Introduction
Trematodes of the family Didymozoidae Monticelli, 1888 typically use marine fish as their definitive hosts (Pozdnyakov, Reference Pozdnyakov1996; Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008). Comprising six subfamilies (Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008), this highly diverse family currently includes over 250 species (Louvard et al., Reference Louvard, Cutmore, Yong, Dang and Cribb2022). Didymozoid body plans are extremely diverse; consequently, body shape and morphological characteristics of the reproductive systems are important for determining taxonomic positions within the family (Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008). However, species-level identification of sexual adults based on morphological characteristics is frequently difficult. In some taxa, this is owing to their soft, slender bodies, which are highly susceptible to damage. In contrast, other members possess large hindbodies packed with extensive, egg-filled uteri that obscure details of the internal organs. Because most didymozoid species were described in the last century (WoRMS Editorial Board, 2026), morphological keying is considered problematic (Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008), prompting calls for systematic revisions based on molecular analyses (Louvard et al., Reference Louvard, Cutmore, Yong, Dang and Cribb2022). Despite recent efforts, adult didymozoids are still often reported without species-level identification, probably because of these persistent morphological bottlenecks (e.g. Louvard et al., Reference Louvard, Cutmore, Yong, Dang and Cribb2022).
In recent years, DNA barcoding has emerged as a standard method for resolving trematode taxonomy including species identifications (e.g. Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Nakao et al., Reference Nakao, Waki, Sasaki, Anders, Koga and Asakawa2017). Effective species identification can be achieved using the nuclear 28S ribosomal RNA (rDNA) gene, internal transcribed spacer (ITS) region, and mitochondrial cytochrome c oxidase subunit 1 (COI) gene. Among these, 28S rDNA is particularly well-suited for resolving phylogenetic relationships across broad trematode lineages (e.g. Ansai et al., Reference Ansai, Sekine, Munakata, Sase, Sasaki, Nitta, Suzuki, Abe, Takano, Fukumori, Nakatsubata, Suzuki and Waki2025; Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Martin et al., Reference Martin, Downie and Cribb2020). However, reference molecular datasets linked to morphologically identified adult specimens are limited for the family Didymozoidae. This critical data gap is largely driven by the technical challenges associated with preparing specimens suitable for detailed morphological identification. Generating coupled molecular and morphological datasets from properly identified adults is thus imperative to establish a robust framework for didymozoid species identification.
In the present study, adult didymozoids were collected from marine fishes and identified using morphological and molecular analyses. Four didymozoid species were characterised, providing novel DNA datasets for each species, including a new species of the genus Annulocystis, which is described in the present study.
As a nomenclatural note, the following family-groups, which have previously been attributed to Ishii (Reference Ishii1935), were in fact described earlier in Ishii (Reference Ishii1934) with brief diagnoses: Nematobothriinae, Gonapodasmiinae, Didymozooinae (= Didymozoinae Monticelli, 1888), and Koellikeriinae. Although Ishii (Reference Ishii1934) was published as a congress abstract, the journal in which it appeared, Jikken Igaku Zasshi (ISSN: 1883-6976), satisfies the criteria for publication under Article 8 of International Code of Zoological Nomenclature (the Code: International Commission on Zoological Nomenclature, 1999) and does not fall under the category of ‘materials issued primarily to participants at meetings’ excluded by Article 9.9 of the Code. Therefore, the family-group names proposed in Ishii (Reference Ishii1934) are considered available and to have priority. Additionally, the following genus-group names are also mentioned by Ishii (Reference Ishii1934), but they are invalid because there was no fixation of type species (Article 13.3 of the Code) or lack of definitions (Article 13.1.1 of the Code): Benedenozoum, Maclarenozoum, Gonapodasmius, Didymocylindrus, Didymoproblema, Lobatozoum, and Koellikerizoum (= Koellikeria Cobbold, 1860).
Material and methods
Sample collection
From 2018 to 2025, marine fish were collected in Japan (Table 1). Host fishes were transported to the laboratory and identified according to Nakabo (Reference Nakabo2013). These hosts were dissected for trematode survey with the naked eye. When didymozoid trematodes were found, the affected organs were documented. Initial attempts to fix didymozoid trematodes from the gills of Scomber spp. in hot 1% saline resulted in severe tissue damage of worms, rendering the specimen unsuitable for morphological observation. Consequently, some specimens were directly fixed in 70% ethanol. However, in these unflattened fixed specimens, the female reproductive organs were obscured, probably because of the superimposition of adjacent organs. Thus, to resolve detailed observations of the organs, selected specimens were flattened and fixed between a glass slide and coverslip in 70% ethanol; this technique successfully permitted the visualisation of the female reproductive system. Based on this optimisation, didymozoid specimens from Auxis thazard and Euthynnus affinis were fixed between coverslips and slides with 70% ethanol, while some specimens from Scomber japonicus were fixed in acetic acid-formalin-alcohol under a coverslip on glass slides. Additionally, didymozoid trematodes collected from the gills of Mola mola, which had been directly fixed and preserved in 70% ethanol, were examined. This specific host fish was previously surveyed for accacoeliids survey and molecularly identified as M. mola sensu stricto (Waki et al., Reference Waki, Nakano, Okawa and Ishikawa2026). The examined specimens were deposited at the Ibaraki Nature Museum (INM), Japan.
Trematodes of Didymozoidae detected from gills of marine fishes in the present study
Table 1. Long description
The table consists of seven columns: Trematode species, Host fish, Sampling locality, Sampling date, Number of hosts examined, Number of hosts infected, and Number of trematode specimens per host.
* Row 1: Annulocystis auxis Yamaguti, 1970; Host: Auxis thazard; Locality: Ofunato City, Iwate Prefecture; Date: 17 September 2025; Hosts examined: 1; Hosts infected: 1; Specimens: 1.
* Row 2: Annulocystis carnata n. sp.; Host: Euthynnus affinis; Locality: Mie Prefecture; Date: 9 May 2025; Hosts examined: 1; Hosts infected: 1; Specimens: 3.
* Row 3: Gonapodasmius squamatus (Pozdnyakov, 1994); Host: Mola mola; Locality: Kyoto Prefecture; Date: 17 March 2025; Hosts examined: 1; Hosts infected: 1; Specimens: 3.
