Macrobiotidae Thulin, 1928

Stec, Daniel, 2022, An Integrative Description of Two New Species (Tardigrada: Eutardigrada: Macrobiotidae) with Updated Genus Phylogeny., Zoological Studies 61 (85), pp. 1-30 : 5-18

Family: Macrobiotidae Thulin, 1928 Genus: Mesobiotus Vecchi, Cesari, Bertolani, Jönsson, Rebecchi and Guidetti, 2016

Mesobiotus diegoi sp. nov. ( Figs. 1–6 ; Tables 3–4) urn:lsid:zoobank.org:act:2C2D67EE-A0CC-46EC-A5E0-30B71554A8C8

Material examined: 68 animals, 28 eggs mounted on microscope slides in Hoyer’s medium (some of the eggs were embryonated), eight eggs examined in SEM and two specimens processed for DNA sequencing.

Type locality: 29°16'5.1"S, 29°30'48.6"E; 1756 m asl: Giants Castle Game Reserve, Drakensberg National Park, KwaZulu-Natal, Republic of South Africa, lichen growing on rock in mountainous grassland, coll. Witold Morek and Bartłomiej Surmacz, 16 September 2018.

Etymology: The species is named after my good friend Diego Fontaneto, a world-known rotiferologist and meiofauna specialist working in the Water Research Institute of the National Research Council (Verbania, Italy).

Type depositories: Holotype (ñ): slide ZA.001.06 with 2 paratypes and 62 paratypes (slides: ZA.001.*, where the asterisk can be substituted by any of the following numbers: 01–04, 07–08) and 20 eggs (slides: ZA.001.*: 09–11) are deposited at the Institute of Systematics and Evolution of Animals , Polish Academy of Sciences , Sławkowska 17, 31-016, Kraków , Poland, whereas 3 paratypes (slide: ZA.001.05) and 6 eggs (slide: ZA.001.12) are deposited at the Department of Animal Taxonomy and Ecology , Adam Mickiewicz University in Poznań , Umultowska 89, Poznań, Poland.

Animals (measurements and statistics in Table 3): Body almost transparent in small specimens and whitish in adults; after fixation in Hoyer’s medium body transparent ( Fig. 1A ). Eyes present in alive animals and dissolved by Hoyer’s medium in approximately 65% of all mounted specimens. Body cuticle smooth, i.e., without pores, body granulation, sculpturing, or tubercles. A fine granulation is present on the external surface of legs I–III ( Fig. 1B ) that extends through the frontal leg surface to the internal surface where it is present mainly in the cuticular fold ( Fig. 1C ). Granulation is also present on the lateral and dorsal surfaces of legs IV ( Fig. 1D ). A cuticular bulge, similar to a pulvinus, is present on the internal surface of legs I–III ( Fig. 1C ). Claws of the Mesobiotus type, with a peduncle connecting the claw to the lunula, a basal septum, and well-developed accessory points situated parallel to the primary branch ( Fig. 2A–B ). Lunulae under claws I–III smooth ( Fig. 2A ) and those under claws IV slightly dentate ( Fig. 2B–C ). A single continuous cuticular bar with shadowed extensions narrowing toward double muscle attachments is present below claws I–III ( Figs. 1C , 2A ), while a horseshoeshaped structure connects the anterior and posterior lunulae on claws IV ( Fig. 2B ).

Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type ( Fig. 3A ), with ventral lamina and ten small peribuccal lamellae. The oral cavity armature well developed and consists of three bands of teeth ( Fig. 3B–C ). The first band of teeth is composed of numerous small granules arranged in several discrete rows located anteriorly in the oral cavity, just behind the bases of the peribuccal lamellae ( Fig. 3B–C ). The second band of teeth is located between the ring fold and the third band of teeth and is composed of ridges parallel to the main axis of the buccal tube that are larger than those in the first band ( Fig. 3B–C ). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the opening of the buccal tube ( Fig. 3B–C ). The third band of teeth is discontinuous and divided into a dorsal and ventral portion. Under PCM, dorsal teeth are visible as two lateral and one median transverse ridges/ crests ( Fig. 3B ) whereas ventral teeth consist of two lateral transverse ridges/crests between which usually one round or trapezoidal ventro-median tooth is present ( Fig. 3C ). Sometimes, additional granular teeth are present between the second and third band of teeth ( Fig. 3C ). Pharyngeal bulb ovoid ( Fig. 3A ), with triangular apophyses, three rod-shaped macroplacoids, and a large, elongated drop-shaped microplacoid placed close to the third macroplacoid ( Fig. 3D–E ). The macroplacoid length sequence is 2 <3 <1. The first macroplacoid is anteriorly narrowed, and the third has a clearly defined subterminal constriction ( Fig. 3D–E ).

