing an ovary containing multiple eggs.

Gonad situated proximally on intestinal loop (Fig. 4C). Ovaries surrounded with male testis (Fig. 4C, D). Oviduct and spermiduct running along secondary loop, opening close to anus (Fig. 4A). Eggs contained in ovaries and oviduct, up to 0.2 mm in diameter (Fig. 4D).


The animal attached itself to a dead sponge in an area with accumulated sand and mud at a depth of 2027 m, where the water temperature was 1.93 °C (Fig. 2A; Suppl. material 1). It opens the oral aperture in the direction facing the water current (Suppl. material 2). An euplectellid sponge was also found attached to the same substrate. Macrobenthos found around this area included other sponges, octocorals, sandy creeplets, sea anemones and sea lilies.

Molecular phylogeny

The clade consisting of four genera in the family Ascidiidae, i.e. Ascidia, Ascidiella, Fimbrora and Phallusia, received high support values (97% bootstrap; 1.00 posterior probability) (Fig. 5). In this clade, F. calsubia was most closely related to Ascidia zara Oka, 1935, but with less-supported values (53% bootstrap; 0.68 posterior probability). The clade of Ascidia + Fimbrora + Phallusia was sister to the genus Ascidiella. The genus Ascidia was recovered as a non-monophyletic group.

Figure 5. 

Phylogenetic relationship of 28 ascidian species; a Maximum-Likelihood tree, based on a concatenated dataset consisting of 18S rRNA (1676 bp) and COI (1136 bp) genes. Bootstrap values and posterior probabilities are indicated if they are higher than 60% and 0.70, respectively. Macrophagous species are indicated with an asterisk (*).

The three macrophagous ascidians included in this analysis—F. calsubia, Megalodicopia hians Oka, 1918 and Oligotrema lyra—were each positioned differently in the phylogenetic tree. As in previous analyses (Kurabayashi et al. 2003; Tatián et al. 2011), M. hians was sister to Corella eumyota Traustedt, 1882; O. lyra was sister to Molgula manhattensis (De Kay, 1843).


Previous studies posited that Fimbrora would belong to Ascidiidae (Monniot and Monniot 1991a; Monniot and López-Legentil 2017) and our phylogenetic analysis supported this view. The morphological characteristics that suggested Fimbrora’s familial affiliation were the longitudinal vessels having papillae and straight stigmata in the pharynx (Monniot and Monniot 1991a; Monniot and López-Legentil 2017), while Monniot and Monniot (1991a) noted the superficial resemblance of the genus with the family Octacnemidae in having an enlarged oral siphon. The phylogenetic position of Fimbrora within Ascidiidae was unresolved in our tree (Fig. 5). The more precise phylogenetic position of Fimbrora in the family would require the inclusion of additional ascidiid taxa in molecular analyses. One such to-be-included taxa is Psammascidia, which shares two characteristics with Fimbrora—having secondary branchial papillae on the longitudinal vessels and lacking primary and intermediate branchial papillae (Monniot and Monniot 1973), features that are not found in other ascidiid genera (cf. Kott (1985); Brunetti and Mastrototaro (2017)). Future molecular analyses may reveal the phylogenetic relationship amongst ascidiid species including Fimbrora.

Monniot and Monniot (1991a) suggested that F. calsubia has a partly carnivorous diet, based on the finding of copepods in its gut contents mixed with unidentified particles, as well as the shape of the oral siphon. The presence of small crustaceans, likely copepods, in the stomach of our specimen supports this assertion. The reports of F. calsubia from Indonesia (Monniot 1993) and Papua New Guinea (Monniot and López-Legentil 2017), however, did not provide any information on gut contents in their specimens. In addition to this, the observed behaviour of F. calsubia, where the individual orientates its oral siphon towards the water flow (Suppl. material 2), is similar to the behaviour found in M. hians as described by Okuyama et al. (2002). This suggests that F. calsubia also utilises water currents for feeding.

While the convergent evolution of macrophagous feeding in Megalodicopia and Oligotrema has already been revealed by Tatián et al. (2011), our phylogenetic tree clearly shows that Fimbrora is also the case: this trait was acquired at least three times independently within the class Ascidiacea (Fig. 5).

