Zoosyst. Evol. 100 (1) 2024, 129-140 | DOI 10.3897/zse.100.113132 eee ee BERLIN Supplemental re-description of a deep-sea ascidian, Fimbrora calsubia (Ascidiacea, Enterogona), with an inference of its phylogenetic position Naohiro Hasegawa‘, Natsumi Hookabe*, Yoshihiro Fujiwara*, Naoto Jimi?*, Hiroshi Kajihara® 1 Department of Natural History Sciences, Graduate School of Science, Hokkaido University, Kita 10 Nishi 8 Kitaku, Sapporo, Hokkaido, 060-0810, Japan 2 Research Institute for Global Change (RIGC), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa 237-0061, Japan 3 Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba, Mie 517-0004, Japan 4 Centre for Marine & Coastal Studies, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia 5 Faculty of Science, Hokkaido University, Kita 10 Nishi 8 Kitaku, Sapporo, Hokkaido, 060-0810 Japan https://zoobank. org/D84B59 15-75 1E-4BAC-BB3A-A45E9282FE29 Corresponding author: Naohiro Hasegawa (hoya.hasegawa.ronbun@gmail.com) Academic editor: Pavel Stoev # Received 22 September 2023 Accepted 11 January 2024 @ Published 26 January 2024 Abstract 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 re- cord, 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 ascid- ian 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 (Muller, 1776) and Ascidiella scabra (Miller, 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 Introduction 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 The ascidiid genus Fimbrora Monniot & Monniot, 1991la is currently monotypic, consisting of the deep-sea ascid- ian 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, a combination of three characteristics: 7) the large, cup- shaped oral siphon with thin, uniformly long, and soft lobes, ii) two large blood vessels running on the oral-si- phon wall and ii7) macrophagous feeding behaviour (cf. Monniot and Monniot (1991a)). The remaining four Copyright Hasegawa, N. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 130 genera are distinguished from each other, based on: 7) whether primary and/or secondary branchial papillae in the pharynx are present and 77) whether accessory open- ings of the neural gland are present (e.g. Kott (1985); Mon- niot 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 Lopez-Legentil 2017); also, the nature of the neural-gland opening (or, whether ac- cessory 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 sur- rounding seawater (Millar 1971), 40 species have hither- to been identified as macrophagous, based on their large oral siphons and unciliated pharynges; direct confirma- tion 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 in- habit deep waters below 200 m with one exception, O/i- gotrema psammites Bourne, 1903, which is also distribut- ed 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 con- vergently, probably due to difficulty in filter-feeding in the deep sea (Millar 1959). This view was confirmed by the phylogenetic study of Tatian 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. The taxonomy of macrophagous molgulids has expe- rienced twists and turns. Historically, Asajirus and Oligo- trema 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 be- tween 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 (Tatian zse.pensoft.net Hasegawa, N. et al.: Supplemental re-description of Fimbrora calsubia 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 Pa- cific 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 (Mon- niot 1993) and eight specimens at 1000-1200 m depth in Papua New Guinea (Monniot and Lopez-Legentil 2017). Meanwhile, during a biodiversity survey in an off-shore submarine nature conservation area around Nishi-Shichi- to Ridge in the western North Pacific, a 14" individual of F. calsubia was obtained. Here, we provide a morpho- logical 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 obser- vation. For detailed examination, the pharynx was stained with haematoxylin. The voucher specimens have been deposited in the Japan Agency for Marine-Earth Sci- ence and Technology (JAMSTEC), Yokosuka, with the catalogue number JAMSTEC No. 111618 for the forma- lin-fixed specimen, JAMSTEC No. 111619 for the lobe in 99% ethanol and JAMSTEC No. 111620 for the lobe in RNAlater. 