Macrobiotus engbergi, 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 : 4-15

Macrobiotus engbergi sp. nov.

urn:lsid:zoobank.org:act:C592B357-37F6-4C92-B16A-C28EDB17A231

Figs 1–9

Etymology

We take great pleasure in dedicating this new species to the friend of the third author, Lars Engberg Hansen, who is a teacher emeritus from Qeqertarsuaq and Alluitsup Paa in Greenland and is always happy to help with collecting samples of mosses and lichens for us.

Material examined

112 animals (including 9 simplex) and 108 eggs. Specimens mounted on microscope slides in Hoyer’s medium (98 animals + 103 eggs), fixed on SEM stubs (10+ 5) and processed for DNA sequencing (4+ 0).

Holotype

GREENLAND • ♀; Alluitsup Paa ; 60°28′1.5″N, 45°34′27.8″W; 25 m a.s.l.; mixed sample of moss and lichen collected from rock in arctic tundra; IZiBB, slide GL.052.22 .

Paratypes

GREENLAND • 107 paratypes; same collection data as for holotype; IZiBB, slides GL.052.17 to 052.24, SEM stub 17.08 • 108 eggs; same collection data as for holotype; IZiBB, slides GL.052.09 to 052.16, SEM stub 17.08 .

Description

Animals (measurements and statistics in Table 2)

Body transparent in juveniles and whitish in adults, after fixation in Hoyer’s medium transparent ( Fig. 1A ). Eyes present, visible also in specimens mounted in Hoyer’s medium. Cuticle porous with two types of pores: large (up to 5.0 μm in diameter) lenticular pores of shape resembling paper wrapped candy, with transversal wrinkles in extremities distributed randomly on entire body cuticle and being the biggest on anterior and posterior dorsal region ( Figs 1 B–C, 2); and small round cuticular pores (0.3–0.7 μm in diameter) scattered in between lenticular pores ( Figs 1C , 2B ). Patches of granulation on all legs present ( Fig. 3 ). A patch of clearly visible granulation is present on the external surface of legs I–III ( Fig. 3 A– B). A pulvinus present on internal surface of legs I–III, together with a faint cuticular fold covered with faint granulation and paired muscles attachments which are present just below claws ( Fig. 3 C–D). Both structures are visible only if the legs are fully extended and well oriented on slide. Granulation on legs IV always visible and consists of a single large granulation patch on each leg ( Fig. 3 E–F).

Claws stout, of the hufelandi type ( Fig. 4 ). Primary branches with distinct accessory points, a common tract and with an evident stalk connecting the claw to the lunula ( Fig. 4 ). Lunulae on all legs smooth ( Fig. 4 ). Cuticular bars under claws are absent. Double muscle attachments are faintly marked under LCM but clearly visible under SEM ( Fig. 4A, C ). The horseshoe structure connecting the anterior and the posterior claw is present and is visible only in PCM ( Fig. 4B ) and sometimes also on legs I–III, but in this case inverted and not connecting the external and the internal claw ( Fig. 4A ).

Mouth antero-ventral, followed by ten peribuccal lamellae and a circular sensory lobe, surrounded by the ring of large pores ( Figs 2A , 5A , 6 ). Bucco-pharyngeal apparatus of the Macrobiotus type ( Fig. 5A ). Under LCM, the oral cavity armature is of the patagonicus type, i.e., with only the 2 nd and 3 rd bands of teeth visible ( Fig. 5 B–C). However, in SEM all three bands of teeth are visible, with the first band situated at the base of peribuccal lamellae and composed of a 1–2 rows of small, cone-shaped teeth arranged around the oral cavity ( Fig. 6 ). 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 5 B–C, 6). 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 5 B–C, 6). 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, whereas the ventral teeth appear as two separate lateral transverse ridges and a median roundish tooth ( Fig. 5 B–C). In SEM, both dorsal and ventral teeth are also clearly distinct ( Fig. 6 ). Under SEM, the margins of the dorsal teeth slightly serrated ( Fig. 6A ). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a small, triangular microplacoid ( Fig. 5A , D–E). The macroplacoid length sequence 2<1. The first macroplacoid has a central constriction, whereas the second macroplacoid is sub-terminally constricted ( Fig. 5 D–E).

Eggs (measurements and statistics in Table 3)

Laid freely, yellowish, spherical ( Figs 7A , 8A ). The surface between processes is of the persimilis type, i.e., with the continuous smooth chorion, never with pores or reticulum ( Figs 7 F–G, 8). Under PCM labyrinthine layer is visible as dark dots/thickenings on the surface between processes, whereas under SEM the surface is smooth ( Figs 7 F–G and 8, respectively). Processes are of the inverted goblet shape, with slightly concave trunks and concave terminal discs ( Figs 7 B–C, 8A–C). Terminal discs are round with margins ranging from only serrated to clearly indented ( Figs 7 D–E, 8). Each terminal disc has a distinct concave central area, which may contain some scattered granulation within, and micro-granulations which are always present on the margins (visible only under SEM; Fig. 8 C–D).

