New deep-sea species of Aborjinia (Nematoda, Leptosomatidae) from the North-Western Pacific: an integrative taxonomy and phylogeny Author Zograf, Julia K. A. V. Zhirmunsky National Scientific Center of Marine Biology, FEB RAS, 690041, Vladivostok, Russia zojulia@yandex.ru Author Semenchenko, Alexander A. Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS, 690022, Vladivostok, Russia Author Mordukhovich, Vladimir V. A. V. Zhirmunsky National Scientific Center of Marine Biology, FEB RAS, 690041, Vladivostok, Russia & Far Eastern Federal University, 690922, Vladivostok, Russia text ZooKeys 2024 2024-01-17 1189 231 256 http://dx.doi.org/10.3897/zookeys.1189.111825 journal article http://dx.doi.org/10.3897/zookeys.1189.111825 1313-2970-1189-231 FCBFC6313C494B98BD021C63654837FE B3209808DF775FA99E2105DDC703973E Aborjinia sp. Figs 7 , 8 Material examined. Anterior and posterior parts of the DESS fixed female. Eastern slope of the Kuril Islands, water depth 3374 m (Fig. 1 , Table 1 ). GenBank accession numbers. OP600454.1 (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 labial 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. Secretory-excretory system consists of two big cells situated 5053 µm from anterior 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. Spinneret very vestigial (Fig. 8D ), caudal glands not observed. Figure 7. Aborjinia sp. A anterior end of female B head end of female C tail of female. Scale bars: 250 µm ( B ); 500 µm ( C ); 1000 µm ( A ). 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.l.s. - outer labial sensilla, ph - pharynx, v - vulva. Scale bars: 50 μm ( C ); 100 μm ( D ); 500 µm ( A, B, E, F-H ). 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 specimens 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 variable sites, 59 parsimony-informative characters). The length of ITS for the genus Aborjinia was 1054-1094 bp (61 variable sites, 9 parsimony-informative characters) whereas for Leptosomatides sp. length was 1267 bp (3 variable sites). To calculate genetic distances, as well as to reconstruct phylogenetic relationships, we used all available sequences of leptosomatids from GenBank (Suppl. materials 2, 3). The BI phylogeny using 18S rRNA reveal Deontostoma Filipjev, 1916 as the earliest branching lineage within Leptosomatidae . 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 Pseudocella 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 Paraborjinia 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 microlobatum (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 respectively if the two non-monophyletic species Thoracostoma trachygaster and Thoracostoma microlobatum Allgen , 1947 as well as Paraborjinia corallicola and the remaining Aborjinia belongs to different genera. Genus Aborjinia (including sequences 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 4SALE, 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 several 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 ). 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. Table 3. Interspecific p-distances (%) between the obtained sequences. Distances for 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 M 10)	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 M 11)	15.85	1.37	1.37	-	3.18	-	24.11
5. Aborjinia sp. (MZ504143)	16.02	1.22	1.83	1.22	-	-	24.11
6. Leptosomatides sp. (HM564855)	15.87	1.25	1.88	1.72	1.57	-	-
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.

Taxon	Aborjinia sp. (specimen M 10)	Aborjinia profunda sp. nov.	Aborjinia sp. (specimen M 11)	Aborjinia _sp. (MZ504143.1)
Aborjinia sp. (specimen M 10)	-	0	0	0
Aborjinia profunda sp. nov.	6	-	0	0
Aborjinia sp. (specimen M 11)	5	3	-	0
Aborjinia sp. (MZ504143)	5	7	4	-

Remarks. To date only three species (including the present material) were originally described in the genus Aborjinia : Aborjinia corallicola , Aborjinia eulagiscae , 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 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 separate 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. corallicola 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 Leptosomatidae , and interspecies p-distances within Aborjinia (Table 3 ). We propose the new genus Paraborjinia gen. nov. for A. corallicola . 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.