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 COL, 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 Biosys- tem, Waltham, MA, USA). The purified products were Zoosyst. Evol. 100 (1) 2024, 129-140 131 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 References macrophagous feeding* Ascidiidae Fimbrora calsubia Monniot & Monniot, 1991 1000-2027 m/c Monniot and Monniot (1991a), Monniot (1993), Monniot and Ld6pez-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 L6pez-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 Lopez-Legentil (201 7) 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 5400 m Sanamyan and Sanamyan (1999) Sanamyan & 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), Slurter (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) *m’ indicates that the species was judged to be macrophagous, based on morphological characteristics; ‘m/c’ indicates that gut contents were also observed in addition to morphological features. zse.pensoft.net Hasegawa, N. et al.: Supplemental re-description of Fimbrora calsubia -abpla oyuolus-lus ~, Hoei Seamount [47° Figure 1. Maps showing the sampling site (red circle), south of Houei Seamount (of which the top is indicated with a red trian- gle). 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. sequenced with an ABI BigDye Terminator ver. 3.1 Cy- cle Sequencing Kit and an ABI 3100 Avant Genetic An- alyzer (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. zse.pensoft.net For phylogenetic analysis, 18S and COI sequences of 27 ascidian species and those of the lancelet Bran- chiostoma floridae Hubbs, 1922 were downloaded from GenBank (Table 2). The dataset of 18S was aligned us- ing MAFFT ver. 7.310 with £-/NS-/ strategy (Katoh and Standley 2013); the aligned 18S dataset was trimmed by using trimAl ver. 1.4. revl15 with gappyout command (Capella-Gutiérrez et al. 2009). An alignment of COI was obtained by using MEGA X (Kumar et al. 2018) follow- ing Hasegawa and Kayihara (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 Branchiosto- ma floridae Hubbs, 1922, used for phylogenetic analysis in this study. Species 18S Col Ascidia ceratodes L12378 MW872268 Ascidia zara LC547325 KY¥230397 Ascidiella aspersa 1¢547321 KF886702 Ascidiella scabra AB811928 MN064599 Botrylloides violaceus LC432326 LC432331 Chelyosoma siboja AF165821 AB104867 Ciona robusta ABO13017 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 LO777585 Halocynthia roretzi AB013016 HM151268 Herdmania momus AF165827 KM411616 Megalodicopia hians ABO75543 AB104866 Molgula manhattensis L12426 MT873565 Oligotrema lyra JN565043 - Perophora japonica AB499607 MNO64600 Perophora viridis FM244849 OM912740 Phallusia fumigata FM244844 KF 309548 Phallusia mammillata AF236803 MN064634 Pycnoclavella diminuta KJ632948 KCO17435 Pyura mirabilis LC432327 LC432332 Styela clava LC432329 LC432334 Symplegma reptans AF 165826 L$992553 Syncarpa composita LC432325 LC432330 Branchiostoma floridae M97571 AB478593 For constructing phylogenetic trees, Bayesian In- ference (BI) and Maximum Likelihood (ML) analyses were performed; MrBayes ver. 3.2.6 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003: Ron- quist et al. 2012) for BI and the ultrafast bootstrap meth- od (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 substitu- tion 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 Zoosyst. Evol. 100 (1) 2024, 129-140 started from a random tree and run for 10’ 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 pos- terior probability (PP) for each node was collected to assess the certainty of the inference. Run convergence was assumed, based on the following values of vari- ables: average standard deviation of split frequencies = 0.002002; average estimated sample size of all param- eters > 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). Results Taxonomy and morphology Order Enterogona Suborder Phlebobranchia Family Ascidiidae Adams & Adams, 1858 Fimbrora calsubia Monniot & Monniot, 1991a Figs 2-4 Fimbrora calsubia Monniot & Monniot, 1991a, p. 384, figs 1-6; Mon- niot (1993), p. 356; Monniot and Lopez-Legentil (2017), p. 531, figs 12s 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). Description. 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 pos- terior end (Fig. 2A, B). Oral siphon enlarged, ca. 10 cm in diameter; single annular muscle strand running on out- er 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 lon- gitudinally running on surface of atrial siphon (Fig. 2D). 