#ZooKeys ZooKeys 1189: 231-256 (2024) DOI: 10.3897/zookeys.1189.111825 Research Article New deep-sea species of Aborjinia (Nematoda, Leptosomatidae) from the North-Western Pacific: an integrative taxonomy and phylogeny Julia K. Zograf', Alexander A. Semenchenko?, Vladimir V. Mordukhovich'? 1 A.V. Zhirmunsky National Scientific Center of Marine Biology, FEB RAS, 690041, Vladivostok, Russia 2 Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS, 690022, Vladivostok, Russia 3 Far Eastern Federal University, 690922, Vladivostok, Russia Corresponding author: Julia K. Zograf (zojulia@yandex.ru) OPEN Qaceess Academic editor: Alexei Tchesunov Received: 29 August 2023 Accepted: 15 November 2023 Published: 17 January 2024 ZooBank: https://zoobank.org/ FCBFC631-3C49-4B98-BD02- 1063654837FE Citation: Zograf JK, Semenchenko AA, Mordukhovich VV (2024) New deep-sea species of Aborjinia (Nematoda, Leptosomatidae) from the North-Western Pacific: an integrative taxonomy and phylogeny. Zookeys 1189: 231-256. https://doi. org/10.3897/zookeys.1189.111825 Copyright: © Julia K. Zograf et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Marimermithid nematodes parasitising invertebrates are mainly found in the deep-sea environments. Several adult and juvenile specimens marimermithids of the genus Abor- jinia have been found in bottom sediments and inside Polychaeta during recent cruises to the Kuril-Kamchatka trench and the Kuril Basin (the Sea of Okhotsk). New species are described based on integrative study. Aborjinia profunda sp. nov. differs from A. eulag- iscae by the location of the ventral gland cell bodies (posterior to the nerve ring vs pos- terior to the cardia), by the smaller body size (23-28 mm vs 103-132 mm) and shorter tail (193-263 um vs 500-850 um). BI and ML phylogenetic analyses based on 18S and 28S rDNA suggest that genus Aborjinia belongs to the family Leptosomatidae. Based on molecular and morphological characters the new genus Paraborjinia gen. nov. is pro- posed for A. corallicola. Within the family Leptosomatidae the new genus differs from all genera except Aborjinia by its endoparasitic lifestyle and hologonic ovaries. Paraborjinia gen. nov. differs from Aborjinia by the position of cephalic sensitive organs (outer labial and cephalic papillae in two separate circles vs outer labial and cephalic papillae in one circle) and by the parasitic adult (vs free-living in Aborjinia). Key words: DNA barcoding, free-living nematodes, Kuril-Kamchatka Trench, Leptosoma- tidae, phylogenetic relationships, scanning electron microscopy Introduction In 1933 Ward described the very unusual nematodes Thalassonema ophiocti- nis Ward, 1933 from the brittle star Ophiocten amitinum Lyman, 1878 with an unclear taxonomical position. Forty years later Rubtzov and Platonova (1974) established a new family Marimermithidae which included two new genera Tro- phomera Rubtzov & Platonova, 1974 and Marimermis Rubtzov & Platonova, 1974. These nematodes resembled Mermithidae Braun, 1883 in lifestyle but differed in digestive system structure, anterior sensilla organization, and reproductive system. Later Rubtzov (1980) raised the family Marimermithidae to the rank of order. However, phylogenetic heterogeneity of the family was demonstrated in Zo. Julia K. Zograf et al.: Aborjinia from the North-Western Pacific the following years. Simultaneously, the family Benthimermithidae Petter, 1980 also comprising internal parasites of marine invertebrates superficially similar to Mermithidae was described by Petter (1980). Later, Benthimermithidae was raised to the rank of order (Tchesunov 1995) and the genus Trophomera was transferred to the family Benthimermithidae, but the position of both orders (Marimermithida and Benthimermithida) in the nematode system remained un- certain. The rarity and peculiarities of the life cycle made studying these taxa difficult. Most species are known on the basis of only few, and often just a single, adult individual from sediments, so the host for most species remains unknown (Miljutin 201 4a, b). Juveniles are found in the body cavities of a wide range of invertebrate hosts (e.g., Nematoda, Polychaeta, Priapulida, Bivalvia, Harpacticoida, Amphipoda, Ilsopoda); however, individuals without reproductive organs are usually unidentifiable (Miljutin 2014a, b). In addition, researchers often received samples fixed in formaldehyde, which limited the possibilities of investigation, particularly molecular studies. Based on the morphological characters, Tchesunov (1997) suggested af- finities between benthimermithids and Plectida and between marimermithids and Enoplia. Phylogenetic analyses based on a Trophomera SSU and LSU rDNA sequences provided support for a relationship between benthimermithids and plectids and resulted in the placement of all Trophomera sequences within the order Plectida (Tchesunov et al. 2009; Mardashova et al. 2011; Holovachov et al. 2013; Leduc and Zhao 2019). Leduc and Zhao (2019) proposed that the family Benthimermithidae be moved to the order Plectida. The phylogenetic relationship of marimermithids remained unclear for many years. Marimermithid nematodes are parasites of invertebrates mainly found in deep-sea environments (Miljutin 2014a). Most marimermithids are large an- imals reaching several centimeters in length. Their morphology is quite simple and characterized by features usual for free-living nematodes, for example the presence of cephalic sense organs, a cylindrical pharynx, and a small cardium with a triradial internal lumen. On the other hand, their alimentary tract is often devoid of rectum and anus, caudal glands are absent or reduced in adults, hy- podermal chords are hypertrophied, and the female genital system is adapted to facilitate the production of a large number of eggs. Such features are related to the parasitic way of life (Miljutin 2003). The results from the phylogenetic analysis based on 18S