A Heterolepidoderma and Halichaetoderma gen. nov. (Gastrotricha: Chaetonotidae) riddle: integrative taxonomy and phylogeny of six new freshwater species from Central Europe Author Križanová, Františka Rataj Author Vďačný, Peter peter.vdacny@uniba.sk text Zoological Journal of the Linnean Society 2024 2023-09-05 200 2 283 335 http://dx.doi.org/10.1093/zoolinnean/zlad079 journal article 10.1093/zoolinnean/zlad079 0024-4082 11240054 88445152-50C8-42E7-A552-CC368E75C2F8 Heterolepidoderma striatum sp.nov. ( Figs 15A–H , 16A–H , 17A, B , 18A–E ; Supporting Information, Table S11 ) ZooBank registration: urn:lsid:zoobank.org:act: 1C883C58- D5D0-48E4-A403-EA8D1CE847C0 . Morphological diagnosis: Body stocky and about 128 µm long. Head almost as wide as neck, separated from trunk by more or less distinct neck constriction. Cephalion large and clearly demarcated, epipleurae and hypopleurae only indistinctly marked in head outline. Ocellar granules absent. Trunk widest at c. U63, abruptly tappers towards furca base (U87). Mouth ventroterminal, without cuticular teeth. Pharynx with three pairs of reinforcements. Intestine straight, with marked anterior section. Dorsal, lateral, and ventrolateral scales keeled, narrowly oblong, partially overlapping, distributed in 37 dorsal alternating columns, 23 scales per central column. Only posterior pair of dorsal sensory bristles anchored on double-keeled, cordiform scales. Furca base short, furcal indentation broadly U-shaped, adhesive tubes short and diverging posteriorly. Furca base and furca appendages dorsally covered with a pair of roughly rectangular and multi-keeled scales, as well as with two pairs of fusiform and keeled scales. Ventral upper-furcal region carries three staggered transversal rows of narrowly oblong scales and a pair of big, broadly oblong, and posteriorly truncated scales. Molecular diagnosis: 18S rRNA gene: 487 A, 489 T, 495 A, 704 T, 708 G, 735 T, 1357 C, 1371 G, 1372 T, 1508 T, 1509 C. 28S rRNA gene: 480 C, 528 C, 611 A, 613 C, 631 G, 633 T, 667 T, 669 G. COI (codon ordinal numbers are followed by the corresponding span of nucleotide positions in parentheses): 32 (94–96) ACC, 40 (118–120) TTG, 49 (145–147) GCG, 121 (361–363) AGG, 122 (364–366) CTA, 188 (562–564) GTC, 189 (565–567) CTT. Reference molecules are shown in Supporting Information, Figs S2 , S 8 . All diagnostic molecular autapomorphies are marked by arrows. Figure 15. Heterolepidoderma striatum sp. nov. A, dorsal overview showing the keel pattern. The body is stocky and about 128 µm long. The head is almost as wide as the neck, separated from the trunk by a more or less distinct neck constriction. The cephalion is large and clearly demarcated, while the epipleurae and hypopleurae are only indistinctly marked in the head outline. B‒D, head, neck, and trunk dorsal scales are keeled and very narrowly oblong. Their size slightly increases in a posterior direction, i.e. from 2.55–5.59 × 0.65–1.65 μm in the head through 4.08–5.20 × 0.75–1.01 μm in the neck region to 4.79–5.75 × 1.29–1.94 μm in the trunk. E, sensory bristle scales are double-keeled and cordiform. F‒H, the dorsal furca base and furca appendages are covered by a pair of roughly rectangular and multi-keeled scales as well as with two pairs of fusiform and keeled scales. at, adhesive tube; ceph, cephalion. Scale bar = 50 µm. Figure 16. Heterolepidoderma striatum sp. nov. A, ventral overview showing the general body organization. B, mouth ring. C, mouth and undermouth region. D, ventrolateral scales are keeled and narrowly to very narrowly oblong. E, the head is roughly five-lobed in the ventral view. F, the pharynx has several reinforcements. G, H, the posterior region of the ventral interciliary field is covered by staggered transversal rows of oblong to very narrowly oblong scales and a pair of broadly oblong and posteriorly truncated