Chaetonotus (Hystricochaetonotus) mirabilis, Križanová & Vďačný, 2022

Križanová, Františka Rataj & Vďačný, Peter, 2022, A huge undescribed diversity of the subgenus Hystricochaetonotus (Gastrotricha, Chaetonotidae, Chaetonotus) in Central Europe, European Journal of Taxonomy 840, pp. 1-93 : 19-27

Chaetonotus (Hystricochaetonotus) mirabilis sp. nov.

urn:lsid:zoobank.org:act:F959427F-762B-4344-A1E4-7D491DD99225

Figs 10‒14 ; Supp. file 1: Table S2

Morphological diagnosis

Body slender and about 83‒107 µm long. Head wider than neck, separated from trunk by a distinct neck constriction. Cephalion clearly demarcated, epipleurae and hypopleurae only inconspicuously marked in head outline. Trunk widest at ca U62, gradually tapers towards furca base (U85). Mouth ventral, with two cuticular teeth. Pharynx with anterior and posterior dilatations. Intestine straight, with a marked anterior section. Scales spined, three-lobed, not overlapping, distributed in 10–12 columns, 17 scales per column. Dorsal surface covered from anterior end of hypopleurae (ca U6) to neck constriction with fairly small scales bearing short spines. Neck caries broader scales with longer spines. Anterior trunk region bears similarly shaped, slightly bigger scales with posterior lobes closer together. Dorsolateral and lateral scales with elongated base and posterior lobes more divergent, spine with an inconspicuous denticle. Mid-trunk to terminal trunk region covered by (i) five horizontal rows of big, anteriorly tapered scales carrying very elongated, massive spines with a denticle and membrane and (ii) two horizontal rows of smaller, anteriorly rounded scales carrying significantly shorter spines with a denticle. Furca base short, lateral margins vaulted, furcal indentation U-shaped, adhesive tubes well-developed, diverging posteriorly. Furca base and branches covered with three-lobed, spined scales and oblong, keeled scales.

Molecular diagnosis

18S rRNA gene: 672 T, 1072 ‒. ITS2: 26 G, 28 A, 40 G, 41 T, 53 G, 60 C, 62 T, 84 A, 96 A, 107 ‒, 117 T, 131 A, 132 A, 133 A, 136 ‒, 137 ‒, 139 C, 156 G, 157 T, 159 T, 168 A, 173 ‒. 28S rRNA gene: 530 G, 608 C, 687 A, 690 G, 717 A, 719 C, 756 C. Cytochrome c oxidase subunit I (codon ordinal numbers are followed by the corresponding span of nucleotide positions in parentheses): 23 (67‒69) ATT, 28 (82‒84) CTT, 44 (130‒132) GTC, 45 (133‒135) GTA, 63 (187‒189) GGA, 72 (214‒216) CTT, 82 (244‒246) CCA, 97 (289‒291) AGT, 98 (292‒294) TTA, 100 (298‒300) CTT, 103 (307‒309) GCG, 129 (385‒387) AGG, 142 (424‒426) GCA, 146 (436‒438) TTG, 155 (463‒465) ACC, 156 (466‒468) CTA, 162 (484‒486) GGT, 165 (493‒495) TTT, 167 (499‒501) CGT, 170 (508‒510) TTA, 179 (535‒537) GTT, 184 (550‒552) CTG, 190 (568‒570) GCC, 212 (634‒636) GGA, 221 (661‒663) CTA.

Reference molecules are shown in Figures 5 , 7 , and Supp. file 1: Fig. S9A . 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 0.22‒4.06% in 18S, 18.19‒34.22% in ITS2, 2.23‒7.12% in 28S, and 11.37‒13.15% in COI. There are 2‒13 CBCs (except for Ch. (H). superbus sp. nov., where there are no CBCs) in the 18S rRNA molecule, 2‒4 CBCs in the ITS2 molecule, and 1‒12 CBCs in the first two domains of the 28S rRNA molecule.

Etymology

The Latin adjective ‘ mirabil · is, - is, - e ’ [m, f, n] (‘marvelous, extraordinary’) refers to the extraordinary long dorsal spines.

Material examined

Holotype SLOVAKIA • adult (photomicrographs, hologenophore); Greenhouse pond , Botanical Garden , Karlova Ves , Bratislava, Podunajská rovina plain (type locality); 48°08′46.8″ N, 17°04′22.6″ E; CU-FNS- 11-09-19 / HO.

