Tenuibiotus zandrae, Stec & Tumanov & Kristensen, 2020

Stec, Daniel, Tumanov, Denis V. & Kristensen, Reinhardt Møbjerg, 2020, Integrative taxonomy identifies two new tardigrade species (Eutardigrada: Macrobiotidae) from Greenland, European Journal of Taxonomy 614, pp. 1-40 : 15-26

Tenuibiotus zandrae sp. nov.

urn:lsid:zoobank.org:act:99D6163D-E8E6-4223-B975-F68AA2268304

Figs 10–19

Etymology

We take great pleasure in dedicating this new species to the friend of the first and third authors. Zandra Maria Skandrup Sigvardt, who recently completed her PhD studies working on crustaceans (Section of Biosystematics) at the Natural History Museum of Denmark in Copenhagen.

Material examined

62 animals (including 18 simplex) and 171 eggs. Specimens mounted on microscope slides in Hoyer’s medium (49 animals + 161 eggs), fixed on SEM stubs (including two extracted buccal apparatuses) (10 +10), and processed for DNA sequencing (3 +0).

Holotype

GREENLAND • Disko Island , Østerlien; 69°15′17″ N, 53°30′46″ W; 30 m a.s.l.; sample of moss collected from the rock in arctic tundra; IZiBB, slide GL.011.17 .

Paratypes

GREENLAND • 58 paratypes; same collection data as for holotype; IZiBB, slides GL.011.08 to 011.09, 011.16 to 011.19, 011.22 to 011.23, 011.27 to 011.28, SEM stubs GL.012.06 to 012.07, 012.13 • 171 eggs; same collection data as for holotype; IZiBB, slides GL.011.10 to 011.15, 011.20 to 011.21, 011.24 to 011.26, 011.29, SEM stub GL.12.13 .

Description

Animals (measurements and statistics in Table 4)

Body transparent in juveniles and whitish in adults, after fixation in Hoyer’s medium transparent ( Fig. 10 ). Eyes present in specimens mounted in Hoyer’s medium. Body cuticle without pores but covered with fine granulation including ventral side of the body and all legs ( Figs 11–13 ). Granulation is distributed uniformly on the body ( Fig. 11 A–B, E) but sometimes, especially in larger specimens, random patches without granulation are present on the body cuticle ( Fig. 11 C–D, F). Patches of dense granulation composed of cushions with aggregated granules present on all legs ( Figs 12–13 ). A patch of clearly visible granulation, is present on the external surface of legs I–III just below the claws ( Figs 12A , 13A ). A pulvinus is absent on the internal surface of legs I–III, whereas a patch of dense granulation is present and wider than the patch on the external leg surface ( Figs 12B , 13B ). A patch of dense granulation on legs IV is always visible and covers the dorsal and the lateral sides of hind legs ( Figs 12C , 13 C–D). Claws slender, of the Tenuibiotus type ( Fig. 14 ). Primary branches with distinct accessory points, a long common tract, and with an evident stalk connecting the claw to the very wide lunula ( Fig. 14 ). Lunulae I–III smooth ( Fig. 14A, C ), whereas lunulae IV exhibit clear dentation ( Fig. 14B, D ). The horseshoe structure connecting the anterior and the posterior claw is present and is visible only in PCM ( Fig. 14B ).

Mouth antero-ventral, followed by ten peribuccal lamellae ( Figs 15A , 16 ). Bucco-pharyngeal apparatus of the Macrobiotus type ( Figs 15A , 17A ). Under LCM, only the second and third bands of teeth visible, with the second band being faintly marked ( Fig. 15 B–C). However, in SEM all three bands of teeth are visible, with the first band being situated at the base of peribuccal lamellae and composed of 1–2 rows of small, cone-shaped teeth arranged around the oral cavity ( Figs 16 , 17B ). The second band of teeth is situated between the ring fold and the third band of teeth and comprises 3–6 rows of small, cone-shaped teeth ( Figs 15 B–C, 16). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the buccal tube opening ( Figs 15 B–C,16). The third band of teeth is discontinuous and divided into dorsal and ventral portions. Under LCM, the dorsal teeth are seen as three distinct transversal ridges of which the median tooth is triangular and is wedged between the lateral teeth ( Fig. 15B ). The ventral teeth under LCM appear as three to four separate roundish teeth, largest than those of the second band ( Fig. 15C ), only sometimes they can be seen as one faintly marked, elongated tooth. In SEM, both dorsal and ventral teeth are also clearly distinct ( Fig. 16 ). Under SEM, the medio-dorsal tooth is the largest within the third band and is positioned anteriorly with respect to the lateral teeth ( Fig. 16A ), whereas the ventral portion consist of cone-shaped teeth of which the lateral ones are larger than the medial ones ( Fig. 16B ). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a small triangular microplacoid ( Fig. 15A , D–E). The macroplacoid length sequence is 2<1. The first macroplacoid exhibits central constriction whereas the second macroplacoid is sub-terminally constricted ( Figs 15 D–E, 17C).