Eggs (measurements and statistics in Table 4): White, laid free, spherical in shape and equipped with large and long conical processes ( Figs. 4A–F , 5A–F ). Egg surface between the processes without areolation. In PCM the egg surface between processes seems to be rough with dark bars/wrinkles and faintly light refracting dots ( Fig. 4A–B ) whereas in SEM the surface is clearly wrinkled with bulging wrinkles radiating out from the process bases ( Fig. 5E–F ). Small pores (up to 0.3 µm) are scattered across the interprocess surface and are mainly distributed in the depression between the bulged wrinkles. The pores are clearly visible in SEM ( Fig. 5A–E ), but under PCM they are seen as the mentioned faintly light-refracting dots ( Fig. 4 ). The bases of egg processes are surrounded by a crown of strong thickenings that are evident only in PCM ( Fig. 4A–B ). The egg processes are evenly spaced, having flexible upper portion often elongated into short filament (only sometimes bifurcation or trifurcation is present; Figs. 4C–F , 5A–C ). Often, within the upper portion of the egg processes, below the flexible part a bubble-like structure is present and visible in the midsection of the process ( Fig. 4C–F ). In SEM only the surface of this upper part of the egg process (about 50% of the entire process length) is punctured with micropores (0.15–0.20 µm in size). The labyrinthine layer is visible under PCM as a reticulum in the process walls, with varying mesh size uniformly distributed within the process walls, except for the ring of basal meshes that are clearly larger than the meshes above them ( Fig. 4A–B ). In SEM, the process walls are evenly annulated at their entire length ( Fig. 5A–F ). The flexible upper portions of the egg processes are smooth and not covered with granules ( Fig. 5C ).

Reproduction: The new species is dioecious. Spermathecae filled with sperm have not been found in gravid females on freshly prepared slides. However, in males, the testes, filled with sperm, are clearly visible under PCM up to 48 hours after mounting in Hoyer medium ( Fig. 6 ). The new species does not exhibit male secondary sexual dimorphism traits such as lateral gibbosities on legs IV.

DNA sequences:

The sequences obtained for all four molecular markers analysed in this study were of good quality and were represented by single haplotypes.

The 18S rRNA sequences (GenBank: OP142526, OP142527), 1020 bp long;

The 28S rRNA sequences (GenBank: OP142520, OP142521), 712 bp long;

The ITS-2 sequences (GenBank: OP142514, OP142515), 354 bp long;

The COI sequences (GenBank: OP143857, OP143858), 658 bp long.

Mesobiotus maklowiczi sp. nov. ( Figs. 7–12 ; Tables 5–6) urn:lsid:zoobank.org:act:15A610D7-7997-4491-92EA-AE9F8D6CAB9A

Material examined: 29 animals, 51 eggs mounted on microscope slides in Hoyer’s medium (some of the eggs were embryonated), 10 eggs examined in SEM and two specimens processed for DNA sequencing.

Type locality: 33°20'32"S, 21°53'31"E; 1004 m asl: Groot Swartberg Nature Reserve, Western Cape, Republic of South Africa, lichen growing on rock, coll. Witold Morek and Bartłomiej Surmacz, 6 September 2018.

Etymology: The species is named after Robert Makłowicz, who is a journalist, historian, and culinary expert that beautifully promotes European cuisine and slow food. He lives in Kraków and runs his own YouTube channel that I enjoy watching. He is also a hat lover, and the egg processes of the new species resemble a funny peaked hat that Robert would be surely eager to try on.

Type depositories: Holotype (ñ): slide ZA.002.01 with 1 paratype and 25 paratypes (slides: ZA.002.*, where the asterisk can be substituted by any of the following numbers: 02–05) and 46 eggs (slides: ZA.002.*: 07–13) are deposited at the Institute of Systematics and Evolution of Animals , Polish Academy of Sciences , Sławkowska 17, 31–016, Kraków , Poland whereas 2 paratypes (slide: ZA.002.06) and 5 eggs (slide: ZA.002.14) are deposited at the Department of Animal Taxonomy and Ecology , Adam Mickiewicz University in Poznań , Umultowska 89, Poznań, Poland.