Photomicrographs of the holotype are available at the Department of Zoology, Comenius University in Bratislava at https://fns.uniba.sk/en/gastrotricha/. The holotype is shown in Fig. 12 .

Paratypes SLOVAKIA • 2 adults (photomicrographs); same collection data as for holotype; CU-FNS- 25-10-19 / PA-1, CU-FNS- 02-10-19 /PA-2 .

Photomicrographs of paratype specimens are available at the Department of Zoology, Comenius University in Bratislava at https://fns.uniba.sk/en/gastrotricha/. Paratypes are shown in Figs 13–14 .

Type material

A DNA sample of the holotype specimen BZs 02 has been deposited in the Natural History Museum, Vajanského nábrežie 2, 810 06 Bratislava, Slovakia (ID Collection Code 01427888).

Type locality

Greenhouse pond, Botanical Garden of Comenius University, Karlova Ves, Bratislava, Podunajská rovina plain, Slovakia, 48°08′46.8″ N, 17°04′22.6″ 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 BZs 02 have been deposited in GenBank under the following accession numbers: OM 421704 , OM 421680 , and OM 424059 , respectively.

Description

HABITUS. Chaetonotus (Hystricochaetonotus) mirabilis sp. nov. is about 83–107 µm long and has a slender body that is tenpin-shaped, with a clearly defined head region, a narrowing neck, and a slightly bulbous trunk ( Figs 10A–B, G , 12A–C ). Body width is 10–19 µm at U10, 11.2–11.6 µm at U50, and 17.5–18.5 µm at U60. The head is relatively wide, with a plate-like, rounded cephalion. The neck (ca U12–U27) is clearly demarcated and smoothly continues to the trunk region, i.e., a distinct neck constriction is formed. The trunk is nearly as wide as the head, gradually dilating from about U39 to U62 where it reaches the maximum width. Then it gradually tapers towards U84 where vaulted margins of the furca branches start to form. Dorsal sensory bristles (setolae) arise from the cuticle in two pairs at U25 and U75 ( Figs 14A , 16C ). The furcal indentation is broadly U-shaped. The furca branches are set apart and diverge posteriorly. Well-developed adhesive tubes are 9.4–10.5 µm long, they are straight and narrow ( Fig. 17G ).

HEAD. The head is roughly five-lobed. The cephalion (U1–U2) is rounded, 0.4–0.9 µm wide, clearly demarcated in the body outline, appears as a lens in the ventral view, and has a free posterior (dorsal) edge ( Figs 10A–B, F , 12B , 13A–C ). The epipleurae are approximately at U3–U5 and the hypopleurae are at ca U6–U9. Notches separating the epipleurae from the hypopleurae are very shallow, causing that they are only inconspicuously marked in the head outline ( Fig. 13A, E ). 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 (U5) ( Figs 10A, F–G , 12A‒C , 13A, E ). The hypostomium is absent and the under-mouth area carries only ciliary patches. Each field is composed of several irregular groups of basal bodies (kinetosomes). The mouth ring is oval, 5.0–5.5 µm in the largest diameter, located subterminally at U2–U4. There are strong but short, rod-like reinforcements lining the walls of the mouth ring as well as an inner pair of cuticular teeth located in the center of the mouth opening ( Figs 10C , 13D ).

INTERNAL MORPHOLOGY. The pharynx extends from ca U5 to U30, is 26–30 µm long and 5.3–7.8 µm wide, sinuous, and has marked anterior and posterior dilatations ( Figs 10F , 13A, C , white arrowheads). The posterior dilatation (ca U20–U30) is larger than the anterior one (ca U5–U8) (Supp. file 1: Table S2 ). The pharynx is connected to the straight intestine through a pharyngeal‒intestinal junction ( Fig. 13B ). The intestine runs from U31 to U84 and has a separate, well-differentiated anterior section (U31–U33) ( Figs 10F , 13A ). There are highly refractive and regularly arranged structures well recognizable in the lateral view of the intestine ( Figs 13F , 14G , double white arrowheads). Transversal bands connected to the base of dorsal scales are recognizable from ca U10 ( Figs 10F , 13A ). The adhesive gland (ca U85– U91) is placed right behind the terminal part of the intestine, it is broadly pyriform forming a short dichotomy at the subtle furca base ( Figs 10F , 13G ).