* Row 4: Nematobothrium filiforme Yamaguti, 1934; Host: Scomber australasicus; Locality: Nagasaki Prefecture; Date: 20 February 2025; Hosts examined: 1; Hosts infected: 1; Specimens: 2.
* Row 5: N. filiforme (continued); Host: S. japonicus; Locality: Ise Bay, Mie Prefecture; Date: 29 March 2018; Hosts examined: 2; Hosts infected: 2; Specimens: 11 and 2.
* Row 6: N. filiforme (continued); Host: S. japonicus; Locality: Muroto City, Kochi Prefecture; Date: 15 May 2025; Hosts examined: 5; Hosts infected: 3; Specimens: 4, 14, and 22.
Footnotes indicate that some specimens were fused and one host fish was used in Waki et al. (2026).
a Two individuals were fused and constructed into one specimen.
b Host fish individual used in Waki et al. (Reference Waki, Nakano, Okawa and Ishikawa2026).
Prior to staining, a small tissue fragment was cut from selected specimens for molecular analyses, as described subsequently. The remaining tissues from these specimens, as well as other fixed ones, were stained with either acetocarmine or Heidenhein’s iron-haematoxylin, dehydrated using a graded ethanol series, cleared in xylene, and mounted on slides with Canada balsam for measurement of body parts. Morphological observations were conducted using an Olympus BX53 light microscope equipped with an AdvanCam-U3II digital camera (Advan Vision). Body part measurements for each developmental stage were extracted from the resulting digital images. Line drawings were created using ArtCAM and Inkscape software. For the didymozoid trematodes from the gills of M. mola, the narrow forebodies were excised, stained, and mounted on slides as described above.
Genomic DNA was extracted from the excised tissue fragments via alkaline lysis, following the same method reported by Nakao et al. (Reference Nakao, Waki, Sasaki, Anders, Koga and Asakawa2017), to serve as templates for polymerase chain reaction (PCR). Partial fragments of the nuclear 28S rDNA gene, ITS region and mitochondrial COI gene were amplified using the following primer sets: for 28S rRNA, dig12 (forward, Tkach et al., Reference Tkach, Kudlai and Kostadinova2016) and 1500R (reverse, Tkach et al., Reference Tkach, Kudlai and Kostadinova2016) or LSU5 (forward, Littlewood et al., Reference Littlewood, Curini-Galletti and Herniou2000) and 1600R (reverse, Williams et al., Reference Williams, Reid and Littlewood2003); for ITS, 81_f (forward, Gustinelli et al., Reference Gustinelli, Caffara, Florio, Otachi, Wathuta and Fioravanti2010) or 3S (forward, Bowles et al., Reference Bowles, Hope, Tiu, Liu and McManus1993) and ITS2.S_r (reverse, Cribb et al., Reference Cribb, Anderson, Adlard and Bray1998); for COI, Dig_cox1Fa (forward, Wee et al., Reference Wee, Cribb, Bray and Cutmore2017) or JB3 (forward, Bowles and McManus, Reference Bowles and McManus1993) and CO1-Rtrema (reverse, Miura et al., Reference Miura, Kuris, Torchin, Hechinger, Dunham and Chiba2005) or trem.cox1.rrnL (reverse, Králová-Hromadová et al., Reference Králová-Hromadová, Špakulová, Horáčková, Turčeková, Novobilský, Beck, Koudela, Marinculić, Rajský and Pybus2008). These PCR primers were also used for sequencing. For 28S rDNA, the following internal primers were used for sequencing: ECD2S (reverse, Williams and Ozawa, Reference Williams and Ozawa2006), D3m (reverse, Takano and Kano, Reference Takano and Kano2014), and 900F (forward, Lockyer et al., Reference Lockyer, Olson, Østergaard, Rollinson, Johnston, Attwood, Southgate, Horak, Snyder, Le, Agatsuma, McManus, Carmichael, Naem and Littlewood2003), when necessary. Sanger sequencing was performed commercially by Eurofins Genomics Inc. (Tokyo, Japan). Sequences for 28S rDNA, ITS, and COI were aligned using BioEdit (Hall, Reference Hall1999). Newly generated sequences were deposited in the International Nucleotide Sequence Database Collaboration (INSDC) archive through the DNA Data Bank of Japan (DDBJ) under accession numbers (LC932360–LC932372). For phylogenetic analyses, reference 28S rDNA sequences of related taxa were retrieved from INSDC. The genetic software MEGA X (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018) was used for computing genetic divergence, estimating substitution models, and inferring maximum likelihood (ML) phylogenies. The ML tree of 28S rDNA (774 sites) was constructed using the Kimura 2-parameter + G + I substitution model (Kimura, Reference Kimura1980), with nodal support determined using 1,000 bootstrap replicates. Sequences of COI were excluded from the phylogenetic analyses because of the lack of closely related reference sequences (>80% similarity) in public databases. Our ITS sequences for the genus Annulocystis and Nematobothrium filiforme and their related sequences retrieved from INSDC were used to calculate pairwise divergence values using MEGA X, facilitating species-level identification of an unidentified trematode previously reported by Louvard et al. (Reference Louvard, Cutmore, Yong, Dang and Cribb2022) and Pitt et al. (Reference Pitt, Browne, Cutmore, Diaz, Nq-x and Cribb2026). The ITS sequence of the trematode obtained from M. mola in this study was not used for molecular analyses owing to the absence of conspecific sequences for the trematode in the online databases (e.g. Didymodiclininae sp. 1 CL-2021 [OL846556] exhibited only 94% similarity across 520 bp).
Results
Trematode survey
In total, 62 trematode specimens were sampled from the gills of five marine fish species (Table 1, Figure 1). Based on morphological analysis, these specimens were assigned to two subfamilies encompassing three genera and identified as four distinct species. One of the four species is described herein as a new species.
Trematodes in situ on the host gills. a. Annulocystis carnata n. sp. from Euthynnus affinis. White arrows represent the clearly observable forebody. b. Gonapodasmius squamatus from Mola mola. c. Nematobothrium filiforme from Scomber australasicus (not to scale). b, c. Dark arrows represent adult worms.
Figure 1. Long description
A three-panel macro photograph labeled a, b, and c.