Reproduction

The new species is dioecious. Spermathecae in females as well as testis in males have been found to be filled with spermatozoa, clearly visible under LCM up to 24 hours after mounting in Hoyer’s medium. The new species exhibits a male secondary sexual dimorphism trait in the form of evident lateral gibbosities on legs IV ( Fig. 9 ).

DNA sequences

We obtained sequences for all four of the above mentioned DNA markers. Sequences of 18S rRNA and 28S rRNA were represented by single haplotypes, whereas sequences of ITS-2 and COI were represented by two (distance: 0.5%) and three (distance: 1.3–1.8%) haplotypes, respectively:

18S rRNA sequence (GenBank: MN443039 ), 1017 bp long;

28S rRNA sequence (GenBank: MN443034 ), 783 bp long;

ITS-2 haplotype 1 sequence (GenBank: MN443036 ), 374 bp long;

ITS-2 haplotype 2 sequence (GenBank: MN443037 ), 374 bp long;

COI haplotype 1 sequence (GenBank: MN444824 ), 638 bp long;

COI haplotype 2 sequence (GenBank: MN444825 ), 638 bp long;

COI haplotype 3 sequence (GenBank: MN444826 ), 638 bp long.

Genus Tenuibiotus Pilato & Lisi, 2011

Figures

Fig. 1. Macrobiotus engbergi sp. nov. Habitus. A. Dorso-ventral projection (holotype, IZiBB, slide GL.052.22, Hoyer’s medium, PCM). B–C. Cuticular pores on the dorso-cephalic and dorso-caudal part of the body seen in PCM, respectively. Arrowheads indicate small oval pores. Scale bars in μm.

Fig. 2. Macrobiotus engbergi sp. nov. Cuticular pores (paratype). A–B. Cuticular pores on the dorsocephalic and dorso-caudal part of the body seen in SEM, respectively. Arrowheads indicate small oval pores Scale bars in μm.

Fig. 3. Macrobiotus engbergi sp. nov. Cuticular structures on legs (paratypes). A–B. External granulation on leg III and II seen in PCM (A) and SEM (B), respectively. C–D. A cuticular bulge (pulvinus) and a faint cuticular fold, covered by granulation, on the internal surface of legs III seen in PCM (C) and SEM (D), respectively. E–F. Granulation on leg IV seen in PCM (E) and SEM (F). Filled flat arrowheads indicate the cuticular bulge, empty flat arrowheads indicate the faint cuticular fold under the claws whereas filled indented arrowhead indicate double muscle attachments under claws. Scale bars in μm.

Fig. 4. Macrobiotus engbergi sp. nov. Claws (paratypes). A–B. Claws III and IV seen in PCM, respectively. C–D. Claws III and IV seen in SEM, respectively. Filled flat arrowheads indicate double muscles attachments under the claws, empty flat arrowhead indicates inverted horseshoe structure under the external and the internal claw, whereas filled indented arrowhead indicates horseshoe structure connecting the anterior and the posterior claw. Scale bars in μm.

Fig. 5. Macrobiotus engbergi sp. nov. Buccal apparatus and the oral cavity armature seen in PCM (holotype, IZiBB, slide GL.052.22). A. Dorso-ventral projection of the entire buccal apparatus. B–C. Oral cavity armature visible in dorsal (B) and ventral (C) view, respectively. D–E. Placoid morphology visible in dorsal (D) and ventral (E) view, respectively. Filled flat arrowheads indicate the 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 subterminal constriction in the second macroplacoids. Scale bars in μm.

Fig. 6. Macrobiotus engbergi sp. nov. The oral cavity armature seen in SEM (paratypes). A–B. The oral cavity armature 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. 7. Macrobiotus engbergi sp. nov. Egg chorion morphology seen in PCM. A. Midsection under 400× magnification. B–C. Midsection under 1000× magnification. D–E. Terminal discs under 1000× magnification. F–G. Surface under 1000× magnification. Scale bars in μm.

Fig. 8. Macrobiotus engbergi sp. nov. Egg chorion morphology seen in SEM. A. Entire egg. B. Magnification of the egg surface. C–D. Details of the terminal discs. Scale bars in μm.

Fig. 9. Macrobiotus engbergi sp. nov. Secondary sexual dimorphism. A. Female without gibbosities on the hind legs. B. Male with gibbosities on the hind legs. Arrowheads indicate gibbosities. Scale bars in μm.