Heterolepidoderma kolickae, Križanová & Vďačný, 2024

Križanová, Františka Rataj & Vďačný, Peter, 2024, A Heterolepidoderma and Halichaetoderma gen. nov. (Gastrotricha: Chaetonotidae) riddle: integrative taxonomy and phylogeny of six new freshwater species from Central Europe, Zoological Journal of the Linnean Society 200 (2), pp. 283-335 : 301-306

Heterolepidoderma kolickae sp.nov.

( Figs 11A–E , 12A–G , 13A–E , 14A–D ; Supporting Information, Table S10)

ZooBank registration: urn:lsid:zoobank.org:act:DCBF766D-2115-4290-8DEA-2D1C37C2C910 .

Morphological diagnosis: Body slender and about 112–136 µm long. Head wider than neck, separated from trunk by a distinct neck constriction. Cephalion large and clearly demarcated, epipleurae and hypopleurae conspicuously marked in head outline. Ocellar granules present. Trunk widest at c. U57, gradually tapers towards furca base (U81). Mouth ventroterminal, with two cuticular teeth. Pharynx without reinforcements. Intestine straight, with marked anterior section. Three pairs of dorsal sensory bristles. Scales partially overlapping, distributed in 20–22 dorsal alternating columns, 23 scales per central column. Dorsal surface covered from anterior end of cephalion to furca base with three different types of scales: (i) spined, three-lobed, and posteriorly rounded; (ii) spined, three-lobed, and posteriorly narrowly rounded or pointed; and (iii) keeled, oblong with truncated posterior end. Lateral to ventrolateral body sides covered from posterior end of epipleurae with spined and narrowly lanceolate scales. Ventrolateral scales keeled and cordiform. Interciliary field scales bowl-shaped. Furca base short, furcal indentation broadly V-shaped, adhesive tubes well-developed, diverging posteriorly. Ventral upper-furcal region covered with big, oblong, and keeled scales.

Molecular diagnosis: 18S rRNA gene: 403 T. 28S rRNA gene: 562 T. Reference molecules are shown in Supporting Information, Figs S1 , S 7 . All diagnostic molecular autapomorphies are marked by arrows.

Type locality: An ephemeral pond in the floodplain area of the Morava river near the foothill of the Devín Castle , Bratislava, Podunajská rovina plain, Slovakia, 48°10ʹ29.4ʹʹN, 16°58ʹ35.8ʹʹE .

Material examined: The holotype (adult, hologenophore, CU-FNS-18-10-19/HO) was destroyed during DNA extraction. The paratype (adult, CU-FNS-22-10-19/PA) is shown in Figures 13A–E and 14A–D . Photomicrographs of the holotype and paratype are available at the Department of Zoology , Comenius University in Bratislava at https://fns.uniba.sk/en/ gastrotricha /.

Type material: A DNA sample of the holotype specimen DB 11 has been deposited in the Natural History Museum, Vajanského nábrežie 2, 810 06 Bratislava, Slovakia (ID Collection Code 01427567) .

Gene sequences: The nuclear 18S and ITS1 - 5.8 S-ITS2-28S rDNA sequences, as well as the mitochondrial COI sequence of the holotype specimen DB 11, have been deposited in GenBank under the following accession numbers: OQ358136, OQ358125, and OQ354328, respectively .

Etymology: Named in honour of Dr Małgorzata Kolicka (Adam Mickiewicz University, Poznań, Poland), as a small token of appreciation for stimulating our research of gastrotrichs and for her constructive criticism and support.

Description: Habitus. Heterolepidoderma kolickae is about 112– 136 µm long and has a slender body that is tenpin-shaped, with a clearly defined head region, a narrowing neck, and a bulbous trunk ( Figs 11A , 12A, G , 13A ). Body width is 23.3 µm at the head (U10), 24.7 µm at U50, and 33.0 µm at U57. The neck (c. U14–U30) is clearly demarcated (i.e. a distinct neck constriction is formed) and smoothly continues to the trunk region. The trunk is nearly as wide as the head, gradually dilating from about U40 to U57, where it reaches its maximum width. Then it gradually tapers towards U80, where the furca base starts to form. Three pairs of dorsal sensory bristles are inserted at U6, U17, and U76 ( Fig. 11A ). The furcal indentation is broadly V-shaped. The furca branches are set apart and bear well-developed adhesive tubes, which are 6.7–8.8 µm long and slightly diverge posteriorly ( Fig. 14C ).