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 133 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 num- ber, almost straight in shape (Fig. 3A) and opening at dor- sal tubercle. Pharynx connected by mesenteries to peripha- ryngeal 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 inter- sections of longitudinal and transverse vessels (Fig. 3C). 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). Gonad situated proximally on intestinal loop (Fig. 4C). Ovaries surrounded with male testis (Fig. 4C, D). Ovi- duct and spermiduct running along secondary loop, open- ing close to anus (Fig. 4A). Eggs contained in ovaries and oviduct, up to 0.2 mm in diameter (Fig. 4D). Habitat. 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. materi- al 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. 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 (Kuraba- yashi et al. 2003; Tatian et al. 2011), MZ. hians was sister to Corella eumyota Traustedt, 1882; O. /yra was sister to Molgula manhattensis (De Kay, 1843). zse.pensoft.net 134 Hasegawa, N. et al.: Supplemental re-description of Fimbrora calsubia thread-like lobes d oral siphon blood vessel f atrial siphon atrial siphon \ thread-like lobe a + 7 4 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 (yel- low arrow); B. Left view in life; C. Inner surface of the oral siphon in fixed state; D. Enlarged view of atrial siphon in life. Discussion _— | -_ ported this view. The morphological characteristics that Previous studies posited that Fimbrora would belong to suggested Fimbrora’s familial affiliation were the lon- Ascidiidae (Monniot and Monniot 1991la; Monniot and _ gitudinal vessels having papillae and straight stigmata Lopez-Legentil 2017) and our phylogenetic analysis sup- in the pharynx (Monniot and Monniot 1991a; Monniot zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 129-140 A neural-gland aperture 3mm “Tay ve dorsal lamina 135 peripharyngeal band dorsal lamina | > * b to R r* % tran§vetse vessels x) VSG & é WR 2 WN eS 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). and Lépez-Legentil 2017), while Monniot and Monniot (1991a) noted the superficial resemblance of the genus with the family Octacnemidae tn having an enlarged oral siphon. The phylogenetic position of Fimbrora with- in Ascidiidae was unresolved in our tree (Fig. 5). The more precise phylogenetic position of Fimbrora in the family would require the inclusion of additional ascidi- id taxa in molecular analyses. One such to-be-included taxa 1s Psammascidia, which shares two characteristics with Fimbrora—having secondary branchial papillae on the longitudinal vessels and lacking primary and inter- mediate 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 relation- ship amongst ascidiid species including Fimbrora. Monniot and Monniot (1991a) suggested that F’ cal- subia 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 re- ports of F’ calsubia from Indonesia (Monniot 1993) and Papua New Guinea (Monniot and Lopez-Legentil 2017), however, did not provide any information on gut con- tents in their specimens. In addition to this, the observed zse.pensoft.net intestine ‘ / ‘iy - ; “sad ' > + oviduct renal vesicle Hasegawa, N. et al.: Supplemental re-description of Fimbrora calsubia stomach Figure 4. Fimbrora calsubia Monniot & Monniot, 1991a (JAMSTEC No. 111618), photographs of fixed specimen. A. Siniste- ro-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. 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 Tatian 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 distri- bution range for about 4000 km northwards, representing zse.pensoft.net 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 be- ing about 1000-2000 m (Monniot and Monniot 1991a; Monniot 1993; Monniot and Lopez-Legentil 2017; pres- ent study). Conclusions We present the first report of F) calsubia from the North Pacific. Our molecular phylogenetic analysis suggested that macrophagous feeding was convergently acquired at Zoosyst. Evol. 100 (1) 2024, 129-140 99/1.00 100/1.00 97/1.00 95/41.00 100/1.00 100/1.00 94/1.00 peta Japonica 137 Ascidiidae Ascia Ascidiella as; Ascidiella sc Ecteinascidia turbinata Phlebobranchia Ecteinascidia herdmanni Perophora viridis 66 Ciona savignyi — Ciona robusta *Megalodicopia hians 100/1.00 100/1.00 Corella eumyota Chelyosoma siboja pate lepadiformis 100/1.00 100 99/1.00 74/4.00 Branchiostoma floridae 2.0 Pycnoclavella diminuta Aplousobranchia Clavelina meridionalis Herdmania momus Pyura mirabilis Halocynthia roretzi Syncarpa composita Botrylloides violaceus Symplegma reptans Styela clava Stolidobranchia Molgula