Macrobiotus vattenrikense, Hulterström & Guidetti & Jönsson & Atherton, 2025

Hulterström, Jens, Guidetti, Roberto, Jönsson, K. Ingemar & Atherton, Sarah, 2025, Integrative taxonomy supports the description of two new species of Macrobiotidae (Tardigrada, Eutardigrada) from Kristianstads Vattenrike Biosphere Reserve in Sweden, European Journal of Taxonomy 1030, pp. 1-52 : 9-18

Macrobiotus vattenrikense sp. nov.

urn:lsid:zoobank.org:act:

Figs 4–8

Diagnosis

Macrobiotus with three bands of teeth in oral cavity armature (OCA): first and second of granules, and third of a transverse crest non-uniform in shape on the dorsal and ventral side, with a dorsal side with one elongated tooth and ventral side with two lateral and two medial teeth. Two macroplacoids, the first with a slight central constriction, the second with a pre-terminal constriction, and a comma-shaped microplacoid. Claws of similar size on all legs, with large, smooth lunulae on legs I–III and serrated on legs IV. Eggs of hufelandi - type with inverse goblet-shaped processes with indented process disks without granulation. Egg chorion with a wrinkled or granulated surface, appears solid in LM but with a ring of small pores around each process visible in SEM.

Etymology

The new species is named after the area of the type locality within Kristianstads Vattenrike Biosphere Reserve, in dedication to its high tardigrade biodiversity.

Material examined

A total of 16 animals and 7 eggs observed, including: 10 animals and 6 eggs mounted in Hoyer’s fluid, 2 animals and 1 egg fixed for SEM, and 4 animals used for DNA extraction.

Type material

Holotype

SWEDEN • Skåne, Sånnarna ; 55°55′41.6″ N, 14°15′11.1″ E; 8 m a.s.l.; 22 Mar. 2021; S. Atherton, R. Guidetti and K.I. Jönsson leg.; moss on calcareous-rich sand and rock; SMNH, slide SMNH-Type-10010 .

Paratypes

SWEDEN • 9 specs, 5 eggs; same collection data as for holotype; GenBank nos: PX093653 to PX093655 (COI), PX093644 (ITS), PX093663 , PX093664 (18S), PX093649 , PX093650 (28S); SMNH, slides SMNH-Type-10011 to SMNH-Type-10015 , SEM stub SMNH-Type-10016 • 2 specs, 2 eggs; same collection data as for holotype; MUSN, slides 24-081, 24-083 .

Description

Morphometric measurements of animals and eggs are given in Tables 1 and 2 , respectively (raw morphometric data in Supp. file 10).

Adult body length 335–530 µm, yellowish with black eyespots visible in live animals ( Fig. 4A ) and animals fixed in Hoyer’s. Cuticle with small (up to 1 µm in diameter), round pores, visible with DIC ( Fig. 4E ) and clearly visible with SEM ( Fig. 6C ), scattered throughout the body cuticle. Very fine granulation clearly visible only with SEM distributed over the entire body, less dense on the ventral side ( Fig. 6B–C ). Medium granulation lateral and external to claws on legs I–III visible with DIC ( Fig. 5B ) and SEM ( Fig. 6B ). Legs IV with a patch of coarse granulation tapering centrally on the caudal segment and around the claws, clearly visible with DIC ( Fig. 5D ), and SEM ( Fig. 6D ). Pulvinus, gibbosities, and garter-like structures on the legs absent.

Rigid buccal tube of Macrobiotus - type ( Pilato & Binda 2010) with ventral lamina, ten peribuccal lamellae and six sensory lobes. OCA of hufelandi - type ( Kaczmarek & Michalczyk 2017) with an anterior band of teeth faintly visible as fine granules with DIC ( Fig. 4C–D ) and as small cones with SEM ( Fig. 6A ); a second band of larger granular teeth and a third row of posterior transverse ridges. Third row non-uniform; ventral side comprising two lateral teeth and a divided median tooth split into two ( Fig. 4D, F ), and dorsal side with a single, fused elongated tooth ( Fig. 4C ). Stylet furca typical of the genus and stylet support insertion point at 75–81% of the buccal tube. Bulbous muscular pharynx with three apophyses and three rows of two rodlike macroplacoids with the length series 1>2 and a microplacoid ( Fig. 4B ). First macroplacoid with slight central constriction. Second macroplacoid with deeper pre-terminal constriction. Comma-shaped microplacoid situated closer than its own length to the macroplacoid row.