Panel a shows three specimens of Annulocystis carnata n. sp. on the purple-red gill filaments of Euthynnus affinis. The parasites are elongated, yellowish, and segmented in appearance. Two white arrows point to the translucent, narrow forebodies of the worms on the left and center specimens. A 1 mm scale bar is located at the bottom right.
Panel b shows Gonapodasmius squamatus on the gills of Mola mola. The gill tissue is pale pink with vertical striations. Three adult worms are visible as oval, yellowish-tan cysts. Three dark arrows point to these adult worms: one in the upper center, one in the lower left, and one in the lower right. A 2 cm scale bar is at the bottom left.
Panel c shows Nematobothrium filiforme on the gills of Scomber australasicus. The gill structure is fan-like and pinkish-purple. Five dark arrows point to various adult worms which appear as small, elongated, yellowish nodules embedded within or resting on the gill filaments. The worms are distributed vertically along the gill arch.
Subfamily Gonapodasmiinae Ishii, 1934
Seven trematode specimens collected from the gills of Auxis thazard, Euthynnus affinis, and M. mola were assigned to this subfamily based on the following morphological characteristics: body divided into two regions (forebody and hindbody); forebody free; hindbody fused; oesophagus long; pharynx present (Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008). Pozdnyakov and Gibson (Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008) reported the absence of a ventral sucker as a diagnostic characteristic for this subfamily; however, Gonapodasmius williamsoni, a member of the type genus, possesses a ventral sucker (Cribb and Williams, Reference Cribb and Williams1992). Consequently, our trematodes from M. mola were assigned to the Gonapodasmiinae based on the aforementioned combination of morphological characteristics, despite the presence of a ventral sucker.
Tribe Gonapodasmiini Ishii, 1934
Specimens from all three host fish species were identified as this tribe based on the forebody not being attached to their hindbody through a common atrium (Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008).
Genus Annulocystis Yamaguti, 1970
Four specimens from Au. thazard and E. affinis were identified as genus Annulocystis based on the following morphological characteristics: hindbody fused only at their anterior and posterior ends; hindbody forming a ring; hindbody not divided into numerous tubular protrusions; two testes present (Pozdnyakov, Reference Pozdnyakov1996; Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008).
Species Annulocystis auxis Yamaguti, 1970
Type locality: Hawaii (Yamaguti, Reference Yamaguti1970).
Type host: Auxis thazard (Lacepède, 1800) (Yamaguti, Reference Yamaguti1970; this study).
Materials examined: One specimen (INM-1-132026) ex Au. thazard, off Iwate Prefecture, North Pacific, Japan, 17 September 2025, stained with acetocarmine and mounted as a permanent slide.
Redescriptions: (Figure 2a–c, based on one flattened specimen) Body yellow in live condition. Forebody narrow and slender. Two forebodies, each situated in opposite positions at the inner margin of the hindbody. Posterior margins of two large hindbodies fused with their respective posterior margins, forming a ring. Inner margin of hindbodies smooth, irregularly with small ridges. Outside margin of hindbodies with nine bulges in total due to uterine coils running in hindbodies. Oral sucker subterminal, long, spindle-shaped. Prepharynx absent. Pharynx subglobular. Posterior margin of oesophagus indistinct. Testis cylindrical, overlapping with uterus. Vas deferens not clearly observed. Ovary tubular, long, winding, present from anterior to posterior positions of hindbody, connecting to shell gland complex at a distance of 1.8 mm from position of forebody. Seminal receptacle unclear. Vitellaria tubular, long and winding, extending along with ovary. Uteruses wide, boundary between the two worms unclear. Metraterm muscular. Genital pore opening at the level of middle of oral sucker. Eggs bean-shaped. Excretory system not well observed. Measurements are in Table 2.
Body and reproductive organs of Annulocystis auxis (INM-1-132026), ventral view. a. Anterior region of the forebody. b. Whole body. Dotted lines represent tissue excised for PCR analysis. c. Female reproductive organs. Abbreviations: c, caecum; gp, genital pore; Mg, Mehlis’ gland; mt, metraterm; o, ovary; od, oviduct; os, oral sucker; ot, ootype; ph, pharynx; rd, ridge; t, testis; u, uterus; v, vitellarium; vd, vitelline duct.
Figure 2. Long description
A multi-panel line drawing of Annulocystis auxis with three sections labeled a, b, and c.
Panel a, top left, shows the anterior region of the forebody with a 100 micrometer scale bar. From the top tip downward, labels indicate the oral sucker, genital pore, pharynx, metraterm, and esophagus.
Panel b, center, shows the whole body in a curved, nearly circular ventral view with a 500 micrometer scale bar. An arrowhead points to the outer tegument. Following the body clockwise from the top, labels include T testis, Rd ridge, O ovary, V vitellarium, and U uterus. A section on the right side is enclosed in dotted lines to indicate tissue excised for PCR analysis.
Panel c, bottom left, provides a detailed view of the female reproductive organs with a 50 micrometer scale bar. Starting from the left, the ovary connects to the oviduct. This leads to a central junction where the vitelline duct and V vitellarium meet. The path continues to the ootype, which is surrounded by small hair-like Mehlis gland.
Comparison of morphological measurements of Annulocystis species. All measurements are in μm unless indicated otherwise
Table 2. Long description
The table compares Annulocystis auxis, An. carnata n. sp., and An. katsuwoni. Measurements are in micrometers unless noted as millimeters.
* Forebody size: An. auxis ranges from 1337 by 87 to 3.4 mm by 0.2 mm. An. carnata is 1850 by 233. An. katsuwoni is 0.18 to 0.4 mm by 0.1 to 0.12 mm.
* Oral sucker: An. auxis is spindle-shaped (34 to 140 length). An. carnata is subglobular (72 by 68). An. katsuwoni is oval (23 to 37 length).
* Pharynx size: An. auxis (20 to 70 diameter). An. carnata (64 by 50). An. katsuwoni (23 to 36 by 20 to 30).
* Hindbody of fused worms: An. auxis and An. carnata are ring-shaped with diameters ranging from 3.5 mm to 11.1 mm. An. katsuwoni is disc-shaped (1.3 mm by 1.0 mm).
* Testis: An. auxis is cylindrical. An. carnata is oval (277 by 192). An. katsuwoni is tubular.