Animals (measurements and statistics in Table 5): Body almost transparent in small specimens and whitish in adults; after fixation in Hoyer’s medium body transparent ( Fig. 7A ). Eyes present in alive animals and dissolved by Hoyer’s medium in approximately 90% of all mounted specimens. Body cuticle smooth, i.e., without pores, body granulation or tubercles, but a fine, poorly visible network-like sculpture is present on the dorsal cuticle ( Fig. 7D ). A fine granulation present on the external surface of legs I–III ( Fig. 7B ), whereas on the internal surface the granulation is absent ( Fig. 7C ). Granulation is also present on the lateral and dorsal surfaces of legs IV ( Fig. 7D ). A cuticular bulge, similar to a pulvinus, is present on the internal surface of legs I–III ( Fig. 7C ). Claws of the Mesobiotus type, with a peduncle connecting the claw to the lunula, a basal septum, and well-developed accessory points situated parallel to the primary branch ( Fig. 8A–B ). Lunulae under all claws smooth ( Fig. 8A–B ). A single continuous cuticular bar with shadowed extensions narrowing toward double muscle attachments is present below claws I–III ( Figs. 7C , 8A ), while a horseshoeshaped structure connects the anterior and posterior lunulae on claws IV ( Fig. 8B ).

Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type ( Fig. 9A ), with ventral lamina and ten small peribuccal lamellae. The oral cavity armature well developed and composed of three bands of teeth ( Fig. 9B–E ). The first band of teeth is composed of numerous small granules arranged in several discrete rows situated anteriorly in the oral cavity, just behind the bases of the peribuccal lamellae ( Fig. 9B–E ). The second band of teeth is located between the ring fold and the third band of teeth and is composed of ridges parallel to the main axis of the buccal tube that are larger than those in the first band ( Fig. 9B–E ). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the opening of the buccal tube ( Fig. 9B–E ). The third band of teeth is discontinuous and divided into a dorsal and ventral portion. Under PCM, dorsal and ventral teeth are visible as two lateral ridges / crests and one median transverse ridge / crest ( Fig. 9B– E ). Sometimes, additional granular teeth are present between the second and third band of teeth ( Fig. 9D–E ) or even below the third band of teeth further toward the pharynx in the buccal tube ( Fig. 9C ). Pharyngeal bulb ovoid ( Fig. 3A ), with triangular apophyses, three rod-shaped macroplacoids, and a drop-shaped microplacoid placed close to the third macroplacoid ( Fig. 9F–G ). The macroplacoid length sequence is 2 <3 <1. The first macroplacoid is anteriorly narrowed and the third has a clearly defined subterminal constriction ( Fig. 9F–G ).

Eggs (measurements and statistics in Table 6): White, laid free, spherical in shape and equipped with large, evenly spaced processes in the shape of wide cones with collars and with the distal part thinned and flexible ( Figs. 10A–F , 11A–F ). Egg surface between the processes without areolation. In PCM and in SEM the egg surface between processes comprises a system of irregularly distributed ridges and small pores between them ( Figs. 10A–D , 11C–D ). The structure may resemble reticulation; however, the ridges are mainly wider than pores diameter (pores diameter range: 0.2–0.6 µm; Figs. 10A–D , 11C–D ). In SEM the ridges have a rough surface, but it cannot be excluded that this might be a preparation artifact or dirt ( Fig. 11C–E ). Basal part of the processes with well-developed collar elevated above the egg surface ( Figs. 10A–D , 11A–E ). From the top view the collar gives the impression that the processes base is slightly pentagonal in shape ( Figs. 10A–D , 11A–E ). Faint dark thickenings are present around the processes bases under the collar and visible only in PCM and only when the collar is folded back ( Fig. 10D ). The labyrinthine layer is visible under PCM as a reticulum in the process walls, with varying mesh sizes uniformly distributed within the process walls ( Fig. 10A–D ). The walls of the processes are punctured with large pores (1.0–3.0 µm in size) that are located around the base of the process just above the collar ( Figs. 10A– D , 11A–F ). The distal portion of the processes is usually strongly elongated and flexible, often containing internal bubble-like structures visible in the process midsection ( Fig. 10F ). The flexible upper portions of egg processes are smooth and not covered with granules ( Fig. 11C–D ), and these top portions rarely can be bi- or trifurcated. The egg surface under the processes is covered by small granulation, which can be visible only in SEM through the large pores perforating the process wall ( Fig. 11F ).

