A huge undescribed diversity of the subgenus Hystricochaetonotus (Gastrotricha, Chaetonotidae, Chaetonotus) in Central Europe Author Križanová, Františka Rataj 80CC56E1-1046-4748-97B6-5FA90E95DFA7 Department of Zoology, Comenius University in Bratislava, 842 15 Bratislava, Slovakia. krizanova66@uniba.sk Author Vďačný, Peter 47A28E80-E04F-40C4-93A3-F7F685C9533A Department of Zoology, Comenius University in Bratislava, 842 15 Bratislava, Slovakia. peter.vdacny@uniba.sk text European Journal of Taxonomy 2022 2022-10-11 840 1 93 http://dx.doi.org/10.5852/ejt.2022.840.1941 journal article 168313 10.5852/ejt.2022.840.1941 6129e7b4-3989-478f-8494-65e0fda5194f 2118-9773 7195216 CE89365D-A3C5-483D-9C80-E5CAECCA740F Chaetonotus ( Hystricochaetonotus ) gulosus sp. nov. urn:lsid:zoobank.org:act: 5B6AE48A-9F1F-4F7A-8054-A01F7AB19E9B Figs 33‒36 ; Supp. file 1: Table S8 Morphological diagnosis Body stocky and around 107 µm long. Head wider than neck, separated from trunk by a distinct neck constriction. Cephalion and epipleurae clearly demarcated, hypopleurae only inconspicuously marked. Trunk widest at ca U50, gradually tapers towards furca base (U80). Mouth ventral, no cuticular teeth. Hypostomium bears two parallel cuticular lamellae. Pharynx without dilatations. Intestine straight, with a marked anterior section. Scales spined, three-lobed, not overlapping, distributed in 14 columns, 12 scales per column. Scales and spines increase gradually in size in a posterior direction. Dorsal surface covered with: (i) small head and anterior neck scales with a small, broadly rounded anterior lobe, equally long posterior lobes, and marked transition between anterior and posterior lobes; (ii) small posterior neck scales with a slightly elongated anterior lobe and an indistinct transition between anterior and posterior lobes; and (iii) comparatively big and triangular trunk scales. Interciliary field covered by (i) small, oval to oblong scales without keels; (ii) a pair of small, oval scales with a keel; (iii) a single distinctly bigger, oblong, and keeled scale; and (iv) a pair of big, very narrowly ovate, and spined scales. Furca branches about as long as adhesive tubes, with lateral margins more or less straight, furcal indentation shallowly U-shaped, adhesive tubes well-developed. Ventral side of furca branches covered with minute, oval to oblong, and keeled scales. Molecular diagnosis 18S rRNA gene: 1378 A. ITS2: 78 T, 128 T, 155 ‒, 172 A, 174 C, 186 ‒. 28S rRNA gene: 503 C. Cytochrome c oxidase subunit I (codon ordinal numbers are followed by the corresponding span of nucleotide positions in parentheses): 20 (58‒60) TTA, 45 (133‒135) GTG, 71 (211‒213) CCG, 74 (220‒222) ATC, 94 (280‒282) CCA, 143 (427‒429) TCA, 151 (451‒453) TTC, 157 (469‒471) AAT, 168 (502‒504) CTC, 178 (532‒534) ACT, 180 (538‒540) GTT, 204 (610‒612) ACA, 209 (625‒627) CCA, 214 (640‒642) GAT. Reference molecules are shown in Supp. file 1: Figs S6 , S 11B, S 18 . 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 0.33‒4.22% in 18S, 6.95‒29.95% in ITS2, 1.46‒8.84% in 28S, and 8.57‒13.14% in COI. There are 1‒15 CBCs in the 18S rRNA molecule, 1‒4 CBCs in the ITS2 molecule (except for Ch. ( H. ) arcanus sp. nov. , Ch. ( H. ) luxus sp. nov. , and Ch. ( H. ) slavicus sp. nov. , where there are no CBCs), and 2‒17 CBCs in the first two domains of the 28S rRNA molecule. Etymology The Latin adjective ‘ gulos · us , - a , - um ’ [m, f, n] (‘gluttonous’) refers to the stocky appearance of the new species. Material examined Holotype SLOVAKIA • adult (the specimen was destroyed during DNA extraction); Vajspeterský potok creek, Rača , Bratislava , Podunajská rovina plain; 48°12′12.8″ N , 17°07′46.9″ E . Paratype SLOVAKIA • adult (photomicrographs); same