The present study expanded the species’ known distribution range for about 4000 km northwards, representing the first record of the species from the North Pacific. Our material also represents the deepest record for the species with the known vertical distribution range being about 1000–2000 m (Monniot and Monniot 1991a; Monniot 1993; Monniot and López-Legentil 2017; present study).


We present the first report of F. calsubia from the North Pacific. Our molecular phylogenetic analysis suggested that macrophagous feeding was convergently acquired at least three times independently in Ascidiacea. Our morphological observation indicated a similarity of Fimbrora to Psammascidia in having secondary papillae and lacking primary and intermediate branchial papillae.


We extend our profound gratitude to the captain and crew of the support vessel Yokosuka, the commander and operation team of the human-occupied vehicle Shinkai 6500 and to both Takao Yoshida (JAMSTEC) and Hiroyuki Yokooka (IDEA Inc.) for their invaluable assistance in sample collection. Without the kind support from Kanta Ochiai and Misato Sako (Nagoya University, Sugashima Marine Biological Laboratory) for experimental work, this paper would not have materialised. We are indebted to all the people who donated to NHa through the academic crowd-funding site “academist”, especially to Shunji Furukuma, Naoki Hayashi, Miyuki Honda, Hitoki Horie, Sho Hosotani, Yoshiki Iwai, Nami Kenmotsu, Moe, Takehiro Nakamura, Ryoma Nishikawa, Yuichi Sasaki, Tatsuya Shimoyama, Makoto Taniguchi, Daiki Wakita, Takaaki Yonekura, amongst others. NHa received financial support from JST SPRING, Grant Number JPMASP2119. This research was partly performed by the Environment Research and Technology Development Fund (JPMEERF20S20700) of the Environmental Restoration and Conservation Agency Provided by the Ministry of Environment of Japan. The cruise YK22-17C of the R/V Yokosuka was funded by an MPA monitoring project outsourced by the Ministry of the Environment of Japan.