manhattensis 100/1.00 *Oligotrema lyra 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 (*). least three times independently in Ascidiacea. Our mor- phological observation indicated a similarity of Fimbrora to Psammascidia in having secondary papillae and lack- ing primary and intermediate branchial papillae. Acknowledgements We extend our profound gratitude to the captain and crew of the support vessel Yokosuka, the commander and oper- ation 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 indebt- ed to all the people who donated to NHa through the academic crowd-funding site “academist’, especially to Shunji Furukuma, Naoki Hayashi, Miyuki Honda, Hito- ki 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 JP- MASP2119. This research was partly performed by the zse.pensoft.net 138 Environment Research and Technology Development Fund (JPMEERF20820700) of the Environmental Resto- ration and Conservation Agency Provided by the Minis- try 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. References Adams H, Adams A (1858) The genera of resent Mollusca; arranged according to their organization. John van Voorst, Paternoster row, London, 661 pp. https://doi.org/10.5962/bh1.title.4772 Bourne GC (1903) Oligitrema psammites, a new ascidian belong- ing to the family Molgulidae. The Quarterly Journal of Micro- scopical Science 47(1): 233-273. https://doi.org/10.1242/jcs. $2-47.186.233 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) Fixa- tion, description and DNA barcode of a neotype for Botryllus schlos- seri (Pallas, 1766) (Tunicata, Ascidiacea). Zootaxa 4353(1): 29-50. https://doi.org/10.11646/zootaxa.4353.1.2 Capella-Gutiérrez S, Silla-Martinez JM, Gabaldon T (2009) trimAl: A tool for automated alignment trimming in large-scale phylogenet- ic analyses. Bioinformatics (Oxford, England) 25(15): 1972-1973. https://doi.org/10.1093/bioinformatics/btp348 Giribet G, Carranza S, Bagufia J, Riutort M, Ribera C (1996) First molecular evidence for the existence of a Tardigrada + Arthropoda clade. Molecular Biology and Evolution 13(1): 76-84. https://doi. org/10.1093/oxfordjournals.molbev.a025573 Hartmeyer R (1923) Ascidiacea, part I. Zugleich eine Ubersicht tiber die arktische und boreale Ascidienfauna auf tiergeographischer Grund- lage. Ingolf-Expedition 2(6): 1-365. Hasegawa N, Kajihara H (2019) A redescription of Syncarpa compos- ita (Ascidiacea, Stolidobranchia) with an inference of its phylo- genetic position within Styelidae. ZooKeys 857: 1-15. https://doi. org/10.3897/zookeys.857.32654 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 Pa- pers 53(2): 215-222. https://doi.org/10.1016/j.dsr.2005.11.005 Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS (2018) UFBoot2: Improving the ultrafast bootstrap approximation. Molec- ular Biology and Evolution 35(2): 518-522. https://doi.org/10.1093/ molbev/msx281 Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogeny. Bioinformatics (Oxford, England) 17(8): 754-755. https://doi.org/10.1093/bioinformatics/17.8.754 Thle 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 _usabili- ty. Molecular Biology and Evolution 30(4): 772-780. https://doi. org/10.1093/molbev/mst010 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. zse.pensoft.net Hasegawa, N. et al.: Supplemental re-description of Fimbrora calsubia 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. Jour- nal of Natural History 43(31-32): 1947-1986. https://doi. org/10.1080/00222930902993708 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Mo- lecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547-1549. https://doi. org/10.1093/molbev/msy096 Kurabayashi A, Okuyama M, Ogawa M, Takeuchi A, Jing Z, Na- ganuma T, Saito Y (2003) Phylogenetic position of a deep-sea ascidian, Megalodicopia hians, inferred from the molecular data. Zoological Science 20(10): 1243-1247. https://doi.org/10.2108/ zs}.20.1243 Lacaze-Duthiers H (1877) Histoire des ascidies simples des cétes de France. Deuxiéme partie: Etude des espéces. 