rRNA of Parabor- jinia corallicola (Westerman et al., 2021) suggest a relationship with the family Leptosomatidae Filipjev, 1916 (Westerman et al. 2021). The recent phylogenet- ic analyses based on the sequences of genes 18S and 28S RNA of Marimermis maritima Rubtzov & Platonova, 1974 and Aborjinia sp. showed placement of these species within the order Enoplida but in the different branches of the tree (Tchesunov et al. 2022). Thus, molecular analyses supported the relationship of marimermithids with the Enoplida and did not justify the order Marimermithi- da as a holophyletic taxon. Although the first representatives of Aborjinia Ozdikmen, 2010 were described 40 years ago, they are rarely found, little-known, and poorly studied; only one species is known. Several adult and juvenile specimens of Aborjinia were found in bottom sediments and inside Polychaeta during recent cruises to the Kuril-Ka- mchatka trench and the Kuril Basin (the Sea of Okhotsk). Here we provide an in- tegrative taxonomic study and determine the phylogenetic position of Aborjinia. ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 232 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific Materials and methods Study sites and sampling Specimens of Aborjinia were collected in several locations during KuramBio | (July-August 2012), SokhoBio (July-August 2015), and KuramBio II (August-Sep- tember 2016) expeditions to the Kuril-Kamchatka Trench and adjacent north- west Pacific at water depths of 3350-9290 m (Fig. 1, Table 1). Samples collect- ed by Agassiz trawl (AGT), epibenthic-sledge (EBS), and giant-box corer (GKG). On deck, the sediment from the AGT was sieved through a 1000-um mesh size, and the upper layer of sediment (0-20 cm) from the GKG was carefully sieved through 1000-, 500-, and 300-um mesh sizes. Immediately after sieving, sam- ples from AGT and GKG were sorted in seawater using stereomicroscopes, and nematodes were removed and fixed in 4% buffered formaldehyde for mor- phological studies and in DESS (solution of 0.25 M disodium EDTA and 20% dimethyl sulfoxide (DMSO), saturated with NaCl, pH 8.0) for DNA studies. On deck, the samples from EBS were immediately transferred into chilled (-20 °C) 96% ethanol and stored in a -20 °C freezer for at least 48 h for subsequent DNA studies. In the laboratories of the ship and home institutes, sorting of the fauna was done on ice in order to avoid DNA decomposition. The NCBI database contains the Leptosomatides sequence HM564626, which has a very high level of similarity to the Aborjinia sequences. There is no unambiguous evidence in the literature of the incorrect identification of the indi- cated sequence, only an assumption (Tchesunov et al. 2022). Moreover, neither for the Leptosomatides TCR192 sample nor for Aborjinia sp. KKT is there any published morphological data. We were able to obtain two individuals (a female and a juvenile) of the genus Leptosomatides for morphological and molecular analysis and comparison with Aborjinia. Bottom sediments with specimens of Leptosomatides sp. were taken by scuba-divers in Ayan Bay (Sea of Okhotsk) in 2019 during a cruise onboard the R/V ‘Academic Oparin’ (Table 1). The se- quences of these specimens are clustered within Leptosomatidae, and based on morphological analysis, specimens belong to genus Leptosomatides (Suppl. material 1). On deck, the sediment was sieved through 1000-, 500-, and 32-um mesh sizes. Immediately after sieving, samples from 1000- and 500-um mesh sizes were sorted in seawater using stereomicroscopes, and nematodes were removed and fixed in 10% formalin for morphological studies and in DESS for DNA studies. Half the samples from 32-um mesh size were fixed with 4% buff- ered formaldehyde and half fixed with DESS. Morphological analysis The male specimens of Aborjinia were picked out from the formaldehyde-fixed samples under a stereoscopic microscope, transferred to glycerin using the Seinhorst’'s (1959) rapid method as modified by De Grisse (1969), and mounted on permanent slides. Drawings and DIC (differential interference contrast) pho- tographs were made on an optical microscope Olympus BX 53 with the aid of a drawing tube and a digital camera, respectively. The female specimen (DESS fixed, voucher M10) and a specimen of Terebel- lides sp. (Polychaeta: Terebellida: Trichobranchidae) with a parasitic juvenile of Aborjinia (ethanol fixed, voucher M11) in the body cavity were picked out from Zookeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 233 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific Bering Sea Sea of Okhotsk 50°N y Ls Sea of Japan | 40°N 30°N ie 5 ; 120°E 130°E 140°E 150°E | 160°E 170°E 180°E Figure 1. Map of the investigated area. Dots indicate sampled stations: A1 — Aborjinia sp. juvenile; A2 — Aborjinia sp. female; A3 - Aborjinia profunda sp. nov. male. the fixative and placed in distilled water in a Petri dish. Photographs were made on an optical microscopes Nikon SMZ25 and Carl Zeiss Axio Observer 7 with the aid of a digital camera. After that, ~ 1 cm from the middle of the body of the nematode was cut off for genetic studies, and the remaining parts were placed back into the fixative. Two male individuals fixed in formalin were cut to obtain pieces of the anterior end, middle part of pharynx, central part of body, and tail end. The specimens were then rinsed in the distilled water. After dehydration in graded ethanol series and ethanol-acetone mixture, the specimens were embedded in Spurr resin (Spur, Sigma). Semi-thin transverse sections (0.5 um) were cut using a Leica Ultracat E Ultratome. The sections were first stained with meth- ylene blue and azure II for 20 min at 60 °C and then with basic fuchsin for 4 min at room temperature (Humphrey and Pittman 1974), and mounted in Spurr resin on permanent glass slides. Photographs were made on an optical micro- scope Carl Zeiss Axiolmager Z.2 with the aid of a digital