* Egg size: An. auxis (12 to 21 by 9 to 12). An. carnata (17 to 19 by 8 to 11). An. katsuwoni (16 to 19 by 9 to 12).
* Host and Habitat: An. auxis and An. carnata are found in the gills of Auxis thazard and Euthynnus affinis respectively. An. katsuwoni is found in the intestine of Katsuwonus pelamis.
* Locality: Sites include Iwate and Mie Prefectures in Japan, and Hawaii, U S A.
Remarks: Our worms were identified as An. auxis based on the following morphological characteristics: forebody long and narrow; hindbodies ring-shaped; oral sucker long and spindle-shaped; testes long and cylindrical (Yamaguti, Reference Yamaguti1970). Genus Annulocystis currently includes two species, An. auxis and An. katsuwoni (Pozdnyakov, Reference Pozdnyakov1996; Yamaguti, Reference Yamaguti1970). Annulocystis auxis can be distinguished from An. katsuwoni because the hindbody formed a ring in An. auxis when matured (disc-shaped in An. katsuwoni: Yamaguti, Reference Yamaguti1970). Annulocystis auxis can be morphologically distinguished from the new species as described later.
Site in host: Gill (Yamaguti, Reference Yamaguti1970; this study).
New Japanese name: Hirasouda-kamon-kyuchu. ‘Hirasouda’ is the standard Japanese name for the host fish; ‘kamon’ is a traditional Japanese family crest or emblem; ‘kyuchu’ translates to ‘trematode’.
DNA markers: 28S rDNA, LC932360; ITS, LC932369; COI, LC932364.
Species Annulocystis carnata Waki & Nitta n. sp.
Type locality: Mie Prefecture, Japan.
Type host: Euthynnus affinis (Cantor, 1849).
Type materials: Holotype (INM-1-132027) and two paratypes (INM-1-132028, 132029) ex E. affinis, off Mie Prefecture, North Pacific, Japan, 9 May 2025, stained with acetocarmine and mounted as permanent slides.
Descriptions: (Figures 1a and 3a–c, based on three flattened specimens) Body yellow in live condition. Forebody wide and robust. Two forebodies, each situated in opposite positions at the inner margin of the hindbody. Posterior margins of two large hindbodies fused with their respective posterior margins, forming a ring. Inner margin of hindbodies smooth, irregularly with small ridges. Outside margin of hindbodies with from 13 to 19 bulges in total due to uterine coils running in hindbodies. Oral sucker terminal, subglobular. Prepharynx absent. Pharynx oval. Posterior margin of oesophagus indistinct. Testis oval, overlapping with uterus. Vas deferens ending posterior to pharynx and connecting to metraterm. Ovary tubular, long, winding, present from anterior to posterior positions of hindbody, connecting to shell gland complex at a distance of 2.5 mm from position of forebody. Seminal receptacle U-shaped. Vitellaria tubular, long and winding, extending along with ovary. Uteruses wide, boundary between two worms unclear. Metraterm muscular. Genital pore opening at level of middle of oral sucker. Eggs bean-shaped. Excretory system not well observed. Measurements are in Table 2.
Body and reproductive organs of Annulocystis carnata n. sp., ventral view. a, c. Holotype (INM-1-132027). b. Paratype (INM-1-132028). a. Anterior region of the forebody. b. Whole body. Dotted lines represent the outlines of the damaged parts. c. Female reproductive organs. Abbreviations: c, caecum; gp, genital pore; hd, hermaphroditic duct; Mg, Mehlis’ gland; mt, metraterm; o, ovary; od, oviduct; os, oral sucker; ot, ootype; ph, pharynx; rd, ridge; sr, seminal receptacle; t, testis; u, uterus; v, vitellarium; vd, vitelline duct; vs, ventral sucker.
Figure 3. Long description
A multi-panel scientific illustration of Annulocystis carnata n. sp. containing three labeled diagrams.
Panel b, located on the right, shows the whole body of the paratype. It is a large, C-shaped organism with a lobed outer margin. A scale bar indicates 3 mm. Labels include Rd for ridge, V for vitellarium, T for testis, U for uterus, O for ovary, and C for caecum. Dotted lines indicate damaged sections of the body wall.
Panel a, at the top left, shows the anterior region of the forebody of the holotype. A scale bar indicates 300 micrometers. Starting from the distal tip and moving proximally, labels identify the os (oral sucker), gp (genital pore), ph (pharynx), hd (hermaphroditic duct), es (esophagus), mt (metraterm), and vd (vitelline duct).
Panel c, at the bottom left, provides a detailed view of the female reproductive organs of the holotype. A scale bar indicates 200 micrometers. From right to left, the figure shows the ovary containing a coiled seminal receptacle. This connects to the oviduct and ootype. The Mehlis’ gland surrounds the ootype area. The vitelline duct and vitellarium are positioned centrally, leading into the coiled uterus at the top.
Remarks: We judged these worms as a new species, An. carnata n. sp., because of the following reasons. Our worms from E. affinis are morphologically distinct from the two known species of this genus because of the following morphological characteristics: forebody wider and robust; two hindbodies forming ring shape; oral sucker subglobular; testes oval. This new species can be distinguished from An. katsuwoni because the new species has ring-shaped hindbodies (disc-shaped in An. katsuwoni: Yamaguti, Reference Yamaguti1970). The new species was morphologically similar to An. auxis, however, the new species is morphologically distinct in the shape of forebody (wide and robust in the new species vs. narrow and long in An.: Yamaguti, Reference Yamaguti1970), oral sucker (subglobular vs. long and spindle shaped: Yamaguti, Reference Yamaguti1970), and testes (oval vs. long and cylindrical: Yamaguti, Reference Yamaguti1970).
Site in host: Gill (this study).
Etymology: Carnata (a Latin adjective [feminine] meaning ‘fleshy’ or ‘fat’) refers to the large and wide body of adult.
New Japanese name: Suma-kamon-kyuchu. ‘Suma’ is the standard Japanese name for the host fish.
DNA markers: 28S rDNA, LC932361; ITS, LC932370; COI, LC932365.
Zoobank Registration: The Life Science Identifier (LSID) for the publication is urn: lsid: zoobank.org:pub:EB13198C-DBAA-4121-B5F2-0F719C96ECD1; for Annulocystis carnata n. sp. is urn: lsid: zoobank.org:act:B22F8447-C048-45FD-93AC-AEEBC26EA294.