  • Brunetti R, Mastrototaro F (2017) Ascidiacea of the European waters. Calderini, Bologna, 447 pp.
  • Brunetti R, Manni L, Mastrototaro F, Gissi C, Gasparini F (2017) Fixation, description and DNA barcode of a neotype for Botryllus schlosseri (Pallas, 1766) (Tunicata, Ascidiacea). Zootaxa 4353(1): 29–50.
  • Capella-Gutiérrez S, Silla-Martinez JM, Gabaldón T (2009) trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics (Oxford, England) 25(15): 1972–1973.
  • Hartmeyer R (1923) Ascidiacea, part I. Zugleich eine Übersicht über die arktische und boreale Ascidienfauna auf tiergeographischer Grundlage. Ingolf-Expedition 2(6): 1–365.
  • Hasegawa N, Kajihara H (2019) A redescription of Syncarpa composita (Ascidiacea, Stolidobranchia) with an inference of its phylogenetic position within Styelidae. ZooKeys 857: 1–15.
  • Havenhand JN, Matsumoto GI, Seidel E (2006) Megalodicopia hians in the Monterey submarine canyon: Distribution, larval development, and culture. Deep-sea Research. Part I, Oceanographic Research Papers 53(2): 215–222.
  • Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS (2018) UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution 35(2): 518–522.
  • Ihle JEW (1935) Octacnemus. Handbuch der Zoologie 35(2): 533–544.
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30(4): 772–780.
  • Kott P (1957) The sessile Tunicata. The John Murray Expedition 1933–34 10(4): 129–149.
  • Kott P (1969) Antarctic Ascidiacea. Antarctic Research Series 13: 1–239.
  • Kott P (1985) The Australian Ascidiacea. Part 1: Phlebobranchia and Stolidobranchia. Memoirs of the Queensland Museum 23: 1–440.
  • Kott P (1989) The family Hexacrobylidae Seeliger, 1906 (Ascidiacea, Tunicata). Memoirs of the Queensland Museum 27(2): 517–534.
  • Kott P (1992) The Australian Ascidiacea, supplement 2. Memoirs of the Queensland Museum 32: 621–655.
  • Kott P (2009) Taxonomic revision of Ascidiacea (Tunicata) from the upper continental slope off north-western Australia. Journal of Natural History 43(31–32): 1947–1986.
  • Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547–1549.
  • Kurabayashi A, Okuyama M, Ogawa M, Takeuchi A, Jing Z, Naganuma T, Saito Y (2003) Phylogenetic position of a deep-sea ascidian, Megalodicopia hians, inferred from the molecular data. Zoological Science 20(10): 1243–1247.
  • Lacaze-Duthiers H (1877) Histoire des ascidies simples des côtes de France. Deuxième partie: Étude des espèces. Archives de Zoologie Expérimentale et Générale 6: 457–673.
  • Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2016) PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution 34(3): 772–773.
  • Madsen FJ (1947) Octacnemus ingolfi n.sp., an Atlantic representative of peculiar tunicate-family Octacnemidae. Videnskabelige Meddelelser fra Dansk naturhistorisk Forening 110: 31–44.
  • Maggioni T, Taverna A, Reyna P, Alurralde G, Rimondino C, Tatián M (2018) Deep-sea ascidians (Chordata, Tunicata) from the SW Atlantic: Species richness with descriptions of two new species. Zootaxa 4526(1): 1–28.
  • Maggioni T, Rimondino C, Taverna A, Reyna P, Largger C, Alurralde G, Calcagno E, Tatián M (2022) Abyssal ascidians (Chordata, Tunicata) from the Weddell Sea, Antarctica, including a new Styela species and stomach content identifications. Zootaxa 5093(3): 296–314.
  • Metcalf MM (1893) Notes upon an apparently new species of Octacnemus, a deep sea, Salpa-like tunicate. John Hopkins University Circulars 12(106): 98–100.
  • Millar RH (1959) Ascidiacea. Galathea Report 1: 189–209.
  • Millar RH (1969) Ascidiacea: Some further specimens. Galathea Report 10: 91–98.
  • Millar RH (1970) Ascidians, including specimens from the deep sea, collected by R.V. ‘Vema’ and now in the American Museum of Natural History. Zoological Journal of the Linnean Society 49(2): 99–159.
  • Millar RH (1988) Deep-sea ascidians from the eastern Pacific collected during the Pacific Ocean Biological Survey Program. Journal of Natural History 22(5): 1427–1435.
  • Minh BQ, Nguyen MAT, von Haeseler A (2013) Ultrafast approximation for phylogenetic bootstrap. Molecular Biology and Evolution 305(5): 1188–1195.
  • Monniot C (1969) Ascidies récoltées par la "Thalassa" sur la pente continentale du golfe de Gascogne: (3–12 août 1967). Bulletin du Muséum National d`Histoire Naturelle 41(1): 155–186.
  • Monniot F (1971) Les Ascidies des grandes profondeurs récoltées par les navires atlantis II et chain, 3e note. Cahiers de Biologie Marine 7: 457–469.