Claws of hufelandi - type ( Bertolani & Pilato 1988) of similar size on all legs. Large lunulae on all claws, and distinctly larger on legs IV (max. width on leg I–III 6–8 µm; leg IV 7–10 µm). Lunulae smooth on legs I–III and serrated on legs IV, visible with DIC ( Fig. 5B–D ), and SEM ( Fig. 6B, D–E ). Cuticular bars absent.

Gonochoristic. Males with sperm-filled testis and females with a spermatheca filled with bundles of sperm observed ( Fig. 5A ).

Ornamented eggs are spherical and laid freely, bare diameter 62.3–78.2 µm. Eggs of hufelandi - type ( Kaczmarek & Michalczyk 2017; Figs 7A , 8A ) with processes shaped as inverted goblets with strait or lightly sigmoidal trunks and terminal disks ( Fig. 7A–C ). Surface of the terminal disks smooth, concave with a deep hollow and cogwheel-shaped edges including 10–11 evenly spaced indentations ( Figs 7D , 8 ). Process height 6.0–7.3 µm; base diameter 6.0–7.2 µm; terminal disk diameter 3.2–4.8 µm; and inter-process distance 2.9–4.2 µm. 23–25 processes on the egg circumference. The egg chorion is wrinkled and appears solid in DIC ( Fig. 7E–F ), but minute (<0.25 µm in diameter) pores present around the bases of the processes are visible with SEM ( Fig. 8B–C ). Larger wrinkles surrounding the base of each process appear as a crown of thickenings in DIC ( Fig. 7E–F ).

DNA sequences

Sequences for M. vattenrikense sp. nov. were attained for all four molecular markers and using four adult animals. ITS2 was represented by a single haplotype; 18S and 28S were represented by two haplotypes (uncorrected p-distance between haplotypes 0.06% and 0.11%, respectively), and COI was represented by three haplotypes (uncorrected p-distances between haplotypes 0.2–2.6%):

– 18S haplotype 1: specimen 22-003; 1769 bp; GenBank accession number PX093663;

– 18S haplotype 2: specimen 22-101; 1769 bp; GenBank accession number PX093664;

– 28S haplotype 1: specimens 22-003, 22-017; 921 bp; GenBank accession number PX093649;

– 28S haplotype 2: specimen 22-101; 921 bp, GenBank accession number PX093650;

– COI haplotype 1: specimen 22-017; 658 bp, GenBank accession number PX093653;

– COI haplotype 2: specimen 22-101; 658 bp;GenBank accession number PX093654;

– COI haplotype 3: specimens 22-003, 22-004-1; 658 bp; GenBank accession number PX093655;

– ITS2: specimens 22-003, 22-017, 22-101; 443 bp; GenBank accession number PX093644.

Morphological differential diagnosis

Macrobiotus vattenrikense sp. nov. has hufelandi - type OCA, serrated lunules on legs IV, and eggs with inverted goblet-shaped processes with terminal disks showing cogwheel-like indented margins and a deep central hollow, wrinkled chorions and very small pores surrounding the processes. Based on these characteristics, M. vattenrikense is most similar to five other species of Macrobiotus (note, the pores on the egg may be easily overlooked with light microscopy/clearly visible only with SEM, and so species with similar egg morphologies that have not been observed with SEM are included):

Macrobiotus halophilus Fontoura, Rubal & Veiga, 2017 – the animals of M. vattenrikense sp. nov. differ from animals of M. halophilus in the wider buccal tube (external width pt 16.7–20.9 compared with pt 13.2–16.5 in M. halophilus ); the lack of a deep constriction in the first macroplacoid; the paired ventromedial teeth and single fused dorsal tooth in the third band of the OCA (compared with an undivided ventromedial tooth and three distinct dorsal teeth in M. halophilus ); the shorter claws (posterior claw IV pt 23.8–33.3 compared to 33.4–42.0 in M. halophilus ); and the lack of male gibbosities. The eggs of M. vattenrikense are differentiated by the larger processes (height 6.0–7.3 µm compared to 3.9–5.9 µm in M. halophilus ). In addition, the very small pores that are present surrounding the egg processes of M. vattenrikense have not been documented for M. halophilus , although eggs of the latter species have not been observed with SEM.