Genus Gonapodasmius Ishii, 1935
Three specimens from the gills of M. mola were identified as a member of genus Gonapodasmius based on the following morphological characteristics: hindbody divided into numerous tubular protrusions; hindbody forming a cocoon-shaped capsule; oesophagus short; genital pore ventral to pharynx (Pozdnyakov, Reference Pozdnyakov1996; Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008). Although the number of testes, a primary diagnostic characteristic for the genus Gonapodasmius, could not be counted in the present study, the cocoon-shaped capsule of the hindbody justifies assignment to the genus Gonapodasmius (Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008). Pozdnyakov and Gibson (Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008) reported that gill tissues are infection sites of members of this genus. Our specimens were found on gills of the host, supporting our identifications at the genus level.
Species Gonapodasmius squamatus (Pozdnyakov, 1994)
Type host: Mola sp. [Mola mola (Linnaeus, 1758) or Mola alexandrini (Ranzani, 1839) (see Sawai et al., Reference Sawai, Yamanoue, Nyegaard and Sakai2018), Mola mola in the original description] (Pozdnyakov, Reference Pozdnyakov1994a).
Other host: Mola mola (Linnaeus, 1758) sensu stricto (this study).
Type locality: East Pacific Ocean (subtropical region) (Pozdnyakov, Reference Pozdnyakov1994a).
Materials examined: Three specimens (INM-1-132035, 132036) ex M. mola, off Kyoto Prefecture, Sea of Japan, Japan, 17 March 2025; two specimens were stained with acetocarmine and mounted as a permanent slide and one specimen was preserved in 70% ethanol.
Redescriptions: (Figures 1b and 4a, based on two forebodies on slides and one entire ethanol-preserved specimen) Forebody narrow and slender. Anterior margin rounded. Oral sucker terminal, rounded. Pharynx rounded, smaller than oral sucker. Ventral sucker rounded, subequal to oral sucker. Genital pore posterior to pharynx. Hindbody large, branched, and tubular. Hindbodies of two worms forming a cocoon-shaped. Eggs oval. Measurements are in Table 3.
Body and reproductive organs of Gonapodasmius squamatus and Nematobothrium filiforme, ventral view. a. Gonapodasmius squamatus (INM-1-132035). b–f. Nematobothrium filiforme (b, INM-1-132037; c–f, INM-1-132038). a–b. Unflattened specimen. c–f. Flattened specimens. a. Anterior region of the forebody of Gonapodasmius squamatus. b, d. Anterior body part of Nematobothrium filiforme. c. Whole body. e. Female reproductive organs. f. Posterior margin of the body. Abbreviations: c, caecum; e, excretory pore; Mg, Mehlis’ gland; mt, metraterm; o, ovary; os, oral sucker; ot, ootype; ph, pharynx; sr, seminal receptacle; sv, seminal vesicle; t, testis; u, uterus; v, vitellarium; vd, vitelline duct; vs, ventral sucker.
Figure 4. Long description
A multi-panel set of line drawings.
Panel a shows the anterior region of the forebody of Gonapodasmius squamatus. At the top is the oral sucker, followed by the pharynx and genital pore. Below these, the metraterm runs centrally, flanked by the caecum. A ventral sucker is located on the right side of the body wall. A 200 micrometer scale bar is provided.
Panel b depicts the anterior body of Nematobothrium filiforme. It features an oral sucker at the top, an esophagus, and a winding metraterm. Further down are the seminal vesicle, vitelline duct, testis, uterus, and ovary. A 200 micrometer scale bar is provided.
Panel c shows the whole body of N. filiforme as a long, thin, thread-like structure with a 2 millimeter scale bar.
Panel d provides a detailed view of the anterior body part of a flattened N. filiforme specimen, showing the convoluted internal ducts. A 200 micrometer scale bar is provided.
Panel e focuses on the female reproductive organs of N. filiforme. From top to bottom, it labels the ovary), Mehlis’ gland, ootype, seminal receptacle, vitelline duct, and vitellarium. A 100 micrometer scale bar is provided.
Panel f shows the posterior margin of the body of N. filiforme, including the vitellarium, uterus, caecum, and excretory pore at the very tip. A 100 micrometer scale bar is provided.
Comparison of morphological measurements of Gonapodasmius species. All measurements are in μm unless indicated otherwise
Table 3. Long description
The table compares Gonapodasmius squamatus (this study and Pozdnyakov 1994), G. haemuli, G. hapuki, G. multilobularis, and G. williamsoni.
* Oral sucker size: 51 times 49 and 59 times 49 for G. squamatus (this study); 0.2 to 0.3 mm for G. haemuli; 0.3 to 0.4 mm for G. hapuki; and 60 times 55 for G. williamsoni.
* Pharynx size: 22 times 19 and 32 times 21 for G. squamatus (this study); smaller than oral sucker for G. squamatus Pozdnyakov, 1994 and G. multilobularis Yamaguti, 1970; 33 times 25 for G. williamsoni.
* Oesophagus length: 195 for G. squamatus (this study); 0.05 to 0.09 mm for G. haemuli; 0.9 to 1.0 mm for G. hapuki; 347 for G. williamsoni.
* Gland-cells around oesophagus: Present only in G. hapuki; absent in all other species.
* Ventral sucker size: 51 times 45 for G. squamatus (this study); 37 times 57 for G. williamsoni; not described for others.
* Cyst size: 2.3 cm for G. squamatus (this study); 18 mm times 15 mm times 5 mm for G. multilobularis.
* Genital pore position: Posterior to pharynx for most; at level of middle of pharynx for G. haemuli; anterior to pharynx for G. hapuki.
* Egg size: 17 times 10 for G. squamatus (this study); 30 times 12 for G. williamsoni.
* Host: Mola mola for G. squamatus; Epinephelus bruneus for G. haemuli; Polyprion oxygeneios for G. hapuki; Xiphias gladius for G. multilobularis; Pagrus auratus for G. williamsoni.
* Habitat: Gill for all species except G. williamsoni, which is found in lateral musculature.
* Locality: Kyoto Prefecture, Japan (this study); East Pacific Ocean; Hawaii, USA; Coral Sea; and Shark Bay, Australia.