Head

The head appears five-lobed only in the ventral view ( Fig. 12A ), while three-lobed in the dorsal view ( Fig. 11A ). Thus, the large size of the epipleurae causes the hypopleurae to be not visible from a dorsal point of view. The cephalion is very conspicuous because comparatively large, i.e. extends from U1 to U6 and is 8.9 µm wide, roughly obtriangular in the dorsal view and biblike in the ventral view, and without free posterior (dorsal) edge ( Figs 11A , 12A, E , 13A–D ). The epipleurae are situated approximately at U3–U8. Notches separating the epipleurae from the hypopleurae (U8–U14) are recognizable only in the ventral view and hence the hypopleurae are difficult to distinguish in the head outline when the dorsal side is observed (cp. Fig. 11A with Fig. 12A ). Two pairs of cephalic ciliary tufts emerge laterally between the cephalion and the epipleurae edge (c. U3), as well as between the epi- and the hypopleurae edge (c. U8) ( Figs 11A , 12A, G , 13A, C, D ). Ocellar granules are situated posterior to the epipleurae edge at U8 and they are only about 1.2 µm across. The mouth ring is oval, approximately 4.4 μm in the largest diameter, and located ventroterminally at U1–U4. There are strong but short, rod-like reinforcements lining the walls of the mouth ring. Two cuticular teeth are clearly visible and placed laterally in the mouth opening ( Figs 12E , 13B ). The hypostomium (c. U4–U7) has a complex morphology, i.e. bears three cuticular structures: one central plate and a pair of posterolateral boomerang-like additional plates. A pair of relatively wide sensoric ciliary patches is placed between the central plate and the posterolateral plates ( Figs 12E , 13B ).

Internal morphology

The pharynx extends from c. U3 to U31, is 35 μm long and 5.9– 8.6 μm wide. The pharynx smoothly continues through the pharyngeal–intestinal junction to the differentiated anterior section (U31–U33) of the intestine, which extends from U33 to U80 ( Figs 12G , 13A, E ). Paired pyriform adhesive glands (c. U79– U83) are placed slightly laterally, right behind the terminal part of the intestine, forming a short dichotomy at the subtle furca base ( Fig. 14C ).

Scales

Almost the entire body is covered by overlapping scales that adhere to the basal cuticle layer along most or all of their perimeter. Scales are distributed in 20–22 longitudinal dorsal alternating columns, with usually 23 scales in the central column. Their size slightly increases in a posterior direction. Central dorsal longitudinal rows of scales begin at the level of the posterior edge of the cephalion (c. U6), while dorsolateral and lateral rows start at c. U8–U11 at the posterior edge of the epipleurae. They run almost along the whole body length (till U87) ( Fig. 11A ). Ventrolateral rows commence at U14 due to the highly developed ventral ciliary bands and terminate near the furcal region at c. U83. Ventral rows commence ahead of ventrolateral rows about at U9 ( Fig. 12A ).

Three main types of dorsal body scales were recognized. (i) Anteriormost head scales are small (1.56–2.20 × 0.78–1.04 µm), three-lobed with a subtle, well-delimited anterior lobe and comparatively long and rounded posterior lobes, the transition between the anterior lobe and the posterior lobes is relatively well marked, Α = 136–164°, and β = 83–94° ( Fig. 11B ). These smallest dorsal scales are arranged in three horizontal rows. (ii) The remaining head and upper-neck scales resemble the first type but are slightly larger (2.50–3.35 × 1.66–1.84 µm), their posterior lobes are narrowly rounded or pointed, and the transition between the anterior lobe and posterior lobes is indistinct and continuous ( Fig. 11C ). They start as a single longitudinal middle row at the posterior edge of the cephalion (U6) and then three or four horizontal rows are added at c. U10. This second type of dorsal scales terminates at about U18. (iii) The most abundant type is represented by oblong scales with a truncated posterior end. They are 2.6–3.0 × 1.12–1.25 µm in size and cover a significant part of the body from U18 to U82 ( Figs 11D , 14A, B ).

Lateral body sides bear 2.76–3.20 × 1.19–1.57 µm-sized and narrowly lanceolate scales from U11 to U85. The transition between the anterior lobe and the posterior lobes is continuous and indistinct ( Figs 11E , 12A ).

Spines

Scales bear spines only in the head region and on the lateral and ventrolateral body sides ( Figs 11A , 13D, E , 14C, D ). The spine emerges rather close to the anterior margin of scale. Spines do not differentiate into various types, only their length slightly increases from 2.85 µm to 3.70 µm in a posterior direction. A lateral denticle is not developed. All remaining scales carry keels, which also begin close to the apical scale margin and increase in length in a posterior direction from 1.93 µm to 3.86 µm ( Figs 11A , 14A, B ).

Ventral ciliary bands and ventral interciliary field

Ventral ciliary bands commence almost right behind the hypostomium. With respect to the arrangement of basal bodies of locomotory cilia, three regions can be distinguished: the bifurcated anterior region, the transition zone, and the main portion of the ciliary band. The anterior region is bifurcated, contains irregularly arranged basal bodies, and extends from U8 to U12 ( Figs 12A , 13B ). In the transition zone (U12–24), the anterior branches are fused but basal bodies are still rather irregularly organized. Locomotory cilia then become regularly arranged in more or less equidistantly spaced horizontal rows. Each row in the main portion of the ciliary band consists of four narrowly spaced basal bodies from c. U25 to U80 ( Figs 12A , 14D ).