scales. das, differentiated anterior section of intestine; epi, epipleurae; hs, hypostomium; hyp, hypopleurae; int, intestine; m, mouth; ov, ovum; ph, pharynx; phr, pharynx reinforcements; rr, rod-like reinforcements. Scale bar = 50 µm. Figure 17. Heterolepidoderma striatum sp. nov. A, B, dorsal and ventral overviews, showing the general body organization and the keel pattern. The body is stocky and tenpin-shaped with a roughly five-lobed head that is almost as wide as the neck, causing the head–neck transition to be indistinct. The trunk is bulbous and distinctly wider than the head. The pharynx extends from U3 to U30, has two weakly marked anterior and posterior dilatations, and continues through the pharyngeal-intestinal junction to the intestine, which extends from U35 to U87. White arrowheads denote the sensory bristle scales, while black arrowheads mark the keeled, broadly oblong, and posteriorly truncated scales placed in the central region of the furca base. ceph, cephalion; m, mouth; ov, ovum; s, sensory bristles; sc, sensory cilia; vcb, ventral ciliary bands. Scale bars = 30 µm. Figure 18. Heterolepidoderma striatum sp. nov. A, overview showing the internal morphology. B, detail of the anterior body region, showing the pharynx and the pharyngeal–intestinal junction. Note that the hypopleurae are only very indistinctly marked in the head outline. C, detail of the posterior body region, showing the narrowly oblong scales, sensory bristle scales (white arrowheads), as well as the paired roughly rectangular and multi-keeled scales at the furca base. These peculiar scales represent a morphological autapomorphy of He. striatum , reliably differentiating it from all congeners. D, the posterior region of the ventral interciliary field is covered by narrowly oblong scales and a pair of broadly oblong scales. E, the furca indentation is broadly U-shaped. at, adhesive tubes; ceph, cephalion; epi, epipleurae; das, differentiated anterior section of intestine; hyp, hypopleurae; m, mouth; pij, pharyngeal–intestinal junction. Scale bars = 5 µm (C, D, E), 15 µm (B), 30 µm (A). 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-13-09-19/HO) is shown in Figures 17A, B and 18A–E . Photomicrographs of the holotype 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 12 has been deposited in the Natural History Museum, Vajanského nábrežie 2, 810 06 Bratislava, Slovakia ( ID Collection Code 01426301) . 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 12, have been deposited in GenBank under the following accession numbers: OQ358140, OQ358129, and OQ354332, respectively . Etymology: The Latin adjective striat · us , -a , -um [m, f, n] (striated) refers to the striated appearance of the new species. Description: Habitus. Heterolepidoderma striatum is about 128 µm long and has a stocky body that is tenpin-shaped ( Figs 15A , 16A , 17A, B , 18A ). Body width is 28.4 µm at U10, 42.1 µm at U50, and 45.0 µm at U63. The head is almost as wide as the neck and hence the head–neck transition is indistinct and continuous. The trunk is wider than the head, bulbous, and gradually dilates from about U34 to U63, where it reaches its maximum width. Then, it gradually tapers towards U87, where a short furca base is formed. Only a posterior pair of dorsal sensory bristles (setolae) present. Sensory bristles arise from cordiform, double-keeled scales at U85 ( Figs 15A, E , 17A , 18C ). The furcal indentation is broadly U-shaped. The furca branches are set apart and bear short but well-developed adhesive tubes, which are 4.7–4.9 µm long and slightly diverged posteriorly ( Figs 15A , 16A , 18A, E ). Head The head appears roughly five-lobed only in the ventral view ( Fig. 16A, E ), while one-lobed in the dorsal view ( Figs 15A , 17A ). The cephalion is very conspicuous because comparatively large, i.e. extends from U1 to U6 and is 7.1 µm wide, oval, and without a free posterior (dorsal) edge ( Figs 15A , 17A ). The epipleurae and hypopleurae are situated ventrolaterally approximately at U3–U6 and U7–U11, respectively, and hence difficult to distinguish in the head outline when the dorsal side is observed. The hypopleurae are slightly more developed than the epipleurae (5.9 μm vs. 5.3 μm long). Notches separating the epipleurae from the hypopleurae are recognizable only in the ventral view. 