A huge undescribed diversity of the subgenus Hystricochaetonotus (Gastrotricha, Chaetonotidae, Chaetonotus) in Central Europe Križanová, Františka Rataj Vďačný, Peter European Journal of Taxonomy 2022 2022-10-11 840 1 93 5876482e-a336-4a3f-b130-d4c53ea6f317 Križanová & Vďačný, 2022 Križanová & Vďačný 2022 [472,1018,818,845] Chaetonotidae Chaetonotus Animalia Chaetonotida 26 27 Gastrotricha species superbus sp. nov. Hystricochaetonotus   urn:lsid:zoobank.org:act: 781619BA-32C2-4336-A729-2BDA3E13C7D4  Figs 15‒17; Supp. file 1: Table S3    Morphological diagnosis Body stocky and about 107 µm long. Head wider than neck, separated from trunk by an inconspicuous neck constriction. Cephalion, epipleurae, and hypopleurae clearly demarcated. Trunk gradually widens from ca U37 to U60 and then gradually tapers towards furca base at U80. Mouth ventral, with delicate protruding structures, no cuticular teeth. Pharynx without dilatations. Intestine straight, with a marked anterior section. Scales spined, three-lobed, not overlapping, distributed in 10–12 columns, 15 scales per column. Spines with a short lateral denticle. Scales and spines increase gradually in size in a posterior direction. Dorsal surface covered by scales from posterior end of cephalion (ca U3) to furca base (ca U93). Furca base short, lateral margins of furca branches more or less straight, furcal indentation deeply U-shaped, adhesive tubes well-developed, almost parallel. Furca base and branches covered with oval, keeled scales.   Molecular diagnosis 18S rRNA gene: 341 C. ITS2: 26 T, 28 G, 40 A, 41 C, 50 T, 62 G, 78 C, 87 A, 96 C, 107 A, 108 A, 112 ‒, 116 G, 117 A, 118 A, 131 T, 139 T, 157 C, 167 T, 168 T. 28S rRNA gene: 317 A, 690 T, 797 T, 902 G. Cytochrome coxidase subunit I (codon ordinal numbers are followed by the corresponding span of nucleotide positions in parentheses): 29 (85‒87) AGT, 44 (130‒132) GTA, 71 (211‒213) CCA, 75 (223‒225) GGT, 81 (241‒243) TTT, 97 (289‒291) TCT, 124 (370‒372) GCT, 126 (376‒378) GCC, 139 (415‒417) CTA, 142 (424‒426) GCC, 155 (463‒465) ACT, 170 (508‒510) CTG, 189 (565‒567) TTC, 196 (586‒588) CTG, 221 (661‒663) CTG. Reference molecules are shown in Supp. file 1: Figs S1, S 9B, S 13. All diagnostic molecular autapomorphies are marked by arrows. Reference alignments with corresponding nucleotide positions are in Supp. file 1: Alignments 1‒4. The p-distance from species described in the present study is 0.22‒4.00% in 18S, 18.19‒34.76% in ITS2, 2.23‒6.70% in 28S, and 9.04‒13.44% in COI. There are 2‒13 CBCs (except for Ch.( H).  mirabilis sp. nov., where there are no CBCs) in the 18S rRNA molecule, 3‒4 CBCs in the ITS2 molecule, and 1‒11 CBCs in the first two domains of the 28S rRNA molecule.    Etymology The Latin adjective ‘ superb· us, - a, - um’ [m, f, n] (‘proud’) refers to the ‘beautiful appearance’ of the new species.    Material examined    Holotype SLOVAKIA• adult(photomicrographs, hologenophore); Zlaté Piesky lake, municipal recreation area, Ružinov, Bratislava, Podunajskárovina plain; 48°11′17.1″ N, 17°11′24.3″ E; CU-FNS- 22-06-20/PA. Photomicrographs of the holotypeare available at the Department of Zoology, Comenius University in Bratislava at https://fns.uniba.sk/en/gastrotricha/. The holotypeis shown in Figs 16A, 17C–G.    Paratype SLOVAKIA• adult(photomicrographs, hologenophore); Kuchajda lake, municipal recreation area, Nové Mesto, Bratislava, Podunajskárovina plain (type locality); 48°10′15.7″ N, 17°08′30.6″ E; CU- FNS- 15-06-20/HO. Photomicrographs of the paratypespecimen are available at the Department of Zoology, Comenius University in Bratislava at https://fns.uniba.sk/en/gastrotricha/. The paratypeis shown in Figs 16B, 17A–B.   Type material A DNA sample of the holotypespecimen ZPvs 55 has been deposited in the Natural History Museum, Vajanského nábrežie 2, 810 06 Bratislava, Slovakia(ID Collection Code 01427126).   