camera. The acquired ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 234 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific Table 1. Localities, depth, and sampling data. Species Aborjinia sp. (specimen M11) Aborjinia sp. (specimen M10) Aborjinia profunda sp. nov. Aborjinia profunda sp. nov. Aborjinia profunda sp. nov. Leptosomatides sp. (specimen L1, L2) Cruise Gear Area | GPS Coordinates Depth | Date [y/m/d] Comments [m] SokhoBio Epibenthic sledge 6 48.0°N, 150.0°E | 3347 2015/07/20 | juvenile in body cavity of Terebellides sp. SokhoBio Agassiz trawl 9 46.2°N, 152.1°E | 3374 | 2015/07/25-27 female KuramBio II Agassiz trawl A6é 45.9°N, 152.8°E | 6114 | 2016/08/25-27 male KuramBio II Box corer, Agassiz trawl | A9 44.7°N, 151.5°E | 8235 | 2016/09/12-17 15 males KuramBio II Epibenthic sledge A10 | 45.0°N, 151.1°E | 5477 2016/09/16 male 56 cruise of RV Scuba-diver 49 56°25.405'N, 4-9 2019/08/02 female and juvenile Akademic Oparin 138°03.879'E images were then adjusted for contrast and brightness using the ImageJ im- age processing software. For the scanning electron microscopy, specimens were gradually dehydrated in a series of baths of increasing ethanol content, dried in a critical-point dryer, sputter-coated with gold, and observed and imaged with a Zeiss SIGMA 300VP scanning electron microscope (SEM). The type material is deposited in the Senckenberg Museum, Frankfurt am Main, Germany (SMF) and in the Museum of the A.V. Zhirmunsky National Sci- entific Center of Marine Biology FEB RAS, Vladivostok, Russia (MIMB). Abbreviations of the measured variables in the tables are as follows: body length divided by maximum body diameter; body length divided by pharyngeal length; body length divided by tail length; tail length divided by corresponding body diameter at cloacal level; body length (um); distance of the vulva from the anterior end (um); (%) distance of the vulva from the anterior end as percentage of body length (%). 0.08 m << FF DNA extraction, sequence processing, phylogenetic inference, and secondary structure predictions Nematodes were picked out from the DESS or ethanol fixed samples under a stereoscopic microscope. Specimens < 3 cm were mounted on temporary slides with sterile distilled water and observed at different magnifications us- ing a light microscope (Olympus BX 53) with differential interference contrast, and equipped with a digital camera. Specimens > 3 cm were observed at differ- ent magnifications using a stereoscopic microscope Nikon SMZ25 equipped with a digital camera. After the vouchering DNA from the middle part of the body (~ 1 cm) was extracted using the Qiagen DNeasy extraction kit according to the protocol. PCR mixture contained 5 ul Go Taq Green Master Mix (Prome- ga Corp., Madison, WI, USA), 0.5 uM of each primer, 3 pl of nuclease-free water (Ambion) and 1 ul of genomic DNA. Fragments of the nuclear ribosomal DNA and internal transcribed spacers (18S rDNA, ITS1, 5.8S rDNA, ITS2 and D2-D3 region of 28S rDNA) were amplified. For 18S rDNA, we used the primer set ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 235 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific SSU_F_03 (f) and SSU_R_81 (r) (Blaxter et al. 1998) which amplifies a fragment of ~ 1800 bp. We used additional primers to sequence 18S rDNA amplicons: SSU_F_24_1 (f) (Meldal et al. 2007) and MN18R (r) (Floyd et al. 2005). The D2- D3 region of the 28S ribosomal DNA region was amplified using the primers D2a (f) and D3b (r) (Nunn 1992). The length of the obtained amplicon was ~ 700 bp. The internal transcribed spacer (ITS, includes ITS1, 5.8S rDNA and ITS2) region was amplified with the primers Vrain2F and Vrain2R (Vrain et al. 1992) which amplifies a fragment of ~ 1200 bp. The length of the obtained amplicon was 700 bp. PCR products were visualized on a 1.5%-TBE agarose gel GelDoc XR+ imaging systems (BioRad). Each PCR fragment was purified using Exonuclease | (Exol) and Thermosensitive Alkaline Phosphatase (Fa- stAP) (Thermo Fisher Scientific Inc., USA). PCR products were bidirectionally cycle sequenced using BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Inc.), and bidirectionally sequenced on an ABI 3130XL automated sequencer using BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Bio- systems, Inc.). Sequences were manually assembled and edited using Finch TV and MEGA 7 (Kumar et al. 2016). Also, MEGA7 was used for calculated inter- and intraspecific p-distances. The 18S and 28S rDNA sequences were checked and aligned at the nucleo- tide level using T-Coffee algorithm (Magis et al. 2014) on a MPI Bioinformatics Toolkit web service (Zimmermann et al. 2018). Bayesian phylogenetic analyses were conducted with MrBayes v. 3.2.7a (Ronquist et al. 2012). For tree recon- struction, we used the obtained sequences, as well as dataset from GenBank belonging to family Leptosomatidae, with lengths longer than 1000 bp for 18S and 650 bp for D2-D3 region 28S rDNA. PartitionFinder 2.1.1 (Lanfear et al. 2012) was used to select the best-fit partitioning scheme and models for each loci using the greedy algorithm with linked branch lengths for the corrected Bayesian Information Criterion as the optimality criterion for model selection. The best models for both ribosomal loci were SYM+I+G. Bayesian Inference was performed with two independent runs of Metropolis-coupled Markov chain Monte Carlo analyses, with each run comprising one cold chain and three heated chains at a default temperature setting of 0.1. The chains were run for 10 million generations and sampled every 100 generations. A burn-in of 2.5 million generations (or 25% of the sampled trees) was used. Moreover, trace files were visually inspected in Tracer 1.7 (Rambaut et al. 2018). We conducted Maximal likelihood (ML) analyses in IQ-Tree v. 2.2.0 (Minh et al. 2020) with 1 million ultra-fast bootstrap replications (Hoang et al. 2018) with model finding (Kalyaanamoorthy et al. 2017) algorithms. FigTree v. 1.4.4 was used to visual- ize phylogenetic trees after analysis. ITS2 boundaries were identified by using hidden Markov models imple- mented in the ITS2 Ribosomal RNA Database (http://its2.bioapps.biozentrum. uni-wuerzburg.de/; Ankenbrand et al. 2015). The common folding pattern of ITS2 molecules for Aborjinia spp. was found by running the multilign and Tur- boFold algorithms on the RNAstructure webserver (http://rna.urmc.rochester. edu/RNAstructureWeb; Reuter and Mathews 2010) using the default parame- ters. We used 4SALE (Seibel et al. 2008) to generate the consensus secondary structure of our dataset after alignment sequence structures in ITS2 Ribosomal RNA Database. CBCAnalyzer (Wolf et al. 2005) was used to detect CBCs and hemi-CBCs (one-sided substitutions before CBCs) from aligned matrix. ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 236 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific Results Taxonomic account Family Leptosomatidae Filipjev, 1916 Diagnosis (Smol et al. 2014; emended). Large nematodes (up to 172 mm). Six inner labial sensilla mostly papilliform, six outer labial and four cephalic sensilla papilliform or setiform. Amphids pocket-shaped. Large number of metanemes with caudal filament usually present: dorsolateral and ventrolateral or only dor- so-lateral orthometanemes and loxometanemes of type |. Many species with ocelli. Buccal cavity narrow, sometimes with tooth-like thickening. Pharynx in- serts into the body cuticle in the region of buccal cavity, the cephalic capsule is variable in the form. Three pharyngeal glands open in the buccal cavity. Pharynx always smooth in outline. Secretory-excretory system, if present, usually restrict- ed to the pharyngeal region, may consists of two cells. Female reproductive sys- tem didelphic-amphidelphic with antidromously reflexed ovaries. Males with two testes opposed. Gonad positions relative to intestine variable in species, with anterior and posterior gonad position reversed. Subventral or ventral precloacal papillae (never tubules) often present. Caudal glands mostly present, extending into the precaudal region. Marine and parasites of marine invertebrates. Aborjinia Ozdikmen, 2010 Diagnosis (emended after Tchesunov and Spiridonov 1985; Miljutin 2003, 2014a). Very large nematodes; at the larval stage parasitize marine inverte- brates. Adult worms are free-living. Three lips. Pharynx cylindrical, muscular, with tri-radial internal lumen. Rectum and anus present. Outer labial and cephal- ic sensilla papilliform, situated in one circle. Amphideal fovea small, pore-like. Cervical setae absent. Excretory-secretory system consists of two cells. Female reproductive system didelphic, amphidelphic, ovaries hologonic. Male reproduc- tive system didelphic, testes outstretched. Tail convex-conoid, broadly rounded. Type species. Aborjinia eulagiscae (Tchesunov & Spiridonov, 1985): Ozdik- men 2010, by original designation [= Australonema eulagiscae Tchesunov & Spiridonov, 1985]. Invalid species. Aborjinia corallicola Westerman, de Moura Neves, Ahmed & Holovachov, 2021. = Paraborjinia corallicola (Westerman, de Moura Neves, Ahmed & Holo- vachov, 2021), comb. nov. Aborjinia profunda sp. nov. https://Zzoobank.org/FA1FD587-B2E2-49BB-996B-57F2D1 5AD74E Figs 2-6 Diagnosis. Body 22.9-27.7 mm long in males. Six outer labial and cephalic sensilla papilliform, situated 19-21 um from anterior end. Amphideal aperture located 37-40 um from anterior end. Pharynx tubular without any valves or bulbs, tightly surrounded by the glandular tissue. Nerve ring situated ~ 40% of ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 237 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific Figure 2. Examined species of Aborjinia, entire bodies, light microscopy A Aborjinia sp., female (assembled panorama) B Terebelus sp. with juvenile of Aborjinia sp. C Aborjinia profunda sp. nov., male. Scale bars: 5000 um. pharynx length from anterior end. Intestine well developed with wide lumen. Spicules slightly bent, 364-372 um long. No pre- or postcloacal sensilla or sup- plements. Spinneret present. ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 238 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific > au (MSL AQ Nw ay poke Sere = See A arc en Rin ae gaat. arm 2s . on Ca a Say . : af Pee LOA a ES _— : pee hte ils tn. ora ofthat ae : tH : Te ' Pe ERE ; ‘ ha Sd ode ee eee ce on ser at tee ng ie es See ee as CER a, Le ers et aaa Ma ee oie Bi pry KenL Ne Oe MER POEM ti eae NS, Ca Se ag Re TRA ee eae PM ehataee a) a rile tend? SLU Oe tea ae es St Ea Pa ee Ser aT es, | cl . Sh SOEs ae > wes a4 oo og sie, a Mel ae tee ee SAS ee 5 OEM stat ay SOS my ty ble Ue okies Bas et Skee Ste ey tee tee Peeks On, EE Arista eli ar a ee Rona I cite eee, ue oiiedty Bese Com eT vgs. S ‘ coke ‘ Pa aS cee at eat ae ea eae) a bagi MD ptatites ce hans So yess weet IMMA M7 ; RG + CUM WAN Sar pat WN EE: 19 GOZO %O POSCst mn ae : Sgeee 20 SS Q00 3/280 aa) cy ANWR eo lee cob? ° e Og 0) \ Figure 3. Aborjinia profunda sp. nov. A anterior end of male B head of male C male reproductive system D male tail. Ab- breviation: c.s — cephalic sensilla. Scale bars: 100 um (B); 200 um (D); 500 um (A); 2000 um (C). Type material examined. Three males (holotype and two paratypes). The ho- lotype (SMF 14457) and paratype (SMF 14458) are deposited in the Sencken- berg Museum, Frankfurt am Main, Germany. Paratype (MIMB 42307) is depos- ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific Tt ace soe east BP pe Pe Pe Figure 4. Aborjinia profunda sp. nov., male. Light microscopy, DIC A-C head D, E cellular bodies of the cervical excretory gland F crystalloid bodies G, H posterior end region I the vesicula seminalis region. Abbreviations: a — amphid, a.t. - an- terior testis, c.s. - cephalic sensillum, c.e.g. — cervical excretory gland, n.r. — nerve ring, p.t. — posterior testis, s. — spin- neret, sp. — spicules, vd — vas deferens. Scale bars: 50 um. ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 240 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific A Figure 5. Aborjinia profunda sp. nov. Light microphotographs of transverse sections A buccal cavity at the upper level of the head (h) B buccal cavity surrounded with pharyngeal glands C pharyngeal region tightly filled with pharyngeal glands bodies D midbody with intestine and gonad E posterior region at the level of distal part of spicules F posterior region close to cloacal opening. Abbreviation: bc — buccal cavity, c - cloaca, cu — cuticle, gc — germinal cells, h — heilostoma, i — intestine, lc — lateral chords, pg — pharyngeal glands, s — spicules, vc — ventral chords. Scale bars: 20 um (A-C); 50 um (E, F); 100 um (D). ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 241 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific Figure 6. Aborjinia profunda sp. nov. SEM A anterior end of male B head of the male C amphideal fovea D posterior end of the male E spicules protruding from cloacal opening F spicules G tail tip with spinneret opening. Scale bars: 2 um (C); 20 ym (B, D, E, F, G); 100 pm (A). ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 242 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific ited in the Zoological Museum of A.V. Zhirmunsky National Scientific Center of Marine Biology, Vladivostok, Russia. Other material examined. One formalin-preserved specimen (male) and two DESS-preserved specimens (males). The Kuril-Kamchatka Trench, water depth 5477 m (45.0°N, 151.1°E), 6114 m depth (45.9°N, 152.8°E) Deposited in the Zoological Museum of A.V. Zhirmunsky National Scientific Center of Marine Biology, Vladivostok, Russia (MIMB 42308). Type locality. The Kuril-Kamchatka Trench, water depth 8235 m (44.7°N, 151.5°E) (Fig. 1, Table 1). Additional locality. The Kuril-Kamchatka Trench, water depth 5477 m (45.0°N, 151.1°E), 6114 m depth (45.9°N, 152.8°E) (Fig. 1, Table 1). Etymology. Species name derived from the Latin profundus that means deepwater and refers to the deepwater habitat of described species. Nucleotide sequences. GenBank accession numbers OP600452.1, OP600453.1 (small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, par- tial sequence); OP407645.1, OP407646.1 (large subunit ribosomal RNA gene, partial sequence). Description. Large nematodes, 22.9-27.7 mm long, with an average diam- eter 0.2-0.4 mm. Body cylindrical, tapering towards both extremities (Figs 2, 3). The cuticle finely striated under SEM, and thin (~ 5 um; Fig. 6). Hypodermis and muscle layers are thin, cords are prominent (Fig. 5). Body pores distinct, irregularly arranged. Measurements tabulated in Table 2. Head narrow, bluntly rounded with three lips. Inner labial sensilla papilliform, hardly visible under light microscope. Papilliform outer labial sensilla and ce- phalic sensilla in one circle, 1-2 um long, situated 19-21 um from anterior end (Fig. 6A, B). Amphideal opening pore-like, located 37-40 um from anteri- or end. Pharynx tubular without any valves or bulbs, tightly surrounded by the Table 2. Morphometrics (um) of Aborjinia profunda sp. nov. and Aborjinia sp. (abbrevia- tions of characters defined in the Materials and methods). Aborjinia profunda sp. nov. Aborjinia sp. HT? 3 3 HT 2 L 27740 22900 25700 172000 Tail length 263 218 193 524 Nerve ring from anterior end 500 513 501 Head diam. at level of cephalic setae 91 83 87 Anal body diam. 289 217 230 467 Maximum body diam. 355 264 290 850 Pharyngeal length 1250 1199 1158 2148 Amphid from anterior end 33 37 43 Spicule length 364 297 335 Renetta cells from anterior end 792 742 725 5053 a 78.1 86.7 88.6 202.3 b 22.2 19.1 22.2. 78.8 Cc 105.5 105 133.2 328.2 C 0.91 1 0.83 1.1 ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 243 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific glandular tissue (Figs 3A, 4). Nerve ring situated ~ 40% of pharynx length from anterior end. Intestine well developed with wide lumen. Ventral gland consists of two cells. Cell bodies ~ 80 um long and 75 um wide (~ 30% of corresponding body diam.), arranged in tandem and situated 725-792 um from anterior end (Fig. 4D, E). Excretory pore not observed. Male reproductive system didelphic, testes outstretched. Spicules paired, symmetrical, slightly bent, 364-372 um long (Figs 3, 4). Gubernaculum not found. No pre- or postcloacal sensilla or supplements. Rectum and anal open- ing functional. Tail conical with terminal spinneret, caudal glands not observed. Remarks. The new species differs from A. eulagiscae by the location of the ventral gland cell bodies (posterior to the nerve ring vs posterior to the cardia), by the smaller body size (23-28 mm vs 103-132 mm), and the shorter tail (193-263 um vs 500-850 um). Aborjinia sp. Figs 7,8 Material examined. Anterior and posterior parts of the DESS fixed female. East- ern slope of the Kuril Islands, water depth 3374 m (Fig. 1, Table 1). GenBank accession numbers. 0P600454.1 (small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence); OP407647.1 (large subunit ribosomal RNA gene, partial sequence). Description. Body opaque, cylindrical, slightly narrowing to both ends, 17.2 cm long (Figs 2, 7). Cuticle smooth under the light microscope. Inner labi- al sensilla papilliform, hardly visible under light microscope. Papilliform outer labial sensilla and cephalic sensilla in one circle (Fig. 8C). Amphideal opening pore-like. Buccal cavity small, narrow. Pharynx tubular without any valves or bulbs, cardia small. Intestinal lumen distinct only in its anterior most part. Se- cretory-excretory system consists of two big cells situated 5053 um from ante- rior end (Fig. 8F). Excretory pore not observed. Reproductive system didelphic, amphidelphic with outstretched ovaries. Uteri large, tubular. Vulva located at midbody, a transverse slit. Tail conico-cylindrical. Anal opening