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 10–12 longitudinal rows, with 17 scales in the central row. Central dorsal and dorsolateral longitudinal rows of scales begin at the level of the anterior edge of the hypopleurae (ca U6), lateral rows start at their posterior end (ca U9). Ventral and ventrolateral rows are hardly visible due to the highly pronounced dorsal spines (for further explanation, see below). Head scales (ca U6–U25) are fairly small, i.e., 3.3–4.1 ×1.9–3.9 µm in size. Two types could be recognized: (i) lateral boomerang-shaped scales with a subtle anterior lobe, the transition between anterior and posterior lobes is indistinct and continuous, α = 170–177°, and β = 70– 85° ( Fig. 11A ) and (ii) dorsal/dorsolateral scales with a distinctly elongated anterior lobe and a smaller angle α ranging from 146 to 169°, angle β spans a wide range of 68–108°, and the transition between anterior and posterior lobes is marked ( Figs 11B , 14B ). Neck dorsal scales are 3.2–5.1 ×2.0–2.7 µm in size. They are anteriorly more broadly rounded, their angle α is slightly smaller (153–163°), β is closer to the right angle (82–91°) than in head scales, and the transition between anterior and posterior lobes is marked ( Figs 11C , 14A ). This type of scale terminates right at the anterior border of the trunk region (ca U26–U39). The trunk region is covered by four types of scales: (i) lateral scales (U37–U50) with a tongue-shaped anterior lobe and comparatively narrow posterior lobes, α = 149–150°, and β = 76–82°, the transition between anterior and posterior lobes is indistinct and continuous ( Figs 11D , 14F ); (ii) dorsolateral scales (U50–U81) with a tongue-shaped anterior lobe and wider posterior lobes being closer together, α = 147–158°, β = 68–76°, the transition between anterior and posterior lobes is indistinct ( Figs 11E , 14A, C ); (iii) dorsal big scales (U50–U78) with a tapered, triangular anterior lobe and narrowly rounded posterior lobes, α = 175–179°, β = 57–68°, the transition between anterior and posterior lobes is also indistinct ( Figs 11F , 14A ); and (iv) dorsal smaller scales (U80–U90) with an anteriorly rounded anterior lobe, the transition between anterior and posterior lobes is marked ( Figs 11G , 14D ).

SPINES. All spines bear a lateral denticle and gradually narrow towards the distal end. Three main types could be distinguished. The most common type of spines emerges from the head (2.7–3.7 µm long), neck (4.5–7.1 µm long), trunk dorsolateral (4.0–6.8 µm long), and lateral scales (6.8–8.8 µm long). The lateral denticle is minute and its tip is distant only 0.7–0.9 µm from the spine apex, which corresponds to a d -ratio of 25.3–31.2%. Lateral spines are, however, slightly thinner (0.32–0.35 µm vs 0.30–0.43 µm) and their subterminal denticle is rather inconspicuous and much closer to the spine apex (d -ratio 8–10%) in comparison with head, neck, and dorsolateral spines. The second type is represented by the prominent, elongated spines carrying a conspicuous denticle (2.6–5.6 µm long) associated with a membrane ( Figs 10D , 11F , 12B–C , 13F , 14A, G ). The denticle is 6.4–7.9 µm distant from the spine apex, which corresponds to a d -ratio of 18.2–22.6%. Type 2 spines are 20.7–35.9 µm long and comparatively wide (1.8–1.9 µm) at the base. Altogether only five horizontal rows of dorsal scales are equipped with this type of spines (U50–U78). The last type could be found only on the two terminal rows of dorsal scales on the furca base and branches. These spines are significantly shorter (13.0–18.4 µm vs 20.7– 35.9 µm) and thinner (0.7–0.8 µm vs 1.1–1.6 µm) than the previous type. They are slightly curved and hair-like tapered distally. Although their denticle is well recognizable, it is not associated with a distinct membrane. The d -value is 2.9–4.4 µm and the d -ratio is 22.0–25.7% ( Figs 10E , 11G , 14D ).

VENTRAL CILIARY BANDS AND VENTRAL INTERCILIARY FIELD. Unfortunately, the ventral side could not be observed in detail due to the very long and strong type 2 spines that precluded turning over and squeezing the worms. Despite that, the following observations could be done. The longitudinal ciliary bands begin at ca U7 and run backward to ca U87. They are somewhat wider on the neck than on the trunk where they narrow slightly from ca U78. The ciliary bands are accompanied by a ventrolateral row of small (2.4–3.0× 1.0–1.5 µm in size), oblong, and keeled scales that start at U7. The upper furcal region (U84– U90) carries two types of scales: (i) three-lobed, spined scales with a broadly rounded anterior lobe, narrowly rounded posterior lobes, α = 137–171°, and β = 59–72° ( Figs 10G , 11H ) and (ii) oblong and keeled scales being 1.5–3.9 ×0.7–1.5 µm in size ( Figs 10G , 11I ).