Reproduction: The new species is dioecious. Spermathecae filled with sperm have not been found in gravid females on freshly prepared slides. However, in males, the testes, filled with sperm, are clearly visible under PCM up to 48 hours after mounting in Hoyer medium ( Fig. 12 ). The new species does not exhibit male secondary sexual dimorphism traits such as lateral gibbosities on legs IV.

DNA sequences:

The sequences obtained for only three out of all four molecular markers analysed in this study were of good quality and were represented by single haplotypes. Several attempts to amplify the ITS-2 marker for the new species failed, which prevented obtaining these sequences.

The 18S rRNA sequences (GenBank: OP142524, OP142525), 989 bp long;

The 28S rRNA sequences (GenBank: OP142518, OP142519), 759 bp long;

The COI sequences (GenBank: OP143855, OP143856), 678 bp long.

Phylogenetic results

Both phylogenetic analyses resulted with trees of similar topology and well-supported nodes in which a monophyletic and paraphyletic clades of non-Antarctic and Antarctic taxa can be distinguished, respectively ( Fig. 13 ). The phylogenetic investigation did not recover the M. harmsworthi and M. furciger morpho-groups to be monophyletic since the respective representatives of these two groups are intermixed in the presented phylogeny ( Fig. 13 ). Both new species described in this study clustered together with other Mesobiotus taxa from tropical and subtropical regions. The analysis recovered Mesobiotus maklowiczi sp. nov. to be the closest relative of Mesobiotus anastasiae Tumanov, 2020 ( Fig. 13 ). While Mesobiotus diegoi sp. nov. stays in sister relationship to the whole clade comprising tropical and subtropical species, namely: Mesobiotus imperialis Stec, 2021 , Mesobiotus philippinicus Mapalo, Stec, Mirano-Bascos and Michalczyk, 2016 , Mesobiotus fiedleri Kaczmarek, Bartylak, Stec, Kulpa, M. Kepel, A. Kepel and Roszkowska, 2020 , M. maklowiczi sp. nov., M. anastasiae , Mesobiotus radiatus ( Pilato, Binda and Catanzaro, 1991) , Mesobiotus ethiopicus Stec and Kristensen, 2017 , Mesobiotus datanlanicus Stec, 2019 , Mesobiotus insanis Mapalo, Stec, Mirano-Bascos and Michalczyk, 2017 , Mesobiotus romani Roszkowska, Stec, Gawlak and Kaczmarek, 2018 . In this study, newly analysed M. peterseni is in a sister relationship with the other three taxa, namely: M. harmsworthi , Mesobiotus occultatus Kaczmarek, Zawierucha, Buda,

Stec, Gawlak, Michalczyk and Roszkowska, 2018 and unspecified species of the M. harmsworthi morpho-group from Russia. The Antarctic part of the tree comprises six monophyletic clades akin to species, but only Mesobiotus hilariae Vecchi, Cesari, Bertolani, Jönsson, Rebecchi and Guidetti, 2016 represents a formally named taxon.

Figures

Fig. 1. Mesobiotus diegoi sp. nov. – PCM image of habitus and leg’s cuticle morphology: (A) dorso-ventral projection (holotype); (B) granulation on the external surface of leg II (paratype); (C) granulation and a pulvinus-like cuticular bulge on the internal surface of leg III (holotype); (D) granulation on the dorsal and lateral surface of leg IV (holotype). Filled flat arrowheads indicate cuticular fold and granulation on the internal leg surface. Scale bar in μm.

Fig. 2. Mesobiotus diegoi sp. nov. – PCM images of claws: (A) claws II with smooth lunulae (paratype); (B) claws IV (paratype); (C) lunula IV with dentate margin (paratype). Filled flat arrowhead indicates a single continuous cuticular bar below the claws, empty flat arrowheads indicate paired muscle attachments, and filled indented arrowhead indicates a horseshoe structure connecting the anterior and the posterior claw. Scale bars in μm.