collection data as for holotype; CU-FNS-28-10-19/PA . Photomicrographs of the paratype specimen are available at the Department of Zoology, Comenius University in Bratislava at https://fns.uniba.sk/en/gastrotricha/. The paratype is shown in Figs 35–36 . Type material A DNA sample of the holotype specimen VP 18 has been deposited in the Natural History Museum, Vajanského nábrežie 2, 810 06 Bratislava, Slovakia (ID Collection Code 01427574). Type locality Vajspeterský potok creek, Rača, Bratislava, Podunajská rovina plain, Slovakia , 48°12′12.8″ N , 17°07′46.9″ E . Gene sequences The nuclear 18S and ITS 1-5.8S-ITS2-28S rDNA sequences as well as the mitochondrial COI sequence of the holotype specimen VP 18 have been deposited in GenBank under the following accession numbers: OM 421721 , OM 421697 , and OM 424076 , respectively. Description HABITUS. Chaetonotus ( Hystricochaetonotus ) gulosus 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 ( Fig. 33A–B ). Body width is ca 18 µm at U10, ca 26 µm at U50, and ca 29 µm at U60. The head is relatively wide, with a plate-like, slightly narrower cephalion. The neck (ca U13–U27) smoothly continues to the trunk, which is distinctly wider than the head, gradually dilates from about U37 to U50 where it reaches the maximum width. Then it gradually tapers towards U80 where the curved margins of the furca branches begin to emerge. Dorsal sensory bristles arise from the cuticle in two pairs at U23 and U60 ( Fig. 33A ). 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 and more or less straight ( Figs 33A–B , 35C , 36C ). HEAD. The head is roughly five-lobed. The cephalion (U1–U2) is rounded, clearly demarcated in the body outline ( Figs 33A–B , 34A ). The epipleurae are formed approximately at U3–U6, the hypopleurae are only inconspicuously marked. Two pairs of cephalic ciliary tufts emerge laterally between the cephalion and the anterior edge of the epipleurae (ca U3) as well as close to the posterior edge of the epipleurae (U6). The mouth ring is oval, ca 4.6 µm in the largest diameter, located subterminally at U2–U4. There are strong, rod-like reinforcements lining the walls of the mouth ring and inner delicate structures directed towards the center of the mouth ring ( Figs 33B , 35B ). Inner cuticular teeth are not present. The hypostomium (ca U6–U9) is in a form of two parallel horizontal cuticular lamellae laterally lined with a few ciliary patches ( Fig. 33B ). Each field is composed of several irregular groups of basal bodies ( Figs 33B , 35B ). INTERNAL MORPHOLOGY. The pharynx extends from ca U5 to U27, is about 28 µm long and 6.5–8.9 µm wide, sinuous, and has no dilatations. The cerebral ganglion appears as a mass surrounding the pharynx along its whole length. The intestine runs from U27 to U81 and has a separate, well-differentiated anterior section (U28–U31). A pair of protonephridia runs from ca U32 to U40 ( Fig. 35C ). Transversal bands connected to the base of dorsal scales are well recognizable. 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 ( Fig. 35C ). Fig. 33. Chaetonotus ( Hystricochaetonotus ) gulosus sp. nov. A . Dorsal overview showing the scale and spine pattern. B . Ventral overview showing the scale and ciliary pattern. Scale bar = 30 µm. Fig. 34. Chaetonotus ( Hystricochaetonotus ) gulosus sp. nov. , schematic drawings of scales. A . Head scale. B . Neck scale. C . Dorsal trunk scales. D–E . Dorsolateral and lateral trunk scales. F . Ventrolateral trunk scales. G . Posteriormost ventral trunk scales. H . Scales of the ventral interciliary field. I . Ventral furcal scales. Fig. 35. Chaetonotus ( Hystricochaetonotus ) gulosus sp. nov. , paratype (CU-FNS-28-10-19/PA), differential interference contrast. A–B . Details of the anterior body region. C . Optical section of the trunk and furca regions. D . Scale and spine pattern of the dorsal trunk region. Scale bars = 15 µm. Fig. 36. Chaetonotus ( Hystricochaetonotus ) gulosus sp. nov. , paratype (CU-FNS-28-10-19/PA), differential interference contrast. A . Scales of the ventral interciliary field. B . Dorsal trunk scales. C . Detail of the posterior body region. D . Posterior ventral keeled and spined scales. Scale bars: A, D = 5 µm; B–C = 10 µm. 