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 analy- ses. Molecular Biology and Evolution 34(3): 772-773. https://doi. org/10.1093/molbev/msw260 Madsen FJ (1947) Octacnemus ingolfi n.sp., an Atlantic representative of peculiar tunicate-family Octacnemidae. Videnskabelige Med- delelser fra Dansk naturhistorisk Forening 110: 31-44. Maggioni T, Taverna A, Reyna P, Alurralde G, Rimondino C, Tatian M (2018) Deep-sea ascidians (Chordata, Tunicata) from the SW Atlan- tic: Species richness with descriptions of two new species. Zootaxa 4526(1): 1-28. https://doi.org/10.11646/zootaxa.4526.1.1 Maggioni T, Rimondino C, Taverna A, Reyna P, Largger C, Alurralde G, Calcagno E, Tatian M (2022) Abyssal ascidians (Chordata, Tuni- cata) from the Weddell Sea, Antarctica, including a new Stvela spe- cies and stomach content identifications. Zootaxa 5093(3): 296-314. https://do1.org/10.11646/zootaxa.5093.3.2 Metcalf MM (1893) Notes upon an apparently new species of Octac- nemus, 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. https://do1.org/10.1111/j. 1096-3642. 1970.tb00732.x Millar RH (1971) The biology of ascidians. Advances in Marine Biolo- gy 9: 1-100. https://doi.org/10.1016/S0065-2881(08)60341-7 Millar RH (1988) Deep-sea ascidians from the eastern Pacif- ic collected during the Pacific Ocean Biological Survey Pro- gram. Journal of Natural History 22(5): 1427-1435. https://doi. org/10.1080/0022293880077085 1 Minh BQ, Nguyen MAT, von Haeseler A (2013) Ultrafast approxima- tion for phylogenetic bootstrap. Molecular Biology and Evolution 305(5): 1188-1195. https://doi.org/10.1093/molbev/mst024 Zoosyst. Evol. 100 (1) 2024, 129-140 Monniot C (1969) Ascidies récoltées par la "Thalassa" sur la pente continentale du golfe de Gascogne: (3-12 aottt 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 pla- teau 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 especes 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 F (2011) A new Octacnemidae (Ascidiacea) from the Mid-At- lantic Ridge. Zootaxa 2864(1): 65-68. https://doi.org/10.11646/zoo- taxa.2864.1.5 Monniot F (2022) Additional records of bathyal ascidians (Tunicata) from the New Caledonia region. Zootaxa 5195(3): 201—223. https:// doi.org/10.11646/zootaxa.5195.3.1 Monniot F, Lopez-Legentil S (2017) Deep-sea ascidians from Papua New Guinea. Zootaxa 4276(4): 529-538. https://doi.org/10.11646/ zootaxa.4276.4.5 Monniot C, Monniot F (1968) Les ascidies de grandes profondeurs récoltées par le navire oceanographique american Atlantis 2 (Pre- miere note). Bulletin de I‘ 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. https://doi.org/10.1016/0011-7471(70)90024-0 Monniot C, Monniot F (1973) Ascidies abyssales récoltées au cours de la campagne océanographique Biacores par le "Jean Charcot". Bul- letin du Muséum National d‘ Histoire Naturelle 121: 389-475. Monniot C, Monniot F (1974) Ascidies abyssales de | Atlantique récoltées par le "Jean Charcot" (Campagnes Nortlante, Walda, Po- lygas A). Bulletin du Muséum National d'Histoire Naturelle 226: 721-786. Monniot F, Monniot C (1976) Tuniciers abyssaux du bassin argentin récoltés par | "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 Francais 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. https://doi.org/10.1029/ ARO032p0095 Monniot C, Monniot F (1983) Ascidies antarctiques et subantarctiques: Morphologie et biogeographic. 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 Madere. Annales de I Institut Océanographique 60(2): 129-142. 139 Monniot C, Monniot F (1984b) Nouvelles Sorberacea (Tunicata) pro- fondes de I’Atlantique Sud et l’?Ocean Indien. Cahiers de Biologie Marine 25: 197-215. Monniot C, Monniot F (1985a) Nouvelles récoltes de tuniciers ben- thiques profonds dans |‘ Ocean Atlantique. Bulletin du Muséum Na- tional d'Histoire Naturelle. Section A. Zoologie, Biologie, et Ecolo- gie Animales A7(1): 5—37. https://doi.org/10.5962/p.285870 Monniot C, Monniot F (1985b) Tuniciers profondes de |’Ocean Indien: Campagnes SAFARI du ‘Marion Dufresne’. Bulletin du Muséum National d' Histoire Naturelle. Section A. Zoologie, Biologie, et Ecol- ogie Animales A7(2): 279-308. https://doi.org/10.5962/p.287567 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 Galapa- gos Islands using the Johnson-Sea-Link research submersible. Pro- ceedings of the Biological Society of Washington 102(1): 14—32. Monniot C, Monniot F (1990) Revision of the class Sorberacea (ben- thic tunicates) with descriptions of seven new species. Zoolog- ical Journal of the Linnean Society 99(3): 239-290. https://doi. org/10.1111/j.1096-3642.1990.tb00562.x Monniot C, Monniot F (1991a) Découverte d'une nouvelle lignée évo- lutive chez les ascidies de grande profondeur: Une Ascididae carni- vore. Comptes Rendus de |’ Académie des Sciences, Série 3. Scienc- es de la Vie 312: 383-388. Monniot C, Monniot F (1991b) Tunicata: Peuplement d'ascidies pro- fondes en Nouvelle-Caledonie. Diversite