Fig. 3. Mesobiotus diegoi sp. nov. – PCM images of the buccal apparatus: (A) an entire buccal apparatus (paratype); (B–C) the oral cavity armature, dorsal and ventral teeth respectively (paratype); (D–E) placoid morphology, dorsal and ventral placoids, respectively (paratype). Filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth, and empty indented arrowheads indicate subterminal constrictions in the third macroplacoid. Scale bars in μm.

Fig. 4. Mesobiotus diegoi sp. nov. – PCM images of the egg. (A–B) egg surface; (C–F) egg processes midsections. Filled flat arrowheads indicate a ring of large basal meshes in the egg process reticulum (labirynthine layer), empty flat arrowheads indicate crowns of thickenings around the processes bases. Scale bars in μm.

Fig. 5. Mesobiotus diegoi sp. nov. – SEM images of eggs: (A–B) entire view of the egg; (C–D) egg processes; (E–F) details of the egg surface between processes. Scale bars in μm.

Fig. 6. Mesobiotus diegoi sp. nov. – reproduction: male with testis filled with spermatozoa. Scale bars in μm.

Fig. 7. Mesobiotus maklowiczi sp. nov. – PCM image of habitus, leg, and dorsal cuticle morphology: (A) dorso-ventral projection (holotype); (B) granulation on the external surface of leg II (holotype); (C) a pulvinus-like cuticular bulge on the internal surface of leg II (holotype); (D) granulation on the dorsal and lateral surface of leg IV and network-like sculpture in dorsal cuticle (holotype). Empty indented arrowheads indicate network-like sculpture in dorsal cuticle. Scale bar in μm.

Fig. 8. Mesobiotus maklowiczi sp. nov. – PCM images of claws: (A) claws II with smooth lunulae (paratype); (B) claws IV with smooth lunulae (paratype). Filled flat arrowhead indicates a single continuous cuticular bar below the claws, empty flat arrowheads indicate paired muscles attachments, filled indented arrowhead indicates horseshoe structure connecting the anterior and the posterior claw. Scale bars in μm.

Fig. 9. Mesobiotus maklowiczi sp. nov. – PCM images of the buccal apparatus: (A) an entire buccal apparatus (paratype); (B–C) the oral cavity armature, dorsal and ventral teeth respectively (bigger paratype); (D–E) the oral cavity armature, dorsal and ventral teeth respectively (smaller paratype); (F–G) placoid morphology, dorsal and ventral placoids respectively (paratype). Filled flat arrowheads indicate the first band of teeth, empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth, empty indented arrowheads indicate subterminal constrictions in the third macroplacoid. Scale bars in μm.

Fig. 10. Mesobiotus maklowiczi sp. nov. – PCM images of the egg. (A–D) egg surface; (E–F) egg processes midsections. Filled flat arrowheads indicate a collar surrounding the egg process, empty flat arrowheads indicate poorly visible crown of thickenings around the processes bases (visible only when the collar is upfolded), filled indented arrowheads indicate big pores puncturing egg process wall above the collar. Scale bars in μm.

Fig. 11. Mesobiotus maklowiczi sp. nov. – SEM images of eggs: (A–B) entire view of the egg; (C–D) egg processes; (E) details of the egg surface between processes; (F) big pore puncturing egg process wall above the collar. Filled flat arrowheads indicate a collar surrounding the egg process, filled indented arrowheads indicate big pores puncturing egg process wall above the collar. Scale bars in μm.

Fig. 12. Mesobiotus maklowiczi sp. nov. – reproduction: male with testis filled with spermatozoa. Scale bars in μm.

Fig. 13. Maximum likelihood (ML) phylogeny constructed from concatenated sequences (18S rRNA + 28S rRNA + ITS-2 + COI) of the genus Mesobiotus. Numbers above branches indicate bootstrap support values, while Bayesian posterior probabilities (pp) are given below branches. Bootstrap <70 and pp <0.95 are not shown. The taxa newly sequenced in this study are marked with bolded font. Taxa of the M. harmsworthi, M. furciger, and M. montanus morpho-groups are indicated by blue, red, and green font, respectively. The outgroup is indicated in gray font. The scale bar represents substitutions per position.