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 14 longitudinal rows, with 13 scales in the central dorsal row. Their size increases in a posterior direction. Central dorsal and dorsolateral longitudinal rows of scales begin at ca U5 (behind the posterior edge of the cephalion), while lateral rows start at the posterior end of the epipleurae (ca U13). Ventral rows start at U10 and exhibit a horizontal zonation pattern. Four main types of scales could be recognized with respect to the shape of the anterior lobe. (i) The head and anterior neck scales (U5–U28) are 3.1–3.5 ×2.0–2.3 µm in size, have a small, broadly rounded anterior lobe and equally long posterior lobes, the transition between the anterior and posterior lobes is marked, α = ~156°, and β = ~123° ( Fig. 34A ). (ii) The posterior neck scales (U29–42) are 2.7–4.3 ×2.4–3.1 µm in size, exhibit a slightly elongated anterior lobe and an indistinct and continuous transition between the anterior and posterior lobes, α = ~167°, and β = ~98° ( Fig. 34B ). (iii) The trunk scales (U37–80) are comparatively big (6.2–7.1 ×4.5–5.4 µm), triangular, and display a continuous and indistinct transition between the anterior and posterior lobes, α = 168–172°, and β = 96–102° ( Figs 34C , 35D , 36B ). Dorsolateral trunk scales are 7.4–8.4 ×4.0–4.4 µm in size and resemble anterior trunk scales ( Fig. 34D ). (iv) The trunk lateral scales are 5.5–5.9×3.7–3.8 µm in size, distinctly three-lobed, all lobes are narrowly rounded to more or less tapered, and the transition between the anterior and posterior lobes is marked ( Fig. 34E ). SPINES. All spines bear a distinct lateral denticle and gradually narrow towards their distal end ( Figs 33A– B , 34A‒F ). Keels start close to the anterior margin of scales. Spines are not straight but distinctly curved ( Fig. 35C–D ). They do not differentiate into various types , only their length increases from 3.1 μm to 14.0 μm in a posterior direction (Supp. file 1: Table S8). The lateral denticle is comparatively distant from the spine apex, i.e., the d -value ranges from 1.0‒3.1 μm, which corresponds to a d ratio of 7.5‒25%. VENTRAL CILIARY BANDS AND VENTRAL INTERCILIARY FIELD. Ciliary bands start at U10 where they are conspicuously broad. However, they begin to narrow from U25 and terminate at U80. The ciliary bands are accompanied by two ventrolateral rows of three-lobed scales that extend from U12 to U80 ( Fig. 33B ). They are 1.5–4.3 ×1.1–3.2 µm in size and have a marked transition between the anterior and posterior lobes, α = 149–160°, and β = 62–100° ( Fig. 34F ). The ventral interciliary field bears four types of horizontally distributed scales: (i) small (2.7–4.3 ×3.1–3.4 µm), oval to oblong, double-edged scales without keels (U10–U73) ( Figs 33B , 34H , 36A ); (ii) a pair of relatively small (4.4–5.1 ×2.6–2.7 µm), oval, double-edged, and keeled scales (U74); (iii) a single oblong (1.3–2.0× 1.0–1.5 µm), centrally positioned, double-edged, and keeled scale (U71); and (iv) a pair of big (11.8–12.3×3.2–3.4 µm), very narrowly ovate, double-edged, and spined scales (U76) ( Figs 34G , 36D ). The furca branches (U81– U90) carry minute (1.1–3.2× 0.5–1.4 µm), oval to oblong, and keeled scales ( Figs 34I , 36C ).