Eggs (measurements and statistics in Table 5)

Laid freely, whitish, spherical or ovoid ( Figs 18 A–B, 19A). The surface between processes is smooth, with thickenings/striae often radiating from the processes bases ( Figs 18 B–D, 19B–C, E–F). Under PCM, these thickenings together with labyrinthine layer within chorion are visible as dark dots and lines on the surface between processes, whereas under SEM they are smooth striae coming out of the process bases ( Figs 18 B–D and 19B–C, E–F, respectively). Under SEM, the surface between processes and between the peribasal striae is covered with micropores ( Fig. 19 E–F). Processes are of conical shape, with elongated apices which are sometimes bi- or trifurcated ( Figs 18 E–H, 19A–D). The labyrinthine layer between the process walls is clearly visible under LCM as a reticular pattern with sinuous margins ( Fig. 18 C–D). The elongated meshes decrease in size from the base to the top of the processes ( Fig. 18 C–D). Under SEM, the surface of the processes is covered with small tubercles, whereas the surface of the elongated apices is smooth ( Fig. 18 B–E).

Reproduction

The examination of specimens freshly mounted in Hoyer’s medium did not revealed any spermathecae or testes filled with spermatozoa. Also, male secondary sexual dimorphism traits such as lateral gibbosities on legs IV were absent. Thus, reproductive mode could not be unambiguously determined.

DNA sequences

We obtained sequences of good quality for all four of the above-mentioned DNA markers. Sequences of each marker were represented by single haplotypes:

18S rRNA sequence (GenBank: MN443040 ), 1035 bp long;

28S rRNA sequence (GenBank: MN443035 ), 780 bp long;

ITS-2 sequence (GenBank: MN443038 ), 439 bp long;

COI sequence (GenBank: MN444827 ), 658 bp long.

Morphological observations of comparative material

Amended description of Tenuibiotus voronkovi

The examination of the holotype and paratype of T. voronkovi under LCM revealed the presence of granulation on the body cuticle. Faint granulation with small and uniformly sized granules is regularly arranged and covers the dorso-medial region of the body, from its cephalic to the caudal end ( Fig. 20A ). On the dorso-lateral surface of the body, granulation is unevenly distributed and resembles patches of granules, within which granule size gradually increases in the dorsal to lateral direction ( Fig. 20B ). The granulation is absent or not visible in LCM on the ventral side of the body. Similarly, the granulation is absent on the legs except a typical dense granulation patch on the external and internal surface of the legs near the claws. However, note that this observation was made on two different specimens (not very well oriented/positioned and stretched on the slide) thus, the certain distribution pattern of body granulation requires a further examination when a new population of T. voronkovi becomes available. As in the original description, the eggs of T. voronkovi have small conical processes with elongated and flexible apices which are often folded and not clearly visible or even broken. ( Figs 21A , C–J, 22). The process walls are smooth, without any obvious thickenings or tubercles ( Fig. 22 ) but with obvious annulation and with the labyrinthine layer within process walls, visible under LCM as roundish polygonal reticulation ( Fig. 21C , E–F), on one egg being abnormally developed and visible more like pores than true reticulation ( Fig. 21D ). Under SEM, the surface between processes is covered with short irregular striae/ridges/wrinkles which often radiate from the process bases, with small micropores randomly scattered in between them ( Fig. 22 ). However, under PCM the surface is visible as being covered with dark dots which are probably the thickenings of the labyrinthine layer within the chorion ( Fig. 21B ).

The morphological analysis conducted on two populations designated as “ T. voronkovi ” by Zawierucha et al. (2016a), from Edgeøya and Nordaustlandet (islands within the Svalbard archipelago, Norway), showed distinct differences in cuticle morphology in comparison to the T. voronkovi type series. Specifically, animals of the Edgeøya population exhibit faint, dense and uniformly distributed granulation on the whole dorso-lateral cuticle from its cephalic to the caudal end (excluding ventral and leg cuticle) ( Fig. 23A ), whereas this granulation is absent or not visible under LCM in animals of the Nordaustlandet population. The morphology of egg processes in both these populations is very similar: specifically, processes are of a conical shape with elongated apices, with the labyrinthine layer between the process walls clearly visible under LCM as a reticular pattern with sinuous margins and elongated meshes decreasing in size from the base to the processes top in most cases ( Figs 23 B–D, 24). Other traits are as described by Zawierucha et. al. (2016a) however, it should be noted that as for the similarly to T. voronkovi , no more conclusions can be made based on this material due to the bad condition of the slides, with bubbles of air and crystalized Hoyer which prevent further investigation and limit the number of specimens suitable for imaging.

Figures

Fig. 10. Tenuibiotus zandrae sp. nov. Habitus. Dorso-ventral projection (holotype, IZiBB, slide GL.011.17, Hoyer’s medium, PCM). Scale bars in μm.

Fig. 11. Tenuibiotus zandrae sp. nov. Body granulation seen in PCM (paratypes). A–B. Uniformly distributed granulation on the dorso-cephalic and dorso-caudal part of the body. C–D. Uniformly distributed granulation on the dorso-cephalic and dorso-caudal part of the body with small, random patches of lacking granulation. E–F. Uniformly distributed granulation on the ventral side of the body without and with small random patches lacking granulation, respectively. A–B, E and C–D, F are from two different paratypes. Scale bars in μm.