Remarks: The genus Gonapodasmius includes five species (Cribb and Williams, Reference Cribb and Williams1992; Pozdnyakov, Reference Pozdnyakov1994a, Reference Pozdnyakovb). Of these, G. williamsoni was the only species to be found in muscle tissue without being encapsulated (Cribb and Williams, Reference Cribb and Williams1992). Of the remaining four species, our specimens were identified as G. squamatus, which was detected on the gills of Mola sp. in the eastern Pacific Ocean (Pozdnyakov, Reference Pozdnyakov1994a), based on the following characteristics: oral sucker larger than pharynx, oesophagus long; genital pore posterior to pharynx; hindbodies cocoon-shaped. Gonapodasmius haemuli can be distinguished from G. squamatus because its pharynx is larger than the oral sucker (vs. smaller in G. squamatus: Pozdnyakov, Reference Pozdnyakov1994b), its oesophagus is short (vs. long in G. squamatus: Pozdnyakov, Reference Pozdnyakov1994b) and its genital pore opens at level of middle of pharynx (vs. posterior to pharynx: Pozdnyakov, Reference Pozdnyakov1994b). Gonapodasmius hapuki differed from G. squamatus because G. hapuki had gland-cells around oesophagus (vs. absent in G. squamatus: Pozdnyakov, Reference Pozdnyakov1994b) and genital pore opening anterior to pharynx (vs. posterior to pharynx). Gonapodasmius multilobularis can also be distinguished from G. squamatus based on the shape of the hindbodies (flattened in G. multilobularis vs. cocoon-shaped in G. squamatus: Yamaguti, Reference Yamaguti1970). Our specimens had ventral suckers in their forebodies, although the original description of this species did not mention presence or absence of ventral sucker in the type specimen (Pozdnyakov, Reference Pozdnyakov1994a). Indeed, some of the original descriptions and redescriptions of other members of Gonapodasmius in past studies did not clearly mention the absence of ventral suckers (Yamaguti, Reference Yamaguti1970; Pozdnyakov, Reference Pozdnyakov1994b), except for Cribb and Williams (Reference Cribb and Williams1992), which clearly reported ventral suckers in their specimens. Despite this issue, we identified our specimens as G. squamatus because the sampling locality of our hosts was close to the type locality (East Pacific Ocean, subtropical region) and the infection site of our worms (gills) was the same as in the original description (Pozdnyakov, Reference Pozdnyakov1994a). The type host of G. squamatus was identified as M. mola in the original description (Pozdnyakov, Reference Pozdnyakov1994a). However, the sunfish, previously identified as ‘M. mola’ is currently known to include two species, M. mola and M. alexandrini, in the North Pacific Ocean (Sawai et al., Reference Sawai, Yamanoue, Nyegaard and Sakai2018). Therefore, the type host of G. squamatus cannot be determined.
The absence of a ventral sucker was one of the morphological characteristics of the subfamily Gonapodasmiinae (Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008). However, G. squamatus in this study had a ventral sucker, as did G. williamsoni reported by Cribb and Williams (Reference Cribb and Williams1992). Seemingly, the presence or absence of a ventral sucker might not be a fine diagnostic characteristic for this subfamily, indicating that the diagnosis of this subfamily may be reconstructed. Alternatively, it is also possible that these two species do not belong to Gonapodasmiinae, but rather represent a different subfamily. In our phylogenetic analyses, G. squamatus formed a clade with members of the subfamily Didymodiclininae (see below). However, G. squamatus does not fit the diagnosis of Didymodiclininae, which requires a filiform or moderately elongate body not divided into regions (vs. hindbody cocoon-shaped and body divided into forebody and hindbody in our specimens). In the present study, our trematodes from M. mola were identified as G. squamatus based on the host species, sites in the host, and the combination of morphological characteristics, despite the presence of a ventral sucker.
Site in host: Gill (Pozdnyakov, Reference Pozdnyakov1994a; this study).
DNA marker: 28S rDNA, LC932363; ITS, LC932372; COI, LC932368.
New Japanese name: Manbou-omatsuri-kyuchu. ‘Manbou’ translates to ‘sunfish’ in Japanese language; ‘omatsuri’ is ‘tangled cord’ in Japanese.
Subfamily Nematobothriinae Ishii, 1934
Trematodes from the gills of Scomber spp. were identified as subfamily Nematobothriinae based on the following morphological characteristics: hermaphroditic; body filiform and not divided into regions; caeca simple and blind; testes parallel; genital pore ventral to oral sucker; ovary and vitellarium elongate; ovary in anterior half of body; uterus long; metraterm well developed; vitellarium in posterior half of body (Pozdnyakov, Reference Pozdnyakov1996; Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008).
Tribe Nematobothriini Ishii, 1934
These trematodes were identified as this tribe based on the following morphological characteristics: ventral sucker absent; two testes present (Pozdnyakov, Reference Pozdnyakov1996; Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008).
Genus Nematobothrium Van Beneden, 1858
The specimens were identified as genus Nematobothrium based on the following morphological characteristics: gland cells around oesophagus or pharynx not observed; testes parallel, located in anterior half of body; pharynx present; body filiform, narrow and slender (Pozdnyakov, Reference Pozdnyakov1996; Pozdnyakov and Gibson, Reference Pozdnyakov, Gibson, Bray, Gibson and Jones2008).
Species Nematobothrium filiforme Yamaguti, 1934
Type locality: Toyama Bay, Japan (Yamaguti, Reference Yamaguti1934)
Type host: Scomber japonicus Houttuyn, 1782 (Yamaguti, Reference Yamaguti1934; Ishii, Reference Ishii1935; this study).
Other host: Scomber australasicus Cuvier, 1832 (this study; Pitt et al., Reference Pitt, Browne, Cutmore, Diaz, Nq-x and Cribb2026, see below).
