Macrobiotidae Thulin, 1928

Yuan, Zhimin, Wang, Yan, Liu, Qiuju, Liu, Lijie & Li, Xiaochen, 2022, , a New Tardigrade Species in the Complex from China., Zoological Studies 61 (86), pp. 1-18 : 8-11

Family: Macrobiotidae Thulin, 1928 Genus: Macrobiotus C.A.S. Schultze, 1834

Macrobiotus hupingensis sp. nov. urn:lsid:zoobank.org:act:DF765A1E-F6C6-4044-AE8B-17FAF9D648F2

Etymology: This species is named after the type locality.

Material examined: 238 animals and 46 eggs. Specimens mounted on microscope slides in Hoyer’s medium (223 animals + 42 eggs), fixed on SEM stubs (11 + 4), and processed for DNA sequencing (4 + 0).

Type locality: 30°02'19.1"N, 110°54'45.2"E, 1,065 m asl, the Hupingshan National Nature Reserve, Shimen Country, Hunan Province, China.

Type repository: Holotype (Slide hp1110202 with 29 paratypes), 183 paratypes (Slides: hp I. II. 02. III, the Roman numerals can be substituted by the following numbers: 1–5, 01–25, 1–10, respectively; SEM stub: 11.02) and 40 eggs (slides: hp–I. II. 02. III; SEM stub: 11.02) were deposited at Xiaochen Li’s tardigrade collection, Molecular Ecology, Department of Biology , College of Life Sciences of Shaanxi Normal University, China. Additional paratypes (10 animals) (slides: Slides: hp 2. II. 02. III, the Roman numerals II– III can be substituted by the following numbers: 01–25, 1–10) and 6 eggs (slides: hp–I. II. 02. III) are deposited at the Department of Invertebrate Evolution , Institute of Zoology and Biomedical Research , Faculty of Biology, Jagiellonian University, Poland.

Description of the new species: Animals (measurements and statistics in Table 6): In live animals, body almost transparent in smaller specimens and whitish in larger animals; transparent after fixation in Hoyer’s medium ( Fig. 3 ). Eyes present in live animals and after fixation in Hoyer’s medium. Small round and oval cuticular pores (0.5–1.5 μm in diameter) visible under both PCM and SEM scattered randomly throughout the entire body ( Figs. 4A–D , 5A–D ). Patches of fine granulation on the external surface of legs I– III as well as on the dorsal and dorsolateral sides of leg IV visible in PCM ( Fig. 4B, D ) and SEM ( Fig. 5B, D ). Only pulvinus is present on the internal surface of legs I–III whereas the granulation on the internal surface is absent ( Figs. 4C , 5C ). In addition to the typical patches of leg granulation, other types of cuticular granulation are absent.

Claws slender, of the hufelandi type. Primary branches with distinct accessory points, a long common tract, and an evident stalk connecting the claw to the lunula ( Fig. 6A–B, 6D–E ). Lunulae on legs I–III smooth, whereas on legs IV usually clearly dentate ( Fig. 6A–F ). Dark areas under each claw on legs I– III are often visible in PCM ( Fig. 6A ). Paired muscle attachments and faintly visible continuous cuticular bars above them on legs I–III are often visible both with PCM and SEM ( Fig. 6A, D ), whereas the horseshoeshaped structure connecting anterior and posterior claw IV is visible only in PCM ( Fig. 6B–C ).