Fig. 12. Tenuibiotus zandrae sp. nov. Patches of dense granulation on legs seen in PCM (paratypes). A. External granulation on leg III (patch of dense granulation encircled). B. Internal granulation on leg III. C. Granulation on leg IV. Scale bars in μm.

Fig. 13. Tenuibiotus zandrae sp. nov. Patches of dense granulation on legs seen in SEM (paratypes). A. External granulation on leg II (patch of dense granulation encircled). B. Internal granulation on leg III. C. Granulation on leg IV. D. Granulation on legs IV and uniformly distributed body granulation on the dorso-caudal region. Scale bars in μm.

Fig. 14. Tenuibiotus zandrae sp. nov. Claws (paratypes). A–B. Claws II and IV seen in PCM, respectively. C–D. Claws I and IV seen in SEM, respectively. Filled indented arrowhead indicates horseshoe structure connecting the anterior and the posterior claw. Scale bars in μm.

Fig. 15. Tenuibiotus zandrae sp. nov. Buccal apparatus and the oral cavity armature seen in LCM. A. Dorso-ventral projection of the entire buccal apparatus (holotype, IZiBB, slide GL.011.17, PCM). B–C. Oral cavity armature seen in NCM, dorsal (B, paratype) and ventral (C, holotype) view, respectively. D–E. Placoid morphology seen in NCM, dorsal (D) and ventral (E) view, respectively (both holotype). Filled flat arrowheads indicate faintly visible second band of teeth in the oral cavity, empty flat arrowheads indicate the third band of teeth in the oral cavity, empty indented arrowheads indicate central constrictions in the first macroplacoids and the subterminal constriction in the second macroplacoid. Scale bars in μm.

Fig. 16. Tenuibiotus zandrae sp. nov. The oral cavity armature seen in SEM (paratype). The oral cavity armature of a single paratype seen in SEM from different angles, dorsal (A) and ventral (B) view, respectively. Filled indented arrowheads indicate the first band of teeth in the oral cavity, filled flat arrowheads indicate the second band of teeth in the oral cavity, whereas empty flat arrowheads indicate the third band of teeth in the oral cavity. Scale bars in μm.

Fig. 17. Tenuibiotus zandrae sp. nov. Buccal apparatus seen in SEM (paratype). A. Entire buccal apparatus. B. Lateral view of the buccal crown. C. Placoids. Filled indented arrowhead indicate the first band of teeth in the oral cavity, empty indented arrowheads indicate the central constriction in the first macroplacoids and the subterminal constriction in the second macroplacoid. Scale bars in μm.

Fig. 18. Tenuibiotus zandrae sp. nov. Egg chorion morphology seen in PCM. A. Midsection under 400× magnification. B. Surface under 400× magnification. C–D. Surface between processes under 1000× magnification. E–H. Midsections of processes of four different eggs under 1000× magnification. Filled flat arrowheads indicate thickenings/striae which are visible as dark dots and lines on the surface between processes. Scale bars in μm.

Fig. 19. Tenuibiotus zandrae sp. nov. Egg chorion morphology seen in SEM. A. Entire egg. B. Magnification of the egg surface. C–F. Details of the egg processes and surface between them. Filled flat arrowheads indicate thickenings/striae on the surface between processes and filled indented arrowheads indicate small tubercles on the process walls. Scale bars in μm.

Fig. 20. Tenuibiotus voronkovi (Tumanov, 2007). Body granulation seen in PCM. A. Uniformly distributed granulation of uniform size on the dorso-medial part of the body (cephalic region, paratype). B. Patch of dorso-lateral granulation composed of granules of different size (holotype). Scale bars in μm.

Fig. 21. Tenuibiotus voronkovi (Tumanov, 2007) Egg chorion morphology seen in PCM. A. Midsection under 400× magnification. B. Surface between processes under 1000× magnification. C–J. Details of egg processes under 1000× magnification. Filled flat arrowhead indicates thickenings/striae/sculpture which are visible as dark dots on the surface between processes and indented empty arrowheads indicate broken apices of the egg processes. Scale bars in μm.

Fig. 22. Tenuibiotus voronkovi (Tumanov, 2007), egg chorion morphology seen in SEM. A. Entire egg. B–D. Details of the egg processes and surface between them. Filled flat arrowheads indicate thickenings/ striae on the surface between processes, filled indented arrowheads indicate elongated and flexible apices of egg processes which are often folded, whereas empty indented arrowheads indicate micro pores on the chorion surface between processes. Scale bars in μm.

Fig. 23. Tenuibiotus cf. voronkovi (Tumanov, 2007) from the Edgeøya population, body cuticle and eggs seen in PCM. A. Uniformly distributed granulation on the dorsal side of the body at the level between leg pairs II and III. B–D. Three different eggs under 1000× magnification. Scale bars in μm.