Richtersius tertius, Pogwizd & Stec, 2022

Pogwizd, Justyna & Stec, Daniel, 2022, An Integrative Description Of A New Richtersius Species From Greece (Tardigrada: Eutardigrada: Richtersiusidae), Acta Zoologica Academiae Scientiarum Hungaricae 68 (1), pp. 1-21 : 6-15

Richtersius tertius sp. n.

( Figs 1–7 )

Richtersius sp. 7 (GR.008; MK214323 –5) in STEC et al. 2020 a

Richtersius sp. (OTU6; MK214323 –5) in KAYASTHA et al. 2020 a

Etymology: The name “ tertius ” refers to the fact that the new species is the third formally described in the genus Richtersius .

Material examined: 171 animals and 73 eggs: specimens mounted on microscope slides in Hoyer’s medium (134 adult animals + 12 hatchlings + 59 eggs), fixed on SEM stub (12 + 9 + 14), and used for DNA extraction and sequencing (4 + 0 + 0; in STEC et al. 2020 a).

Description of the new species – Adults (measurements and statistics in Table 2).

Body yellow, after fixation in Hoyer’s medium, all specimens become transparent ( Fig. 1A ). Eyes present both in live animals and specimens mounted in Hoyer’s medium. Body and leg cuticle without granulation ( Figs 1A , 3A–D ).

Claws slender, primary branches with distinct accessory points ( Fig. 3A–D ) and a system of internal septa as described by LISI et al. (2020). Secondary branches approximately 80% of the length of the primary branches. An evident stalk system connecting the claws to the lunulae is visible under PCM and SEM ( Fig. 3A–D ). The stalk system consists of a thin laminar stalk connecting the claw to the lunula and two posterior lateral extensions ( Fig. 3A–D ). The distal tips of these lateral extension under PCM appear to be connected to the stalk where it contacts the lunula ( Fig. 3A–B ) whereas under SEM the stalk system is visible as a cuticular plate with a protuberant laminar stalk ( Fig. 3C–D ). Lunulae large, smoothly unified with the leg cuticle and with a crown of long, numerous and densely arranged spikes (2.0–3.1 μm long) ( Fig. 3A–D ). Lunulae I–III trapezoidal, whereas lunulae IV ovate ( Fig. 2A–F ). Divided cuticular bars, double muscle attachments, and horseshoe structures are visible in PCM ( Fig. 3A–B ).

Mouth antero-ventral. Sensory lobes merged into a single circular sensory field surrounding the mouth ( Fig. 4A ). Anteriorly, the mouth begins with fused peribuccal lamellae forming a circular velum/lamina which is posteriorly folded into a pre­mouth ventricle ( Fig. 4A–B ). With the exception of the second band of teeth, which is rarely and only faintly visible in larger specimens, the oral cavity armature is not visible under PCM. ( Fig. 5B–C ). Under SEM, the oral cavity armature is clearly seen to be composed of three bands of teeth ( Fig. 4A–B ). The first and the second band form continuous rings around the axis of the mouth, whereas the third band is divided into a dorsal and a ventral portion ( Fig. 4A–B ). The first band of teeth lies on the inner surface of the velum and is composed of numerous small granular cones forming about twenty irregular rows with slightly bigger teeth positioned on the edge closest to the velum ( Fig. 4A–B ). The second band of teeth consists of about ten irregular rows of densely packed, elongate, sharp cones which lie on a cuticular fold protruding from the pre-mouth ventricle ( Fig. 4A–B ). The discontinuous third band of teeth is situated between the second band of teeth and buccal tube opening and is divided into a dorsal and a ventral portion, both in the form of a single large tooth, resembling a beak ( Fig. 4A–B ). The ventral tooth resembles an isosceles trapezium standing on its longer base, with a ragged upper edge. The dorsal tooth is semicircular in shape, with a crescentshaped indentation in its middle ( Fig. 4 A–B ).

mens; SD = standard deviation).

Buccal apparatus massive. The oral cavity is followed by a system of massive apophyses forming a buccal crown ( Fig. 5A–C ). Anteriorly, the system consists of dorso-lateral and ventro-lateral triangular apophyses ( Fig. 5A, C ). The dorsal and ventral apophyses, are composed of anteriorly positioned large cuticular hooks followed by longitudinal crests which in case of ventral apophyses might be homologous to the ventral lamina found in ordinary macrobiotids ( Fig. 5B ). The hook in the ventral apophyses is smaller than the dorsal hook ( Fig. 5B ). The buccal tube wall exhibits variable thickness, but the internal diameter of the buccal tube is almost uniformly narrow ( Fig. 5A ). From mouth opening to the stylet support insertion point, the thickens of the buccal tube wall increases only slightly before also slightly expanding to its greatest thickness beyond this point before then shrink posteriorly ( Fig. 5A ). Pharynx spherical, with bilobed apophyses, three anterior cuticular spikes (typically only two are visible in any given plane) and two granular macroplacoids (2<1). The first and the second macroplacoid with a faint constriction positioned centrally and subterminally, respectively ( Fig. 5A–E ).