Mouth antero-ventral: Buccal apparatus of the Macrobiotus type ( Fig. 7A ), with the ventral lamina and ten peribuccal lamellae ( Fig. 8A–B ). The oral cavity armature (OCA) was well developed and composed of three bands of teeth, from which only the second and third bands were always clearly visible under PCM ( Fig. 7B–C ), whereas the first band was only visible under SEM ( Fig. 8A–B ). The first band of teeth is composed of numerous small teeth visible under SEM as cones ( Fig. 8A–B ), arranged in several rows, situated anteriorly in the oral cavity, just behind the bases of the peribuccal lamellae. The second band of teeth is situated between the ring fold and the third band of teeth and comprises 3–4 rows of teeth visible with PCM as granules ( Fig. 7B–C ), and as cones in SEM ( Fig. 8A– B ) but larger than those in the first band. The posterior row of teeth within the second band seems to comprise larger teeth than the previous anterior rows ( Fig. 8A– B ). 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. 7B–C , 8A–B ). The third band of teeth is divided into the dorsal and ventral portions. Under PCM, the dorsal teeth are seen as three distinct transverse ridges, whereas the ventral teeth appear as two separate lateral transverse ridges, between which one large tooth (circular in PCM) is visible ( Fig. 7B–C ). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids (2 <1) and a microplacoid positioned close to them (i.e., the distance between the second macroplacoid and the microplacoid is shorter than the microplacoid length; Fig. 7A, D ). The first macroplacoid is anteriorly narrowed and constricted in the middle, whereas the second has a subterminal constriction ( Fig. 7D–E ).

Eggs (measurements and statistics in Table 7): Laid freely, white, spherical with conical processes surrounded by one row of areolae ( Fig. 9A–B ). In SEM, multiple rings of faintly visible annulation were visible on the entire process ( Fig. 10B, E ), although in some processes, annulation was present only in the upper portion of the process ( Fig. 10A–F ) (annulation not visible in PCM because it was obscured by the eminent labyrinthine layer). The upper parts of the processes are smooth and not covered with granulation ( Fig. 10B, C, E–F ). The labyrinthine layer between the process walls is present and visible as reticulation with circular/ellipsoidal meshes throughout the entire process ( Figs. 9A–B , 10A–F ). Small areas without reticulation are rarely present in some processes ( Fig. 9B ). The upper part of the process is often elongated into short flexible apices ( Figs. 9C–F , 10A–C, E–F ), which are occasionally absent or bifurcated and sometimes have bubble-like structures ( Figs. 9C–F , 10A–F ). The base of the processes extends into the six (only sometimes five) arms that form areolae rims ( Fig. 9A–B ). Each process is surrounded by six (only sometimes five) hexagonal areolae ( Figs. 9A–B , 10A–C ), which are occasionally falsely subdivided in the middle into two areolae by a thin thickening perpendicular to the process base ( Figs. 9A–B , 10B ). Areolae rims (walls) thick and usually flat ( Fig. 10A, C ), with the labyrinthine layer inside the rims visible as bubbles in PCM ( Fig. 9B ). Areolae rims also delimit the areolae at the bases of processes, which forms an irregular collar around process bases ( Figs. 9B , 10A, C ) and makes the process bases penta- or hexagonal in the top view ( Figs. 9A–B , 10A–C ). The areola surface has wrinkles that are faintly visible under PCM ( Fig. 9A–B ) but clearly visible under SEM ( Fig. 10B–D ). Micropores are present within the areolae, but they are distributed only around the areolae rims and usually absent in the central part of the areola ( Fig. 10B–D ).

Remarks: The wall of egg processes is perforated with a small number pores, which can only be seen in SEM ( Fig. 10C–F ).

Reproduction: The species is dioecious. Spermathecae in females as well as testes in males have been found to be filled with spermatozoa, clearly visible under PCM up to 24 hours after mounting in Hoyer’s medium ( Fig. 11A–B ). The species exhibits secondary sexual dimorphism in the form of clearly visible lateral gibbosities on hind legs in males ( Fig. 11B ).

DNA sequences and intraspecific genetic distances

We obtained sequences for all four molecular markers amplified in this study. All sequenced fragments were represented by two haplotypes except the 18S rRNA, in which only one single haplotype was present:

18S rRNA sequences (GenBank: MW183923); 1749 bp long; 1 haplotype was found.

28S rRNA sequences (GenBank: MZ470349–50); 811 bp long; 2 haplotypes were found, separated by a p -distance of 0.1%.