present. Spin- neret very vestigial (Fig. 8D), caudal glands not observed. Molecular analysis. In total, six specimens were sequenced for the 18S rDNA, 28S rDNA (D2-D3 region), and ITS (including ITS1, 5.8S rDNA, and ITS2) genes. Of these, two specimens belong to Leptosomatides Filipjev, 1918, two speci- mens to Aborjinia profunda sp. nov., and one female and one juvenile belong to Aborjinia sp. The sequence length of 18S rDNA was 1694-1716 bp (14 variable sites, 11 parsimony-informative characters), 28S rDNA — 659-671 bp (66 vari- able sites, 59 parsimony-informative characters). The length of ITS for the genus Aborjinia was 1054-1094 bp (61 variable sites, 9 parsimony-informative char- acters) whereas for Leptosomatides sp. length was 1267 bp (3 variable sites). To calculate genetic distances, as well as to reconstruct phylogenetic re- lationships, we used all available sequences of leptosomatids from GenBank (Suppl. materials 2, 3). The BI phylogeny using 18S rRNA reveal Deontosto- ma Filipjev, 1916 as the earliest branching lineage within Leptosomatidae. ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 244 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific 6 b] rt > Ww (starrer, (CC (UCTS ( (( UCC eT ((((( ((((( (ACC ACCC (AC Figure 7. Aborjinia sp. A anterior end of female B head end of female C tail of female. Scale bars: 250 um (B); 500 um (C); 1000 um (A). ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 245 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific Figure 8. Light microscopy, DIC. Aborjinia sp., female A anterior end B head C anterior end with cephalic sensilla D tail end with spinneret E pharynx-intestine connection F cellular bodies of the cervical excretory gland G ovary H vulva region. Abbreviation: c.e.g. — cervical excretory gland, h.s. — cephalic sensilla; i — intestine, o — ovary, o.I.s. — outer labial sensilla, ph — pharynx, v — vulva. Scale bars: 50 um (C); 100 um (D); 500 um (A, B, E, F-H). ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 246 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific However, this genus was shown to be polyphyletic which occupied two of the branches of the polytomous clade. A well supported clade (Bayesian PP BPP = 1; ML bootstrap value percent, ML = 89) united the four samples of Pseudo- cella Filipjev, 1927. Another moderately supported clade uniting Thoracostoma microlobatum and two species of genus Proplatycoma Platonova, 1976 was uncovered (BPP = 0.78). Two species of Platonova Mordukhovich et al., 2019 and Synonchus Cobb, 1894 were placed as monophyletic clade with moderate support (BPP = 0.96; ML = 98), whereas the placement of Cylicolaimus de Man, 1889 was unsupported. Thoracostoma trachygaster Hope, 1967 was sister to obtained sequences and Paraborjinia corallicola (BPP = 0.99, ML = 86) and not monophyletic to Thoracostoma microlobatum and Thoracostoma sp., rendering that genus polyphyletic. A clade containing two samples of Leptosomatides (vouchers L1, L2) and a clade uniting samples of Aborjinia (excluding Parabor- jinia corallicola) and Leptosomatides (HM564626) were high supported (BPP = 1; ML = 94 and BPP = 0.99; ML = 96, respectively). Paraborjinia corallicola was sister to previous clade with high Bayesian support (BPP = 0.99, ML = 72). The phylogenetic relationships using 28S rRNA reveals opposite topology compared with 18S rRNA. Paraborjinia corallicola, the rest Aborjinia species and Leptosomatides (vouchers L1, L2) were the earliest branching lineages but supports of these clades were moderate or low. Genus Thoracostoma was also polyphyletic. Deontostoma was placed in one clade with Thoracostoma microlo- batum (BPP = 0.99, ML = 83). Pseudocella and one out of three Thoracostoma were sister to Platonova (including Synonchus) (BPP = 0.95, ML = 88). The average intergeneric p-distances within Leptosomatidae were 1.96% (0.57%-4.66%) and 13.30% (9.15%-17.12%) for 18S rDNA and 28S rDNA respec- tively if the two non-monophyletic species Thoracostoma trachygaster and Tho- racostoma microlobatum Allgén, 1947 as well as Paraborjinia corallicola and the remaining Aborjinia belongs to different genera. Genus Aborjinia (including se- quences HM564626 and HM564855, excluding Paraborjinia corallicola) differed from other genera of the family by 1.49% and 11.57% in average for 18S rDNA and 28S rDNA, respectively. Same values for genus Leptosomatides were 1.71% and 13.84% and for Paraborjinia corallicola were 3.78% and 16.15%, respectively. The interspecific p-distance for 18S rDNA between Aborjinia profunda sp. nov. and Aborjinia sp. (voucher M10) was 0.24%, for 28S rDNA this value was 1.82%, and for ITS2 5.78% (Table 3). Using the programs RNA structure and ASALE, homologous regions of Aborjinia ITS2 as well as Aborjinia sp. MZ504143 sequences were generally folded as comparable secondary structural motifs. Analyses revealed single secondary structure for all sequences contained four universal helices (Fig. 9). Comparison of sequences across taxa identified sev- eral hemi-compensatory base changes (hemi- CBCs, Table 4, Fig. 9) which in turn belonged to different types of changes (H1-H3). Various comparison pairs of Aborjinia species gave 3-7 hemi-CBCs while no double-sided changes (CBC) were found (Table 4). Remarks. To date only three species (including the present material) were originally described in the genus Aborjinia: Aborjinia corallicola, Aborjinia eulag- iscae, and Aborjinia profunda sp. nov. but Aborjinia corallicola is here transferred to Paraborjinia sp. nov. Both species (A. eulagiscae and A. profunda sp. nov.) are characterized by the outer labial and cephalic sensilla situated in one circle and the presence of two cells of secretory-excretory system. In the description ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 247 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific Table 3. Interspecific p-distances (%) between the