  • Monniot C (1972) Dicopia antirrhinum n.sp. Ascidie de la pente du plateau continental du Golfe de Gascogne. Interprétation nouvelle de la famille des Octacnemidae. Cahiers de Biologie Marine 13: 9–20.
  • Monniot C (1993) Tunicata: Sur trois espèces d`ascidies bathyales récoltées au cours de la campagne franco-indonésienne Karubar. Mémoires du Museum National d`Histoire Naturelle 158: 355–359.
  • Monniot C (1998) Abyssal ascidians collected from the proximity of hydrothermal vents in the Pacific Ocean. Bulletin of Marine Science 63(3): 541–558.
  • Monniot C (2001) Ascidiacea and Sorberacea. In: Costello MJ, Emblow C, White R (Eds) European register of marine species: a check-list of the marine species in Europe and a bibliography of guides to their identification. Muséum national d’histoire naturelle, Paris, 352–355.
  • Monniot C, Monniot F (1968) Les ascidies de grandes profondeurs récoltées par le navire oceanographique american Atlantis 2 (Premiere note). Bulletin de l`Institut Océanographique 67(1379): 1–48.
  • Monniot C, Monniot F (1970) Les ascidies des grandes profondeurs récoltées par les navires Atlantis, Atlantis II et Chain (2éme note). Deep-Sea Research and Oceanographic Abstracts 17(2): 317–336.
  • Monniot C, Monniot F (1973) Ascidies abyssales récoltées au cours de la campagne océanographique Biacores par le "Jean Charcot". Bulletin du Muséum National d`Histoire Naturelle 121: 389–475.
  • Monniot C, Monniot F (1974) Ascidies abyssales de l`Atlantique récoltées par le "Jean Charcot" (Campagnes Nortlante, Walda, Polygas A). Bulletin du Muséum National d`Histoire Naturelle 226: 721–786.
  • Monniot F, Monniot C (1976) Tuniciers abyssaux du bassin argentin récoltés par l "Atlantis II". Bulletin du Muséum National d`Nistoire Naturelle 387(269): 629–662.
  • Monniot C, Monniot F (1977) Quelques ascidies abyssales du Sud-Ouest de l’Ocean Indien. Comité National Français des Recherches Antarctiques 42: 305–327.
  • Monniot C, Monniot F (1982) Some Antarctic deep-sea tunicates in the Smithsonian collections. In: Biology of the Antarctic Seas. 10. Antarctic Research Series 32: 95–130.
  • Monniot C, Monniot F (1983) Ascidies antarctiques et subantarctiques: Morphologie et biogeographie. Mémoires du Muséum National d`Histoire Naturelle Série A. Zoologie 125: 1–168.
  • Monniot C, Monniot F (1984a) Tuniciers benthiques récoltées au cours de la campagne Abyplaine au large de Madère. Annales de l`Institut Océanographique 60(2): 129–142.
  • Monniot C, Monniot F (1984b) Nouvelles Sorberacea (Tunicata) profondes de l’Atlantique Sud et l’Ocean Indien. Cahiers de Biologie Marine 25: 197–215.
  • Monniot C, Monniot F (1985a) Nouvelles récoltes de tuniciers benthiques profonds dans l`Ocean Atlantique. Bulletin du Muséum National d`Histoire Naturelle. Section A. Zoologie, Biologie, et Écologie Animales A 7(1): 5–37.
  • Monniot C, Monniot F (1985b) Tuniciers profondes de l’Ocean Indien: Campagnes SAFARI du ‘Marion Dufresne’. Bulletin du Muséum National d`Histoire Naturelle. Section A. Zoologie, Biologie, et Écologie Animales A 7(2): 279–308.
  • Monniot C, Monniot F (1985c) Ascidies profondes au large de Mayotte (Archipel des Comores). Cahiers de Biologie Marine 26(1): 35–52.
  • Monniot C, Monniot F (1989) Ascidians collected around the Galapagos Islands using the Johnson-Sea-Link research submersible. Proceedings of the Biological Society of Washington 102(1): 14–32.
  • Monniot C, Monniot F (1991a) Découverte d`une nouvelle lignée évolutive chez les ascidies de grande profondeur: Une Ascididae carnivore. Comptes Rendus de l`Académie des Sciences, Série 3. Sciences de la Vie 312: 383–388.
  • Monniot C, Monniot F (1991b) Tunicata: Peuplement d`ascidies profondes en Nouvelle-Caledonie. Diversite des strategies adaptives. Mémoires du Muséum National d`Histoire Naturelle, Série A. Zoologie 151: 357–448.
  • Monniot F, Monniot C (2003) Ascidies de la pente externe et bathyales de l’ouest Pacifique. Zoosystema 25(4): 681–749.
  • Monniot C, Monniot F, Gaill F (1975) Les Sorberacea: Une nouvelle classe des tuniciers. Archives de Zoologie Expérimentale et Générale 116: 77–122.
  • Monniot C, Monniot F, Laboute P (1991) Coral reef ascidians of New Caledonia. ORSTOM, Paris, 247 pp.