Hosts of metacercariae: Aegina citrea Eschscholtz, 1829 and Aldersladia magnificus Gershwin, 2006 (Pitt et al., Reference Pitt, Browne, Cutmore, Diaz, Nq-x and Cribb2026, see below)
Synonym: Nematobothrium sabae Ishii, 1935 (Ishii, Reference Ishii1935); Didymozoid Species K (Pitt et al., Reference Pitt, Browne, Cutmore, Diaz, Nq-x and Cribb2026, see below)
Materials examined: two unflattened specimens (INM-1-132037) ex S. australasicus, off Nagasaki Prefecture, Japan, 20 February 2025, stained with acetocarmine and mounted as permanent slides; 13 flattened specimens (INM-1-132038–132042) ex. S. japonicus, off Matsusaka City (34°38′02″N, 136°35′21″E), Mie Prefecture, Ise Bay, Japan, 29 November 2018, stained with Heidenhain’s iron-haematoxylin and mounted as permanent slides.
Additional specimens deposited: Nine unflattened specimens (INM-1-132030–132034) ex S. japonicus, Muroto City, Kochi Prefecture, Japan, 15 May 2025, stained with acetocarmine and mounted as permanent slides.
Redescriptions: (Figure 4b–f, based on two and four unflattened and flattened specimens, respectively) Body of a worm narrow and slender. Anterior margin of body conical. Oral sucker at apical of anterior body, small and cylindrical. Prepharynx absent. Pharynx muscular. Oesophagus long and narrow, bifurcation of caeca not well observed in our specimens. Caeca reaching to posterior margin of body. Ventral sucker absent. Two testes tubular, coiled, parallel, and overlapped, and difficult to completely distinguish each other. Testes between posterior margin of seminal vesicle and nearly anterior margin of uterus. Seminal vesicle well-coiled. Ovary elongate, tubular, between testes and middle of body. Position of female reproductive complex not observed in unflattened specimens, possibly due to presence of internal organs including large uterus containing numerous eggs. Vitellarium elongate, tubular, starting posterior end of ovary and reaching nearly posterior margin of uterus. Uterus long, looping at its anterior and posterior regions. Uterus not reaching posterior margin of body. Metraterm muscular. Seminal receptacle oval, situated between ovary and vitelline. Genital pore opening at middle of oral sucker. Excretory pore opening at posterior margin of body. Measurements are in Table 4.
Comparison of morphological measurements of Nematobothrium species. All measurements are in μm unless indicated otherwise
Table 4. Long description
A detailed table comparing morphological measurements of Nematobothrium filiforme, N. atlanticum, and N. scombri.
Columns include:
* This study (unflattened) for N. filiforme.
* This study (flattened) for N. filiforme.
* Yamaguti, 1 9 3 4 for N. filiforme.
* Ishii, 1 9 3 5 for N. filiforme.
* Mordvinova, 1 9 8 9 for N. atlanticum.
* Skrjabin, 1 9 5 5 for N. scombri.
Key morphological rows include:
* Body size: Ranges from 15 mm in this study to 133.5 mm in N. atlanticum.
* Anterior margin shape: Conical for N. filiforme and N. atlanticum, but oval for N. scombri.
* Oral sucker: Measured at 41 by 29 mu m in this study (unflattened) compared to 0.3 to 0.4 mm diameter in N. scombri.
* Pharynx: 30 by 27 mu m in this study (unflattened).
* Oesophagus: 56 to 61 mu m in this study, reaching 1.60 mm in N. atlanticum.
* Testes size: 700 by 103 mu m in this study (unflattened).
* Seminal vesicle: Present in most except N. atlanticum.
* Ovary: 7.6 mm by 42 mu m in this study (flattened).
* Eggs: Approximately 12 to 13 by 8 mu m across N. filiforme studies.
* Host: Scomber australasicus and S. japonicus for N. filiforme; Cheilopogon species for N. atlanticum; S. scombrus for N. scombri.
* Habitat: Primarily gills, except orbital cavity for N. atlanticum.
* Locality: Japan for N. filiforme, Atlantic Ocean for N. atlanticum, and Mediterranean Sea for N. scombri.
a Sizes of two testes.
b Sizes of smaller and larger testes, respectively.
Remarks: The worms on the gills of the fish hosts were identified as N. filiforme based on the following characteristics: body narrow and slender; anterior margin of body conical; oral sucker small; oesophagus long and narrow; ventral sucker absent; testes between posterior margin of seminal vesicle and nearly anterior margin of uterus; uterus not reaching posterior margin of body; vitelline reaching nearly posterior margin of uterus (Ishii, Reference Ishii1935; Yamaguti, Reference Yamaguti1934). This species morphologically similar to N. atlanticum because of the following characteristics: body narrow and slender; anterior margin of body conical; oral sucker small; ventral sucker absent (Mordvinova, Reference Mordvinova1989). However, these two species can be distinguished based on positions of posterior margin of uterus (not reaching posterior margin of body in N. filiforme vs. reaching in N. atlanticum: Mordvinova, Reference Mordvinova1989), posterior margin of testes (nearly anterior margin of uterus vs. exceeding anterior margin of uterus), and presence of seminal vesicle (present vs. absent: Mordvinova, Reference Mordvinova1989). Nematobothrium filiforme and N. scombri were also similar to each other in the following characteristics: body narrow and slender; ventral sucker absent (Skrjabin, Reference Skrjabin1955). However, these two species can be distinguished based on shape of anterior margin of body (conical in N. filiforme vs. wide and oval in N. scombri: Skrjabin, Reference Skrjabin1955), oesophagus (long vs. short: Skrjabin, Reference Skrjabin1955), and oral sucker (small vs. large: Skrjabin, Reference Skrjabin1955).
Site in host: Gill (Ishii, Reference Ishii1935; Yamaguti, Reference Yamaguti1934; this study).
DNA marker: 28S rDNA, LC932362; ITS, LC932371; COI, LC932366, LC932367.
New Japanese name: saba-era-dama-kyuchu. ‘Saba’ is the standard Japanese name for ‘Scomber fish’ in the Japanese language; ‘era’ means ‘gill’; ‘dama’ translates to ‘ball’.