obtained sequences. Distances for aot ITS and 28S are above and below the diagonal, respectively (“-“ — data absent). Taxon 1 2 3 4 5 6 7 1. Aborjinia corallicola - - = = = - 2. Aborjinia sp. (specimen M10) 15.85 5.78 4.62 4.34 - 24.11 3. Aborjinia profunda sp. nov. 16.46 1.82 3.47 4.34 23.21 4. Aborjinia sp. (specimen M11) 15.85 1.37 1.37 3.18 24.11 5. Aborjinia sp. (MZ504143) 16:02" | 122% | 1:83: | S122 - 24.11 6. Leptosomatides sp. (HM564855) 15.87 1.25 1.88 AZ ys = 7. Leptosomatides sp. (Specimen L1,L2) | 16.07 9.02 8.72 8.72 9.04 9.15 Table 4. Number of CBCs (above diagonal) and hemi-CBCs (under diagonal) in the ITS2 secondary structure. Aborjinia sp. Aborjinia sp. Aborjinia_sp. Faxon (specimen M10) | AD™inia profunda sp. nov. | (| inen M11) | (MZ504143.1) Aborjinia sp. (specimen M10) 0 0 0 Aborjinia profunda sp. nov. 6 0 0 Aborjinia sp. (specimen M11) 5 3 0 Aborjinia sp. (MZ504143) 5 7 4 u °° or G e®°e, vu cat e 6 Torc AorG ® “cre0e er as é 2 Tl 3, — fe e oe, Py “0° = ? x i ‘a ‘ @ “H1: UG-CG %, 00, : : mn ood gf Coru OH2: GC-GU $ pannnneey 8 2 %eecce =o @ OH3: UG-UA eo Cort e e ovece coe” ; e “cog?” “ee Phe A © r ° ’ r Coeceecse®” or U ll ® o GorU ) e 6 CorT ® CorT AorG ®o,0° | Figure 9. Consensus ITS2 secondary structure and the hemi compensatory base change (hemi-CBCs) derived from Aborjinia species. The four stems are labelled. Each type of base change (H1-H3) indicates by unique color. CU and GU change that relates to H1 or H2 depending on comparison pair. of P corallicola provided by Westerman et al. (2021) it is mentioned that outer labial sensillae and cephalic sensilla are situated in one circle. However, on the photograph provided in that paper these sensilla are situated in two sepa- rate circles. In addition, in P corallicola the secretory-excretory system was not found, contrasting with the two giant and clearly visible cells in other Aborjinia. It should also be noted that, unlike other representatives of the genus, P cor- allicola is parasitic as an adult. Based on above we assume that P corallicola belongs to another genus. Our conclusion is also supported by the molecular phylogenetic tree (Fig. 10), values of intergeneric p-distances within Leptoso- matidae, and interspecies p-distances within Aborjinia (Table 3). We propose the new genus Paraborjinia gen. nov. for A. corallicola. ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 248 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific 100 100 Enoplus sp. DBA21 HM564551 HM564759 Phanodermatidae gen. sp. K2 MZ474687 MZ476005 Thoracostoma trachygaster 2S20H7 FN433905 FN433906 Thoracostoma sp. C-Tsp MZ504146 Deontostoma sp. 1S2F8 FN433899 FN433915 , 87 1 Pseudocella sp. 2S22E8 FN433900 FN433909 91 4| 100 Pseudocella sp. 4S26E8 FN433902 FN433912 98), Pseudocella sp. 3S26E8 FN433901 FN433910 1 100 Pseudocella trichodes C-Pt MZ504145 97 a Platonova magna SB57 MK007570 MK007567 0.85 Platonova magna SB56 MK007571 MK007568 4 Platonova magna KB20 MK007572 MK007569 1 100 Synonchus sp. TCR206 HM564630 HM564859 4} Platonova verecunda KB13 MK007573 MK007567 99} Platonova verecunda KB14 MK007574 MK007568 Platonova verecunda KB1 MK007575 MK007569 4, Leptosomatides sp. L1 OP600456 OP407649 100 Leptosomatides sp. L2 OP600457 OP407650 Leptosomatides sp. TCR192 HM564626 HM564855 0.89 5 Aborjinia sp. KKT MZ504143 #3 Bs Aborjinia sp. M10 OP600454 OP407647 1} 1, Aborjinia sp. M11 OP600455 OP407648 88 2 A Aborjinia profunda sp. nov. M7 OP600452 OP407645 95 4 yh Aborjinia profunda sp. nov. M8 OP600453 OP407646 100 1 Paraborjinia corallicola 100 MW916764-MW916782 MW916745-MW916763 0.02 Figure 10. Bayesian phylogeny of the family Leptosomatidae, using concatenated 18S and 28S rDNA and SYM+ G model of nucleotide substitution. Enoplus sp. (Enoplidae) and Phanodermatidae gen. sp. were used as outgroup to root tree. Bayesian posterior probabilities (PP) are given above tree nodes and bootstrap support values found in the ML analysis are shown below nodes. Specimens obtained in this study are in bold. ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 Paraborjinia gen. nov. https://zoobank.org/5662ABEE-8888-4C29-ACF2-6EDDB2A63F0B Type species. Paraborjinia corallicola (Westerman, de Moura Neves, Ahmed & Holovachov, 2021). Type locality: Atlantic Ocean, Labrador Shelf (60.6083°N, 61.7428°W), 426 m depth. Type host: Acanella arbuscula. Diagnosis. Parasitic life style. Distinct body pores along the body. Outer labi- al and cephalic sensilla papilliform, situated in two circles. Amphideal aperture pore-like. Muscular and uniformly cylindrical pharynx. Intestine not modified into trophosome. Hologonic ovaries in females. Presence of caudal glands. Differential diagnosis. Within the family Leptosomatidae the new genus dif- fers from all genera except Aborjinia by having and endoparasitic lifestyle and hologonic ovaries. Paraborjinia gen. nov. differs from Aborjinia by the position 249 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific of cephalic sensitive organs (outer labial and cephalic papilla in two separate circles in Paraborjinia vs outer labial and cephalic papilla in one circle in Abor- jinia). Paraborjinia gen. nov. differs from Aborjinia, Ananus, and Thalassonema by the parasitic adult (vs free-living in Aborjinia, Ananus, and Thalassonema). Paraborjinia gen. nov. differs from Ananus by the presence of rectum and anus. In addition, in all described species of Aborjinia and Ananus the secretory-ex- cretory system is well developed and consists of two prominent cells while in Paraborjinia the secretory-excretory system was not found. Discussion These results of the phylogenetic analyses are only preliminary due to the low number of sequences available. The different sets of species and genera for con- structing the SSU and LSU phylogenetic trees, as well as the small number of se- quences relative to the total number of species affect the different topologies. It is premature