  • Moseley HN (1876) On two new forms of deep-sea ascidians, obtained during the voyage of H.M.S. “Challenger”. Transactions of the Linnean Society of London, 2nd Series. Zoology : Analysis of Complex Systems, ZACS 1: 287–294.
  • Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268–274.
  • Nishikawa T (1991) The ascidians of the Japan Sea. II. Publications of the Seto Marine Biological Laboratory 35(1–3): 25–170.
  • Oka A (1913) Zur Kenntnis der zwei aberranthen Ascidiengattungen Dicopia Sluit. und Hexacrobylus Sluit. Zoologischer Anzeiger 43: 1–10.
  • Oka A (1918) Megalodicopia hians n.g., n.sp., eine sehr merkwurdig Ascidie aus dem japanischen Meere. Annotationes Zoologicae Japonenses 9(4): 399–406.
  • Okuyama M, Saito Y, Ogawa M, Takeuchi A, Jing Z, Naganuma T, Hirose E (2002) Morphological studies on the bathyal ascidian, Megalodicopia hians Oka, 1918 (Octacnemidae, Phlebobranchia), with remarks on feeding and tunic morphology. Zoological Science 19(10): 1181–1189.
  • Ritter WE (1906) Octacnemus. Bulletin of the Museum of Comparative Zoology at Harvard College 46(13): 233–252.
  • Ritter WE (1907) The ascidians collected by the United States Fisheries Bureau steamer Albatross on the coast of California during the summer of 1904. University of California Publications in Zoology 4(1): 1–52.
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MRBAYES 3.2: Efficient Bayesian phylogenetic inference and model selection across a large model space. Systematic Biology 61(3): 539–542.
  • Sanamyan K (2014) Deep-sea fauna of European seas: An annotated species check-list of benthic invertebrates living deeper than 2000 m in the seas bordering Europe. Ascidiacea. Zoologia Bespozvonocnyh 11(1): 13–24.
  • Sanamyan K, Sanamyan N (1998) Some deep-water ascidians from the NW Pacific (Tunicata: Ascidiacea). Zoosystematica Rossica 7(2): 209–214.
  • Sanamyan K, Sanamyan N (2002) Deep-water ascidians from the south-western Atlantic (RV Dmitry Mendeleev, cruise 43 and Academic Kurchatov, cruise 11). Journal of Natural History 36(3): 305–359.
  • Sanamyan K, Sanamyan N (2006) Deep-water ascidians (Tunicata, Ascidiacea) from the northern and western Pacific. Journal of Natural History 40(5–6): 307–344.
  • Seeliger O (1906) Tunicata: Mantelthiere. Klassen und Ordnungen des Tierreichs 3(Suppl. 68–80): 1041–1280.
  • Sluiter CP (1905a) Zwei merkwurdige Ascidien von der Siboga-Expedition. Tijdschrift der Nederlandsche Dierkundige Vereeniging 9(2): 325–327.
  • Sluiter CP (1905b) Die Tunicaten der Siboga-Expedition. Supplement zu der I Abteilung: Die socialen und holosomen Ascidien. Siboga-Expeditie 56a: 129–139.
  • Tatián M, Lagger C, Demarchi M, Mattoni C (2011) Molecular phylogeny endorses the relationship between carnivorous and filter-feeding tunicates (Tunicata, Ascidiacea). Zoologica Scripta 40(6): 603–612.
  • Tokioka T (1953) Ascidians of Sagami Bay. Iwanami Shoten, Tokyo, 315 pp.
  • Van Name WG (1945) The north and south American ascidians. Bulletin of the American Museum of Natural History 84: 1–476.
  • Vinogradova NG (1969) On the finding of a new aberrant ascidian in the ultrabyssal of the Kuril-Kamchatka Trench. Bulletin de la Société des Naturalistes de Moscou. Section Biologique 74(3): 27–43.
  • Vinogradova NG (1975) On the discovery of two new species of an aberrant deep-water ascidiacean genus Situla in the South-Sandwich trench. Trudy Instituta Oceanologii 103: 289–306.
  • Wessel P, Luis JF, Uieda L, Scharroo R, Wobbe F, Smith WHF, Tian D (2019) The Generic Mapping Tools version 6. Geochemistry, Geophysics, Geosystems 20(11): 5556–5564.
  • Whiting MF, Carpenter JC, Wheeler QD, Wheeler WC (1997) The Strepsiptera problem: Phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Systematic Biology 46(1): 1–68.

Supplementary materials

Supplementary material 1 

Video 1. A close encounter with the deep-sea ascidian

Naohiro Hasegawa, Natsumi Hookabe, Yoshihiro Fujiwara, Naoto Jimi, Hiroshi Kajihara

Data type: mov

Explanation note: Video of the moment the specimen was discovered at a depth of 2027 m.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (13.98 MB)
Supplementary material 2 

Video 2. Grabbing the ascidian with the manipulator of Shinkai 6500

Naohiro Hasegawa, Natsumi Hookabe, Yoshihiro Fujiwara, Naoto Jimi, Hiroshi Kajihara

Data type: mov

Explanation note: Video of the moment the specimen used in this study was collected by Shinkai 6500.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (18.97 MB)
login to comment