Macrobiotus marlenae Kaczmarek & Michalczyk, 2004 – the animals of M. vattenrikense sp. nov. differ from the animals of M. marlenae by the granulation present on all legs (compared to granulation only on legs IV in M. marlenae ); the shallower constriction of the first macroplacoid; the subterminal constriction in the second macroplacoid; and the claws with larger lunules in M. vattenrikense The eggs of M. vattenrikense have more numerous (23–25 on the circumference compared to 16 in M. marlenae ) processes that are shorter (6.0–7.3 µm compared to 8.4–8.8 µm in M. marlenae ), more closely spaced (inter-process distance 2.9–4.2 µm compared with 4.5–6.5 µm in M. marlenae ), and with much smaller terminal disks (diameter 3.2–4.8 µm compared to 9.5–11.4 µm in M. marlenae ). In addition, the very small pores that are present surrounding the egg processes of M. vattenrikense have not been documented for M. marlenae , although eggs of the latter species have not been observed with SEM.

Macrobiotus ovovittatus Stec, 2024 – the animals of M. vattenrikense sp. nov. are smaller than animals of M. ovovittatus (335–530 µm compared to 570–879 µm in M. ovovittatus ) and have a shorter first macroplacoids ( pt 21.4–27.8 compared to pt 28.4–34.4 in M. ovovittatus ) with shallower median incision. The new species is further distinguished by the paired ventromedial teeth and single fused dorsal tooth in the third band of OCA (compared with an undivided ventromedial tooth and three distinct dorsal teeth in M. ovovittatus ), and the lack of cuticular bars below the claws of the first three pairs of legs (bars present in M. ovovittatus ). The eggs of M. vattenrikense are smaller than those of M. ovovittatus (diameter without the processes 62.3–78.2 µm compared to 100.6–129.8 µm in M. ovovittatus ) with fewer processes on the circumference (23–25 compared to 28–34 in M. ovovittatus ), and smaller processes (height 6.0–7.3 µm compared to 9.5–13.5 µm in M. ovovittatus ; diameter at the base 6.0–7.2 µm compared to 9.4–13.6 µm in M. ovovittatus ) with smaller (diameter 3.2–4.8 µm compared to 6.1–8.7 µm in M. ovovittatus ), solid terminal disks (covered by multiple light-refracting dots in M. ovovittatus ).

Macrobiotus persimilis Binda & Pilato, 1972 (following the redescription of Bertolani et al. 2023) – the animals of M. vattenrikense sp. nov. differ from animals of M. persimilis in the lack of a deep constriction in the first macroplacoid; the shorter macroplacoids (first/second macroplacoid pt 21.4–27.8/16.7–20.9 compared to pt 32.0–33.8/ 22.8–24.2 in M. persimilis ) and macroplacoid row ( pt 37.1–52.8 compared to pt 54.7–57.2 in M. persimilis ); the longer microplacoids ( pt 8.6–11.6 compared to pt 7.7–8.0 in M. persimilis ); the paired ventromedial teeth and single fused dorsal tooth in the third band of the OCA (compared with an undivided ventromedial tooth and three distinct dorsal teeth in M. persimilis ); and the shorter claws (external claw III/IV pt 23.3–33.3/25.6–30.6 compared to 35.9–38.3/ 35.9–37.9 in M. persimilis ). The eggs of M. vattenrikense are differentiated by the larger processes (height 6.0–7.3 µm compared to 3.7–5.3 µm in M. persimilis ; diameter at the base 6.0–7.2 µm compared to 3.6–5.1 µm in M. persimilis ). In addition, the very small pores that are present surrounding the egg processes of M. vattenrikense have not been documented for M. persimilis , although eggs of the latter species have not been observed with SEM.

Macrobiotus polonicus Pilato, Kaczmarek, Michalczyk & Lisi, 2003 – the animals of M. vattenrikense sp. nov. differ from the animals of M. polonicus by the presence of granulation lateral to the claws on legs I–III (absent in M. polonicus ); the absence of lateral gibbosites on the hind legs (present in M. polonicus ); the absence of sclerotized areas near the lunulae of legs I–III (present in M. polonicus ). The eggs of M. vattenrikense differ by the smaller diameter of the process terminal disks (3.2–4.8 µm compared with 4.9–6.3 µm in M. polonicus ) and by the minute pores surrounding each process (lack of egg pores for M. polonicus confirmed with SEM).