Molecular analyses
The phylogenetic tree of 28S rDNA showed that the two species of the genus Annulocystis formed a well-supported clade with other sequences of this genus retrieved from the online databases (bootstrap value = 98) (Figure 5). Furthermore, the sequences generated for the new species were identical to those of An. cf. auxis (OL336059), previously reported from Au. thazard in Moreton Bay, Australia (Louvard et al., Reference Louvard, Cutmore, Yong, Dang and Cribb2022), suggesting that they are conspecific. The sequence of N. filiforme clustered in a tight clade with another N. species (bootstrap value = 100); however, members of the superfamily Nematobothriinae were divided into multiple clades (Figure 5). Moreover, the sequence of G. squamatus (Gonapodasmiinae) nested in a clade with 11 members of the subfamily Didymodiclininae, positioning it distantly from the Annulocystis clade (Gonapodasmiinae). Pairwise interspecific divergence values of ITS sequences (440 bp-overlapping region) in the genus Annulocystis ranged from 0.0049 to 0.0149. Intraspecific divergence values could not be calculated, because only a single sequence was available for each species. The divergence value between the new species and An. cf. auxis in Louvard et al. (Reference Louvard, Cutmore, Yong, Dang and Cribb2022) was zero, supporting their conspecificity. Pairwise intraspecific divergence values based on the ITS sequences (433-bp overlapping region) from the databases of trematodes closely related to N. filiforme ranged from zero to 0.0069. The divergence value between our N. filiforme and metacercariae of Didymozoid Species K detected from cnidarian jellyfish, Ae. citrea and Al. magnificus, in Pitt et al. (Reference Pitt, Browne, Cutmore, Diaz, Nq-x and Cribb2026) was zero, indicating their conspecificity.
Maximum likelihood phylogenetic tree of didymozoid trematodes inferred from partial nuclear 28S ribosomal DNA (774 sites), with Derogenes spp. (LC586089 and PX068002) as outgroup taxa. Bootstrap values above 50% are shown. Database accession numbers are indicated after species names. Asterisks represent newly obtained sequences. Grey represents Gonapodasmiinae and Nematobothriinae. Scale bar indicates the number of substitutions per site.
Figure 5. Long description
A phylogenetic tree with horizontal branches of varying lengths representing genetic substitutions per site. The outgroup at the bottom consists of Derogenes lacustris LC5 86 08 9 and Derogenes varicus PX0 68 00 2.
Moving from top to bottom, the major clades are labeled with vertical bars on the right:
* The top section is a large cluster of Didymozoinae, which includes a sub-clade of Koellikeriinae at the very top. Species include Wedlia, Koellikeriinae species, Didymocystis, Didymosulcus, Lobatozoum, and Didymozoon.
* Below this is a small clade of Gonapodasmiinae highlighted in grey, containing Annulocystis species, including newly sequenced Annulocystis auxis LC9 32 36 0 asterisk and Annulocystis caranata n. sp. LC9 32 36 1 asterisk.
* A second Didymozoinae clade follows, containing Didymozoidae and Didymocystis species.
* A grey-shaded Nematobothriinae clade includes Sarcocheilichthys variegatus and several Nematobothriinae species.
* A third Didymozoinae clade follows, featuring Helicodidymozoon, Indodidymozoon, Neometadidymozoon, Neodidymozoon, Maccallumtrema, and Metadidymozoon.
* The lower section contains alternating clades of Didymodiclininae, a single Gonapodasmius squamatus LC9 32 36 3 (Gonapodasmiinae) asterisk in grey, and a final Nematobothriinae clade at the bottom containing Nematobothrium filiforme LC9 32 36 2 asterisk.
Bootstrap values are indicated at the nodes, such as 100 at several terminal bifurcations. A scale bar at the bottom-left represents 0.050 substitutions per site.
Discussion
The detailed morphological observation of adult didymozoids is often hindered by their fragile bodies and large uterus filled with eggs (e.g. Yamaguti, Reference Yamaguti1970). The novel DNA markers generated herein for the four didymozoid species will help facilitate future taxonomic identification. For example, the 28S rDNA sequence of An. carnata n. sp. was identical to that of An. cf. auxis (OL336059), previously reported from Auxis thazard in Australia (Louvard et al., Reference Louvard, Cutmore, Yong, Dang and Cribb2022). This genetic identity supports their conspecificity, indicating that the new species is widely distributed across the Western Pacific and Oceania. Moreover, our molecular analyses revealed that three metacercariae of Didymozoid Species K from cnidarian jellyfishes in Pitt et al. (Reference Pitt, Browne, Cutmore, Diaz, Nq-x and Cribb2026) were conspecific with N. filiforme. Pitt et al. (Reference Pitt, Browne, Cutmore, Diaz, Nq-x and Cribb2026) also detected Didymozoid Species K from Scomber australasicus in Moreton Bay, Australia, but the specimen was not identified morphologically. Therefore, our morphological and molecular analyses clarify a part of the life cycle of this species, demonstrating that DNA markers of morphological identified worms are invaluable tools for linking the life cycles of members of this family. On the other hand, although unidentified didymozoid metacercariae are frequently reported in the literature (Louvard et al., Reference Louvard, Cutmore, Yong, Dang and Cribb2022, Reference Louvard, Yong, Cutmore and Cribb2024; Pitt et al., Reference Pitt, Browne, Cutmore, Diaz, Nq-x and Cribb2026), their species-level identities remain entirely elusive. This underscores the current deficit of reference DNA markers for adult didymozoids. Expanding this molecular database is essential not only for accurate adult identification but also for improving our understanding of the complex life cycles of didymozoid trematodes, a strategy that has been successful in several other trematode lineages (e.g. Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024).
Our molecular phylogeny suggests that the currently accepted macrotaxonomy of the family Didymozoidae may be problematic for certain subfamilies and genera. Notably, the subfamily Nematobothriinae was divided into multiple clades. Moreover, G. squamatus, a member of the subfamily Gonapodasmiinae, nested in the Didymodiclininae clade as described above. Although the limited taxon sampling in this study precludes a definitive systematic overhaul, these topological inconsistencies strongly emphasise the need for taxonomic revisions as broader molecular phylogenetic data are accumulated for this family in the future.
Acknowledgements
The present study was kindly helped by Yasufusa Hatakeyama, Rintaro Shigeno, and Sho Shirakashi. We would like to express our sincere thanks to all participants in the research cruise of TRV Seisui-maru, Mie University, for their cooperation at sea.
Financial support
This work was partially supported by JSPS KAKENHI Grant (No. JP18J00466) to MN.
Competing interests
The authors declare none.
Ethical standards
Formal ethical approval was not required for this study because it involved unregulated fish species.