Type locality Zlaté Piesky lake, Ružinov, Bratislava, Podunajská rovina plain, Slovakia, 48°11′17.1″ N, 17°11′24.3″ E.   Gene sequences The nuclear 18S and ITS1-5.8S-ITS2-28S rDNA sequences as well as the mitochondrial COI sequence of the holotypespecimen ZPvs 55 have been deposited in GenBank under the following accession numbers:  OM421708,  OM421684, and  OM424063, respectively.    Description HABITUS.  Chaetonotus( Hystricochaetonotus) superbus sp. nov.is about 107 µm long and has a stocky body that is tenpin-shaped, with a clearly defined head region, a narrowing neck, and a rather bulbous trunk ( Figs 15A, H, 16A). Body height in lateral view is 14.0–15.0 µm at U10, 15.0–15.4 µm at U50, and 16.1–17.0 µm at U60. The head is relatively wide, with a plate-like, rounded cephalion. The neck (ca U12–U27) smoothly continues to the trunk, which is significantly wider than the head, gradually dilates from about U37 to U60 where it reaches the maximum width. Then it gradually tapers towards U80 where curved margins of the furca branches begin to emerge. Dorsal sensory bristles arise from the cuticle in two pairs at U12 and U79 ( Fig. 19A). The furcal indentation is deeply U-shaped. The furca branches are set apart and diverge posteriorly. Well-developed adhesive tubes are approximately 9 µm long, more or less straight, and run almost in parallel ( Figs 15A, H, J, 16A). HEAD. The head is five-lobed. The cephalion (U1–U2) is rounded, clearly demarcated in the body outline, appears as a lens in the ventral view, and has a free posterior (dorsal) edge ( Figs 15A, H, J–K, 16B, 17C). The epipleurae are formed approximately at U3–U5 while the hypopleurae at ca U6–U13. The latter structures are well recognizable in the head outline ( Figs 15A–B, 16A, black stars). Two pairs of cephalic ciliary tufts emerge laterally between the cephalion and the epipleurae edge (ca U3) as well as between the epi- and the hypopleurae edge (U6) ( Figs 15A, H, 16A). The hypostomium (ca U4–U6) is free of structures, lined only with ciliary patches. The mouth ring is oval, 4.4–4.9 µm in the largest diameter, located subterminally at U3–U6. There are strong, rod-like reinforcements lining the mouth walls and delicate structures protruding from the mouth ring ( Figs 15H, K, 17C). Inner cuticular teeth are not present.   Fig. 15.  Chaetonotus( Hystricochaetonotus) superbus sp. nov. A, H. Dorsal and lateral overviews showing the scale and spine pattern. B. Head scale. C. Neck scale. D. Trunk scale. E. Ventral scales. F. Upperfurcal dorsal scales. G. Lateral view of a trunk spined scale. I. Lateral view showing the connection of a transversal band to the base of a scale. J. Lateral overview showing the internal morphology. K. Lateral view of the anterior body region. Scale bars = 30 µm.   Fig. 16.  Chaetonotus( Hystricochaetonotus) superbus sp. nov., differential interference contrast. A. Holotype (CU-FNS-15-06-20/HO), lateral overview showing the scale and spine pattern. B. Paratype (CU-FNS-22-06-20/PA), optical section showing the internal morphology in lateral view. Black stars in (A) mark the epipleurae and hypopleurae. White star in (B) marks the cerebral ganglion. Arrows mark the lateral denticle of spines. Scale bars = 30 µm.   Fig. 17.  Chaetonotus( Hystricochaetonotus) superbus sp. nov., differential interference contrast. A–B. Paratype (CU-FNS-22-06-20/PA). C–G. Holotype (CU-FNS-15-06-20/HO). A. Detail showing the salivary gland, pharyngeal-intestinal junction, and differentiated anterior section of the intestine. B. Detail showing the connection of transversal bands to the base of scales. C. Detail showing the anterior portion of the digestive tract. D. Head scales. E. Neck scales. F. Spines. G. Trunk spined scales. Scale bars: A–B, D‒G = 5 µm; C = 10 µm. INTERNAL MORPHOLOGY. The pharynx extends from ca U5 to U34, is 28–32 