Macrobiotus trunovae Biserov, Pilato & Lisi, 2011 – the animals of M. vattenrikense sp. nov. differ from animals of M. trunovae by the paired ventromedial teeth and single fused dorsal tooth in the third band of the OCA (compared with an undivided ventromedial tooth and three distinct dorsal teeth in M. trunovae ); the more anteriorly inserted stylet supports ( pt 75.0–81.0 compared to 81.7–82.4 in M. trunovae ); the smaller first macroplacoid ( pt 21.4–27.8 compared to 28.3–31.4 in M. trunovae ) with a much narrower constriction; and the similar sizes of the claws on all legs (compared to distinctly smaller claws on legs I–III than on legs IV in M. trunovae ). The eggs of M. vattenrikense are smaller (diameter without processes 62.3–78.2 µm compared to 134.3 µm in M. trunovae ) with fewer (23–25 on the egg circumference compared to 32 in M. trunovae ) and smaller (length 6.0–7.3 µm compared to up to 10.9 µm in M. trunovae ; diameter at the base 6.0–7.2 µm compared to 9.1–9.9 µm in M. trunovae ) processes. In addition, the very small pores that are present surrounding the egg processes of M. vattenrikense have not been documented for M. trunovae , although eggs of the latter species have not been observed with SEM.

Figures

Fig. 4. Macrobiotus vattenrikense sp. nov. A. DIC photograph of paratype in water (SMNH, SMNH- Type-10013); whole body. B. DIC photograph of holotype in Hoyer’s fluid (SMNH, SMNH-Type-10010); pharynx and placoids. C. DIC photograph of holotype in Hoyer’s fluid (SMNH, SMNH-Type-10010); dorsal OCA. D. DIC photograph of holotype in Hoyer’s fluid (SMNH, SMNH-Type-10010); ventral OCA. E. DIC photograph of holotype in Hoyer’s fluid (SMNH, SMNH-Type-10010); body cuticle with small pores. F. DIC photograph of paratype in Hoyer’s fluid (MUSN, 24-081); second and third rows of the ventral OCA. White arrowhead indicates slight constriction of the first macroplacoid; white indented arrowhead indicates constriction of the second macroplacoid; white full arrows indicate pores in the body cuticle; black arrowheads indicate first row of OCA; black full arrows indicate second row of OCA; black indented arrowheads indicate third row of OCA.

Fig. 5. Macrobiotus vattenrikense sp. nov. A. DIC photograph of paratype in Hoyer’s fluid (SMNH, SMNH-Type-10011); spermatheca with sperm. B. DIC photograph of paratype in Hoyer’s fluid (MUSN, 24-081); claws of leg I. C. DIC photograph of holotype in Hoyer’s fluid (SMNH, SMNH-Type-10010); claws of leg IV. D. DIC photograph of paratype in Hoyer’s fluid (MUSN, 24-081); legs IV. White full arrow indicates pores in the body cuticle; asterisks indicate leg granulation.

Fig. 6. Macrobiotus vattenrikense sp. nov., SEM photographs of paratype (SMNH, SMNH-Type-10016). A. Mouth opening. B. Claws of leg II. C. Details of the body cuticle. D. Leg IV. E. Serrated lunules of the claws of leg IV. White full arrows indicate pores in the body cuticle; empty full arrows indicate very fine granules on the body cuticle; white arrowhead indicates first row of OCA.

Fig. 7. Macrobiotus vattenrikense sp. nov., DIC photographs of egg in water (SMNH, 22-040) A. Whole egg. B–C. Examples of egg processes. D. Terminal disk of egg process. E–F. Base of processes and egg chorion. Black arrowheads indicate the crown of thickenings at the base of each process.

Fig. 8. Macrobiotus vattenrikense sp. nov., SEM photographs of egg (SMNH, SMNH-Type-10016). A. Whole egg. B–C. Egg processes and chorion. White arrowheads indicate examples of the very small pores that surround the base of each process.