Each ciliary band is laterally accompanied by two or three ventrolateral rows of cordiform scales (1.3–3.6 × 1.1–2.4 µm) ( Figs 12A, B , 14D ). They start at U18, the rightmost and leftmost rows terminate at U80, while the middle scale row ends at U84. The ventral interciliary field bears minute (1.20–1.52 × 1.02– 1.43 µm), bowl-shaped scales, which start posterior to the edge of the hypostomium at U9 ( Figs 12A, F , 13B ). The upper-furcal region carries a pair of relatively big (4.7–5.4 × 2.1–2.2 µm), oblong, and keeled scales ( Figs 12A, D , 14D ).

Figures

Figure 1. Secondary structure of the 18S rRNA molecule of Halichaetoderma fluviatile sp. nov.Thick arrows mark species-specific diagnostic molecular characters, while thin arrows denote diagnostic characters of the genus Halichaetoderma. Thus, there are five genus-specific molecular characters (thin arrows) localized in the double-stranded regions of helix 21es6c from the variable V4 region, helix 33 from the V6 region, and helix 41 from the V8 region. On the other hand, there are as many as 31 species-specific nucleotide characters (thick arrows) scattered over the whole 18S molecule both in the single- and double-stranded regions. Note the long-range tertiary contacts between the V2 and V4 regions (highlighted in grey). The helix number system follows Lee and Gutell (2012) and Petrov et al. (2014). 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).

Figure 7. SIMMAP reconstruction of scale morphologies in chaetonotids based on 10 000 stochastic character maps simulated under the ER model. The present reconstruction analyses indicated that scales became oblong in the last common ancestor of the Halichaetoderma + Halichaetonotus clade as well as three times independently within the Heterolepidoderma s.l. clade. Relative proportions of character states were mapped onto the best scoring ML tree.

Figure 11. Heterolepidoderma kolickae sp. nov. A, dorsal overview showing the spine and keel pattern. B, head dorsal scales are spined and three-lobed with a subtle anterior lobe and a well-marked transition between the anterior lobe and the posterior lobes. C, upper-neck dorsal scales resemble head scales but the transition between the anterior lobe and posterior lobes is indistinct. D, trunk dorsal scales are keeled and oblong with a truncated posterior end. E, trunk dorsolateral scales are spined and narrowly lanceolate. ceph, cephalion; epi, epiplaeurae.Scale bar = 30 µm.

Figure 12. Heterolepidoderma kolickae sp. nov. A, ventral overview showing the scale and spine pattern. B, ventrolateral scales are keeled and cordiform. C, trunk lateral scales are spined and narrowly lanceolate. D, upper-furcal ventral scales are relatively big, oblong, and keeled. E, mouth region. F, interciliary field scales are minute, bowl-shaped, and spineless. G, internal morphology. ceph, cephalion; ct, cuticular teeth; das, differentiated anterior section of intestine; epi, epipleurae; hs, hypostomium; hyp, hypopleurae; int, intestine; ph, pharynx. Scale bar = 30 µm.

Figure 13. Heterolepidoderma kolickae sp. nov.A, overview showing the general body organization.B, C, details of the anterior body region, showing a pair of cuticular teeth in the mouth centre and the hypostomium. D, detail of the anterior dorsal body region, showing the cephalion, epipleurae, ocellar granules, and spined head scales. E, optical section showing the internal morphology.ceph, cephalion; ct, cuticular tooth; das, differentiated anterior section of intestine; epi, epipleurae; hs, hypostomium; hyp, hypopleurae; int, intestine; m, mouth; oc, ocellar granules; ov, ovaries; phr, pharynx reinforcements; pij, pharyngeal‒intestinal junction; pn, protonephridia; sc, sensory cilia; vs, ventral scales. Scale bars = 10 µm (C, D), 15 µm (B), 30 µm (E), 50 µm (A).

Figure 14. Heterolepidoderma kolickae sp. nov.A, dorsal view of the posterior neck and the upper trunk region, showing keeled and oblong scales with a truncated posterior end. B, dorsal view of the posterior body region, showing the scale pattern. C, the furca indentation is broadly U-shaped.Adhesive glands are paired and placed right behind the terminal part of the intestine, forming a short dichotomy at the subtle furca base.D, ventral view of the posterior body region, showing the small cordiform scales (arrows) and the paired big oblong keeled scales (arrowheads). ag, adhesive glands; at, adhesive tubes. Scale bars = 10 µm.