µm long and 5.9–8.3 µm wide, sinuous, and has no dilatations ( Figs 15J, 16B, 17C). The cerebral ganglion appears as a mass surrounding the pharynx along its whole length ( Fig. 15K). The salivary glands are recognizable as small hyaline balls situated at the dorsal side of the pharynx around at U33 ( Figs 15J, 16B, 17A). The intestine runs from U35 to U82 and has a separate, well-differentiated anterior section (U35–U38) ( Figs 15J, 16B). Transversal bands connected to the base of dorsal scales are well recognizable under DIC and secondary magnification of 2500 × ( Figs 15J, 16B, 17A–B). The adhesive gland (ca U85–U91) is placed right behind the terminal part of the intestine, forming a short dichotomy at the subtle furca base. SCALES. Almost the entire body is covered by not overlapping three-lobed scales that adhere to the basal cuticle layer along their whole perimeter. Scales are distributed in a minimum of 12 longitudinal rows, with 10 scales in the central row. Their size increases gradually in a posterior direction. Central dorsal and dorsolateral longitudinal rows of scales begin at the level of the anterior edge of the epipleurae (ca U5), while lateral rows start at the posterior end of the hypopleurae (ca U13). Ventral rows are hardly visible due to the thick, elongated dorsal spines (for further explanation, see below) and long locomotory cilia. Three main typesof scales could be recognized with respect to the shape of the anterior lobe of scales: (i) head and upper-neck scales (U5–U28) with a small, rounded anterior lobe and elongated posterior lobes, α= 160–170°, and β= 89–98° ( Figs 15B, 17E), (ii) lower-neck and anterior trunk scales (U29–42) with a truncated anterior lobe and elongated posterior lobes, α= 160–175°, and β= 76–93° ( Figs 15C, 17D), and (iii) posterior trunk scales (U45–80) with a more broadly rounded anterior lobe and a larger angle β(101‒110°) formed by the posterior lobes ( Figs 15D, 17G). The transition between anterior and posterior lobes is indistinct and continuous in all scales. SPINES. All spines bear a distinct lateral denticle and gradually narrow towards their distal end. Keels start near the anterior margin of scales. Spines are not straight but distinctly curved ( Figs 15A, G‒J, 16A–B, 17D‒G). They do not differentiate into various types, only their length changes in a posterior direction (Supp. file 1: Table S3). The most pronounced change occurs at the beginning of the trunk ( Figs 15H, 16A), where dorsal spines increase significantly from 6.7–10.7 µm to 10.8–13.0 µm. The lateral denticle is comparatively distant from the spine apex, i.e., d-value ranges from 1.7‒5.1 μm, which corresponds to a dratio of 21.4‒34.7%. VENTRAL CILIARY BANDS AND VENTRAL INTERCILIARY FIELD. Unfortunately, the ventral side could not be observed in detail due to the thickness of the dorsal spines that precluded turning over and squeezing the worms. Moreover, the ventral locomotory cilia also hampered detailed observations of the ventral side. Despite these problems, the following observations could be done. The longitudinal ciliary bands begin at ca U10 and run backward to ca U85. The ciliary bands are accompanied by two ventrolateral rows of small (2.0–2.9 ×1.9–2.2 µm in size), three-lobed scales that start at U13. The interciliary field bears small (1.7–2.6 ×0.9–1.2 µm in size), oval scales without spines or keels ( Fig. 15E). The upper furcal region (U84–U90) carries two typesof scales: (i) lateral three-lobed, spined scales with a broadly rounded anterior lobe, narrowly rounded posterior lobes, α= ~140°, and β= ~73° and (ii) oblong and keeled scales being 0.84–1.2×1.47–1.72 µm in size ( Fig. 15F). 3926158309 Slovakia Podunajska 48.188084 Zlate Piesky lake 1 17.190083 Bratislava 27 28 1 holotype 3926158302 Slovakia Podunajska 48.17103 Kuchajda lake 1 17.141832 Bratislava 27 28 1 paratype