ITS-2 sequences (GenBank: MZ474842–3); 432 bp long; 2 haplotypes were found, separated by a p -distance of 0.2%.

COI sequences (GenBank: MW186952 and MW187003); 684 bp long; 2 haplotypes were found, separated by a p -distance of 0.8%.

Figures

Fig. 3. Macrobiotus hupingensis sp. nov. from China seen in PCM (holotype, Hoyer’s medium) – habitus, adult specimen in dorso-ventral projection. Scale bar in μm.

Fig. 4. Macrobiotus hupingensis sp. nov. from China seen in PCM (paratypes) – body and leg cuticle morphology seen with PCM: A, cuticle on the last body segment without caudal band of granulation; B, granulation on the external surface of leg II; C, internal surface of leg III with evident pulvinus; D, granulation on dorsal surface of leg IV. Filled indented arrowheads indicate granulation on the legs, arrow indicates pulvinus on the III leg. Scale bar in μm.

Fig. 5. Macrobiotus hupingensis sp. nov. from China seen in SEM (paratypes) – body and leg cuticle morphology seen with SEM: A, cuticle on the last body segment without caudal band of granulation; B, granulation on the external surface of leg III; C, internal surface of leg II with evident pulvinus; D, granulation on dorsal surface of leg IV. Scale bar in μm.

Fig. 6. Macrobiotus hupingensis sp. nov. from China (paratypes) – claw morphology: A–B, claws II and IV seen with PCM; C, magnification of lunulae IV seen with PCM; D–E, claws I and IV seen with SEM; F, magnification of lunulae IV seen with SEM. Indented arrowhead indicates dark circular areas under lunulae on the first three pairs of legs, double arrowheads indicate double muscle attachments under claws, arrows indicate cuticular bar above muscle attachments, bent arrows indicate horseshoe structure connecting the anterior and the posterior claw. Scale bars in μm.

Fig. 7. Macrobiotus hupingensis sp. nov. from China (paratype) – buccal apparatus seen with PCM: A, an entire buccal apparatus; B–C, the oral cavity armature, dorsal and ventral teeth, respectively; D–E, placoid morphology, dorsal and ventral placoids, respectively. Arrows indicate the second band of teeth, indented arrowheads indicate the third band of teeth, double arrowhead indicates central and subterminal constrictions in the first and second macroplacoid. Scale bars in μm.

Fig. 8. Macrobiotus hupingensis sp. nov. from China (paratype) – the oral cavity armature seen with SEM: A–B, the oral cavity armature of a single specimen seen with SEM from different angles showing dorsal and ventral portion, respectively. Arrows indicate the first band of teeth, indented arrowheads indicate the second band of teeth, double arrowhead indicates the third band of teeth. Scale bars in μm.

Fig. 9. Macrobiotus hupingensis sp. nov. from China (paratypes) – eggs seen with PCM: A–B, surface under ×1000 magnification of egg; C–F, midsections of four different egg processes.Arrows indicate thickening perpendicular to the process base that divides the areola in the middle, double arrowhead indicates areas of the egg processes without reticulation/labyrinthine layer, and indented arrowheads indicate irregular collar around process bases. Scale bars in μm.

Fig. 10. Macrobiotus hupingensis sp. nov. from China (paratypes) – eggs seen with SEM: A, entire view of the egg (missing a process); B–F, details of the egg surface between processes, areolation and egg processes. The arrows indicate thickening perpendicular to the process base, which divides the areola in the middle, indented arrowheads indicate irregular collar around process bases, and double arrowheads indicate pores on the surface of egg processes. Scale bars in μm.

Fig. 11. Macrobiotus hupingensis sp. nov. from China (paratypes) – reproduction (PCM): A, spermatheca (seminal vesicle) filled with spermatozoa and visible in females freshly mounted in Hoyer’s medium; B, testis filled with sperm visible in a male freshly mounted in Hoyer’s medium. The indented arrowhead indicates the female spermathecae, double arrowhead indicates the testis, and the arrows indicate gibbosity on the IV leg. Scale bars in μm.