to make solid conclusions about the relationships of genera within Leptosomatidae based on the available data; however, concatenated 18S and 28S rDNA phylogenetic tree showed relatively high support values (Fig. 10). Recent studies have shown that the genus Aborjinia belongs to the family Leptosomatidae based on both molecular and morphological characters (Tchesunov et al. 2022) and our SSU and D2-D3 of LSU phylogenetic trees confirm the previous analyses. The males of Aborjinia profunda sp. nov. and female Aborjinia sp. (specimen M10) have pronounced morphological differences and p-distances (28S and ITS). Moreover, for all known Aborjinia isolates, differences in the nucleotide sequenc- es of LSU and ITS are observed (Table 4). The presence of sexual dimorphism is known for nematodes, including leptosomatids, and the values of p-distanc- es are relatively small. In addition to the commonly used phylogenetic analysis and genetic distances, we used Compensatory Base Changes (CBCs) in Internal Transcriber Spacer 2 (ITS2) for species delimitation. ITS2 is useful locus for cal- culation of lower-level phylogenetic trees in many eukaryotic lineages (Young and Coleman 2004; Ahvenniemi et al. 2009) to predict the ability to interbreed success- fully between putative biological species. Organisms that differ by even one CBC in the conserved ITS2 regions (helices 2 and 3) are unable to interbreed (Coleman 2009). At the same time, changes in single stranded region (hemi-CBC) do not contribute to the appearance of CBCs (Caisova et al. 2011) and lead to failure in sexual reproduction (Coleman 2009). Based on this evidence, when CBCs occur among species, Wolf et al. (2013) developed a generalized ‘CBC species concept’. Double-sided changes (CBC) were not found; therefore, there are no strict reasons for classifying the studied individuals as different species, but resolving the issue of the species status of some individuals requires further research. Our results indicate a rather wide distribution of representatives of the ge- nus Aborjinia in the deep-sea communities of the northwestern Pacific, includ- ing depths of more than 8000 m. Molecular and morphological (in particular, the two-celled renette, the presence of a spinerette, minute sensory sensilla, normal muscular pharynx) data support the assignment of Aborjinia to Lepto- somatidae. Analysis of molecular data confirms the independence of the gen- era Aborjinia and Paraborjinia and demonstrates clearly supported differences from Leptosomatides. We agree with Tchesunov et al. (2022) that morphologi- cal uniformity can lead to misidentification of Aborjinia specimens, especially if ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 250 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific fixed in ethanol. This may lead to an underestimation of the frequency of occur- rence, abundance, and diversity of both representatives of the genus Aborjinia and parasitic leptosomatids in general. Acknowledgements The scanning electron microscopy investigations were done in the A.V. Zhir- munsky National Scientific Centre of Marine Biology FEB RAS. The authors are grateful to D.V. Fomin for assistance with the SEM facilities. The light micros- copy investigations were done in the Laboratory of Ecology and Evolutionary Biology of Aquatic Organisms of the Far Eastern Federal University. We are very grateful to Prof. Dr. A. Brandt (Senckenberg Research Institute and Natural History Museum, and Goethe University Frankfurt, Frankfurt) and to Dr. M.V. Malyutina (NSCMB FEB RAS) for invitation to join international projects and the deep-sea expeditions. We thank the crews of the RVs ‘Sonne’, ‘Akade- mik M.A. Lavrentyev’, ‘Akademik Oparin’ and the scientific teams of the expedi- tions for their assistance on board. The authors are also grateful to Dr. Inna Alalykina (NSCMB FEB RAS) for help with taxonomic identifications of Polychaeta and Dr. Alexey Chernyshev, who found a sample of Aborjinia in the bottom sediments from the AGT. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding We thank the Ministry of Science and Higher Education of the Russian Federation for funding this study (grant 13.1902.21.0012, contract No. 075-15-2020-796). Author contributions All authors have contributed equally. 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Copyright notice: This dataset is 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 users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1189.111825.suppl1 Supplementary material 2 Bayesian 18S rDNA phylogeny of the family Leptosomatidae, using the SYM+I+G model of nucleotide substitution Authors: Julia K. Zograf, Alexander A. Semenchenko, Vladimir V. Mordukhovich Data type: pdf Explanation note: Enoplus taipingensis (Enoplidae) and Phanoderma sp. (Phanoderma- tidae) were used as outgroups to root the tree. Bayesian posterior probabilities (PP) are given above tree nodes and bootstrap support values found in the ML analysis are shown below nodes. Copyright notice: This dataset is 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 users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1189.111825.suppl2 ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 255 Julia K. Zograf et al.: Aborjinia from the North-Western Pacific Supplementary material 3 Bayesian 28S rDNA phylogeny of the family Leptosomatidae, using the SYM+I+G model of nucleotide substitution Authors: Julia K. Zograf, Alexander A. Semenchenko, Vladimir V. Mordukhovich Data type: pdf Explanation note: Enoplus sp. (Enoplidae) and Phanoderma sp. (Phanodermatidae) were used as outgroups to root the tree. Specimens obtained in this study are indicated in bold. Copyright notice: This dataset is 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 users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1189.111825.suppl3 ZooKeys 1189: 231-256 (2024), DOI: 10.3897/zookeys.1189.111825 256