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. U6) ( Figs 15A , 16A, E , 17A, B , 18A, B ). Ocellar granules are absent. The mouth ring is oval, approximately 5.7 μm in the largest diameter, and located ventroterminally at U1–U4 ( Figs 16A, B, E , 17B , 18B ). There are strong but short, rod-like reinforcements lining the walls of the mouth ring. Mouth with no cuticular teeth. The hypostomium (U3–U7) is a thin plate with two transversal reinforcements bars ( Fig. 16C ). Internal morphology The pharynx extends from c. U3 to U30, is 37 μm long and 6.3– 8.7 μm wide. It has two weakly marked anterior and posterior dilatations. The posterior dilatations are wider than the anterior ones. There are several anterior pharyngeal reinforcements, which are visible only at higher magnifications ( Figs 16F , 18A ). The pharynx smoothly continues through the pharyngeal–intestinal junction to the differentiated anterior section (U31–U35) of the intestine, which extends from U35 to U87 ( Figs 16A , 17B , 18A, B ). Paired adhesive glands ( c. U85–U92) are placed right behind the terminal part of the intestine, forming a short dichotomy at the subtle furca base ( Fig. 18A ). Adhesive tubes are relatively short, just 4.7–4.9 μm long ( Fig. 18E ). 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 37 longitudinal dorsal alternating columns, with usually 23 scales in the central column. Central dorsal longitudinal rows of scales begin at the level of the posterior edge of the cephalion ( c. U6). They run almost along the whole body length (till U96) ( Figs 15A , 17A ). Ventrolateral rows commence at U12 due to the highly developed ventral ciliary bands and terminate at the furca branches at c. U97 ( Figs 16A , 17B ). There are about seven to nine ventrolateral rows of scales on each body side. The most abundant type of body scales is very narrowly oblong with a slight size increase in a posterior direction (head: 2.55–5.59 × 0.65–1.65 μm, neck: 4.08–5.20 × 0.75–1.01 μm, trunk:4.79–5.75 × 1.29–1.94 μm) ( Figs 15B–D , 17A ). Narrowly oblong scales cover the dorsal, dorsolateral, and ventrolateral body sides. The only other type of scales on the dorsal side of the trunk is represented by a pair of cordiform, double-keeled, sensory bristle scales (2.03–2.21 × 1.79–1.91 μm), which are placed at U85 ( Figs 15A, E , 17A , 18C ). The dorsal furca base and branches are covered by a pair of roughly rectangular and multi-keeled scales as well as with two pairs of fusiform and keeled scales ( Figs 15F–H , 18C ). Ventral ciliary bands and ventral interciliary field Ventral ciliary bands commence at U7 and they start to diverge posteriorly from U70 to U85, very likely due to the developing eggs. Each ciliary band is laterally and ventrolaterally accompanied by one (U8–U20), two (U21–U31), and up to six (U37–U70) alternating rows of very narrowly oblong scales (1.4–6.6 × 0.8–1.3 µm). From U73 to U85, the number of rows gradually decreases to one row due to the posteriorly diverging ciliary bands ( Figs 16A, D , 17B ). The ventral interciliary field is almost naked except for the posterior trunk end (U84–U87), which bears three staggered transversal rows of narrowly oblong scales (2.0–4.6 × 0.6–13 µm) ( Figs 16A, G , 18D ). The last pair of the ventral interciliary field scales is located on the furca base. These scales (5.7 × 3.3 µm) have an oblong shape with a posteriorly truncated edge ( Figs 16A, H , 17B , 18D ).