des strategies adaptives. Mémoires du Muséum National d‘ Histoire Naturelle, Série A. Zo- ologie 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 nou- velle 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, ob- tained during the voyage of H.MS. “Challenger”. Transac- tions of the Linnean Society of London, 2" Series. Zoology : Analysis of Complex Systems, ZACS 1: 287-294. https://doi. org/10.1111/j.1096-3642.1877.tb00443.x Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maxi- mum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268-274. https://doi.org/10.1093/molbev/msu300 Nishikawa T (1991) The ascidians of the Japan Sea. II. Publications of the Seto Marine Biological Laboratory 35(1—3): 25-170. https://doi. org/10.5134/176172 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 Ja- ponenses 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. https://doi.org/10.2108/zsj.19.1181 zse.pensoft.net 140 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 sum- mer of 1904. University of California Publications in Zoology 4(1): 1-52. https://doi.org/10.5962/bhl.title. 1573 Rocha RM, Zanata TB, Moreno TR (2012) Keys for the identifica- tion of families and genera of Atlantic shallow water ascidians. Biota Neotropica 12(1): 270-302. https://doi.org/10.1590/S1676- 06032012000100022 Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phyloge- netic inference under mixed models. Bioinformatics (Oxford, En- gland) 19(12): 1572-1574. https://doi.org/10.1093/bioinformatics/ btg180 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna 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. https://doi.org/10.1093/sysbio/sys029 Sanamyan K (1998) Ascidians from the north-western Pacific region. 5. Phlebobranchia. Ophelia 49(2): 97-116. https://doi.org/10.1080/007 85326.1998.10409376 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 Bespozvo- nocnyh 11(1): 13—24. https://doi.org/10.15298/invertzool.11.1.04 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 (1999) Some benthic Tunicata from the southern Indo-Pacific Ocean. Journal of Natural History 33(12): 1835-1876. https://doi.org/10.1080/002229399299761 Sanamyan K, Sanamyan N (2002) Deep-water ascidians from the south-western Atlantic (RV Dmitry Mendeleev, cruise 43 and Ac- ademic Kurchatov, cruise 11). Journal of Natural History 36(3): 305-359. https://do1.org/10.1080/00222930010004232 Sanamyan K, Sanamyan N (2006) Deep-water ascidians (Tunicata, Asci- diacea) from the northern and western Pacific. Journal of Natural His- tory 40(5—6): 307-344. https://doi.org/10.1080/002229306006284 16 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-Expe- dition. 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. Sibo- ga-Expeditie 56a: 129-139. Tatian M, Lagger C, Demarchi M, Mattoni C (2011) Molecular phylog- eny endorses the relationship between carnivorous and filter-feeding tunicates (Tunicata, Ascidiacea). Zoologica Scripta 40(6): 603-612. https://doi.org/10.1111/).1463-6409.2011.00493.x Tokioka T (1953) Ascidians of Sagami Bay. Iwanami Shoten, Tokyo, 315 pp. Van Name WG (1945) The north and south American ascidians. Bulle- tin of the American Museum of Natural History 84: 1-476. zse.pensoft.net Hasegawa, N. et al.: Supplemental re-description of Fimbrora calsubia 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. Geochemis- try, Geophysics, Geosystems 20(11): 5556-5564. https://doi. org/10.1029/2019GC008515 Whiting MF, Carpenter JC, Wheeler QD, Wheeler WC (1997) The Strepsiptera problem: Phylogeny of the holometabolous insect or- ders inferred from 18S and 28S ribosomal DNA sequences and mor- phology. Systematic Biology 46(1): 1-68. https://doi.org/10.1093/ sysbio/46.1.1 Supplementary material | Video 1. A close encounter with the deep-sea ascidian Authors: Naohiro Hasegawa, Natsum1 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. Copyright notice: This dataset 1s made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers 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. Link: https://doi.org/10.3897/zse.100.113132.suppl1 Supplementary material 2 Video 2. Grabbing the ascidian with the manipulator of Shinkai 6500 Authors: Naohiro Hasegawa, Natsum1 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. Copyright notice: This dataset 1s made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers 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. Link: https://doi.org/10.3897/zse.100.113132.suppl2