Hatchlings (measurements and statistics in Table 3). Same as adults, except for small- er body size ( Fig. 1B ) and round pores (1.2–4.1 μm in diameter) with wavy edges, clearly visible both under PCM and SEM, scattered randomly on the entire body cuticle ( Figs 1B , 2A–E ), with a PD range of 3–6.

anterior and posterior claws. Scale bars in Μm

standard deviation).

Eggs (measurements and statistics in Table 4). Large, oval, light yellow, laid freely ( Figs 6A–B , 7A ). The surface between processes is smooth but with refracting dots faintly visible only under PCM, but difficult to observe because of the amount of debris that is typically attached to the egg surface ( Figs 6C , 7A–D ). Processes in the shape of elongated, thin, cones with a ragged surface ( Figs 6 D–E , 7B–D ). Terminal portions of the egg processes are flexible, sometimes divided into two or three short filaments ( Figs 6D–E , 7A–D ). Terminal discs or spatulas absent.

Reproduction – The new species is dioecious: both males with testes and females with ovaries were recorded within the examined population ( STEC et al. 2020 a).

third band are marked with D and V, respectively. Scale bars in Μm

DNA sequences – The DNA sequences of four molecular markers (18S rRNA, 28S rRNA, ITS-2 and COI) associated with the species description have been previously published by STEC et al. (2020 a). The respective GenBank accession numbers are given in Table 1.

Locality: 35°16’16’’N, 23°57’41’’E; Greece, Crete, Chania, Omalos ; forest; moss and lichen from a tree in a forest; coll. 04.07.2015 by Małgorzata Mitan and Małgorzata Osielczak .

Type depositories: The holotype (slide GR.008.15 with 4 paratypes), as well as 162 paratypes (slides: GR.008.*, where the asterisk can be substituted by any of the following numbers, 03–29; SEM stub: 19.12) and 73 eggs (slides: GR.008.* 30–33; SEM stub: 19.12) are deposited at the Institute of Zoology and Biomedical Research , Jagiellonian University , Gronostajowa 9, 30–387, Kraków, Poland whereas 51 paratypes (slides: GR.008.*, where the asterisk can be substituted by any of the following numbers, 20–29) and 31 eggs (slides: GR.008.* 34–37) are deposited at the Institute of Systematics and Evolution of Animals , Polish Academy of Sciences , Sławkowska 17, 31­016, Kraków, Poland .

egg surface is often covered by dirt). Scale bars in Μm

Figures

Fig. 1.Richtersius tertius sp. n. habitus. A = adult, dorso­ventral projection (holotype, PCM); B = hatchling, dorso­ventral projection (PCM). Scale bars in μm

Fig. 2. Richtersius tertius sp. n. pores. A = pores on the hatchling dorsal cuticle (PCM); B–E = pores on the hatchling dorsal cuticle (SEM). Scale bars in Μm

Fig. 3. Richtersius tertius sp. n. claws. A–B = claws II and IV (PCM); C–D = claws III and IV (SEM). Filled indented arrowheads indicate lateral expansions positioned posteriorly to the laminar stalk connecting the claw to the lunula, empty indented arrowheads indicate the double muscle attachments under the claws, empty flat arrowheads indicate the divided cuticular bar, filled flat arrowhead indicates the horseshoe structure connecting the

Fig. 4. Richtersius tertius sp. n. mouth opening with peribuccal structures and oral cavity armature of an adult (SEM). A = general view of the mouth opening; B = details of the oral cavity armature. Asterisk indicates the circular sensory field, filled arrow indicates the divided peribuccal velum/lamina, filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the teeth of the second band, dorsal and ventral teeth of

Fig. 5. Richtersius tertius sp. n. buccal apparatus (PCM). A = dorsal projection of the entire buccal apparatus; B = later view of the buccal crown; C = ventral view of the buccal crown; D = stylet support insertion point of the buccal apparatus; E = macroplacoid morphology visible in ventral view. Filled arrows indicate the ventro-lateral triangular apophysis, filled indented arrowheads indicate dorsal spikes, empty indented arrowheads indicate constrictions in the macroplacoids, the empty arrow indicates the cuticular hook on the dorsal apophysis, the black filled arrow indicates the cuticular hook on the ventral apophysis, filled flat arrowheads indicate the second band of teeth in the oral cavity. Scale bars in Μm

Fig. 6. Richtersius tertius sp. n. egg chorion morphology (PCM). A–B = egg midsection un- der 400× magnification; C = egg surface under 1000× magnification; D–E = egg processes midsection under 1000× magnification. Filled flat arrowheads indicate flexible terminal portion of the egg process, filled indented arrowheads indicate the refracting dots covering the egg surface between processes (in the majority of eggs this character is not visible as the

Fig. 7. Richtersius tertius sp. n. egg chorion morphology (SEM).A – entire egg; B–C = Details of egg surface and processes; D = magnification of the egg process. Filled flat arrowheads