Figures

Fig. 10. Chaetonotus (Hystricochaetonotus) mirabilis sp. nov. A‒B. Dorsal and lateral overviews showing the scale and spine pattern. C. Detail showing the cuticular teeth protruding from the mouth ring. D. Elongated dorsal spines with a membrane. E. Terminal dorsal spines. F. Internal morphology of a strongly squeezed specimen (body shape hence not representative). G. Ventral overview. Scale bars: A = 30 µm; F–G = 20 µm.

Fig. 11. Scales of Chaetonotus (Hystricochaetonotus) mirabilis sp. nov. A. Head lateral scales. B. Head dorsal scales. C. Neck dorsal scales. D. Trunk lateral scale. E. Trunk dorsolateral scale. F. Trunk dorsal scale. G. Terminal dorsal scale. H. Furcal ventral three-lobed scales. I. Furcal ventral oblong scales.

Fig. 12. Chaetonotus (Hystricochaetonotus) mirabilis sp. nov., holotype (CU-FNS-11-09-19/HO), differential interference contrast. A‒B. Optical sections showing the general body organization. C. Lateral overview. Arrows in (B) and (C) mark the lateral denticle of spines. Scale bars = 30 µm.

Fig. 13. Chaetonotus (Hystricochaetonotus) mirabilis sp. nov., differential interference contrast. A, C, E, G. Paratype (CU-FNS-25-10-19/PA-1). B, D, F. Paratype (CU-FNS-02-10-19/PA-2). A. Internal morphology. Opposed arrowheads denote posterior pharyngeal dilatations. B. Lateral view of the anterior body region. White star marks the cerebral ganglion. C. Anterior pharyngeal dilatations (opposed arrowheads). D. A pair of cuticular teeth is situated in the center of the mouth ring. E. Ventral view of the head region. Asterisks mark the beginning of the ventral ciliary bands. F. The intestine contains highly refractive and regularly arranged structures (white double arrowheads). G. Furca with a U-shaped indentation and adhesive glands. Scale bars: A = 15 µm; B, E–G = 10 µm; C–D = 5 µm.

Fig. 14. Chaetonotus (Hystricochaetonotus) mirabilis sp. nov., differential interference contrast. A–B, E. Paratype (CU-FNS-25-10-19/PA-1). C–D, F–G. Paratype (CU-FNS-02-10-19/PA-2). A. Dorsal view showing the scale and spine pattern. B. Head scales. C. Neck and upper trunk dorsolateral scales. D. Terminal dorsal trunk scales with a lateral denticle (arrows). E. Ventral ciliary bands. White dashed lines mark upper-furcal scales. F. Lateral trunk scales. G. Dorsal trunk spines with a membrane. White double arrowheads mark the highly refractive bodies in the intestine. Scale bars: A = 30 µm; B–F = 5 µm; G = 10 µm.

Fig. 5. Secondary structure of the 18S rRNA molecule of Chaetonotus (Hystricochaetonotus) mirabilis sp. nov. The single diagnostic molecular autapomorphy (red arrow) is situated in the terminal loop of helix 21es6c in the V4 region of the C domain. Note the long-range tertiary contacts between the V2 and V4 regions (highlighted in pink). The reference 18S secondary structure map of Saccharomyces cerevisiae Meyen ex E.C.Hansen (inset) is from http://apollo.chemistry.gatech.edu/RibosomeGallery (Petrov et al. 2014).

Fig. 7. Secondary structure of the first two domains of the 5.8‒28S rRNA molecule of Chaetonotus (Hystricochaetonotus) mirabilis sp. nov. Diagnostic molecular autapomorphies are marked by red arrows. The reference 28S secondary structure map of Saccharomyces cerevisiae Meyen ex E.C.Hansen (inset) is from http://apollo.chemistry.gatech.edu/RibosomeGallery (Petrov et al. 2014).

Fig. 9. Phylogenetic subtree based on 18S, 28S, and COI, showing the systematic positions of the nine new species within the Chaetonotus (Hystricochaetonotus) cluster. Arrows mark the same node in the subtree and the inset phylogram. The whole tree is presented in Fig. 8. The Chaetonotus (Hystricochaetonotus) cluster is highlighted in the inset phylogram. ML bootstrap values and posterior probabilities were mapped onto the best ML tree.

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.