Tardigrades of Kristianstads Vattenrike Biosphere Reserve with description of four new species from Sweden

Taxonomic and morphological results

Four species new to science (Mesobiotus emiliae sp. nov., Xerobiotus gretae sp. nov., Itaquascon magnussoni sp. nov., and Thulinius gustavi sp. nov.) were found in the Kristianstad Vattenrike Biosphere Reserve, and their formal descriptions follow.

Morphometric data for the animals and the egg of these species are reported in the Tables 1 and 2 respectively (Supplementary Table S1 for the raw and the Thorpe’s normalized data). The morphometric data used in differential diagnoses of the species were not Thorpe’s normalized. After Thorpe’s normalization, only the lengths of very few characters in the new species resulted not isometric with respect to the length of the buccal tube (Supplementary Table S1). For these characters, the range (min–max) of pt indexes calculated on the Thorpe’s normalized data and the range (min–max) of pt indexes calculated on the non-normalized data did not change significantly (Supplementary Table S1).

Mesobiotus emiliae sp. nov.

ZooBank: lsid:zoobank.org:act:3DA2F1C0-BEC8-4D9A-B111-5D5F8F1FCCA0.

Type locality

Sånnarna, west of the nature reserve (Kristianstad, Skåne, Sweden). Sandy soil with grass (55.928931 N, 14.246299 E), collected on June 10th, 2014; sample SVC22 (C4342 in Bertolani’s Collection). The species was also found in two other localities (SVC34, 35) and it is probably present in further four localities in which only animals were found (SVC2, 8, 27, 30; Supplementary Table S2).

Type repositories

The holotype (SVC22 s11o), 34 paratypes, and an egg (SVC22 s1, s5, s7, s8, s11) are at Kristianstad University [HKR], 19 paratypes and an egg (SVC22 s4, s10) are in the collection of the Swedish Museum of Natural History [SMNH], and 3 paratypes and two eggs (C4342 s3, s9) are in the Bertolani’s Collection of University of Modena and Reggio Emilia [Unimore].

Description

Body whitish, 96.8–342.0 µm in length (Fig. 1a). Eye-spots absent in mounted specimens. Cuticle smooth, with sparse granules on the posterior side of the legs IV (visible with Light Microscopy [LM]; Fig. 1g,i), and with granules covered with 1–5 dots on the external side of the legs I-III (Fig. 1h; visible with Scanning Electron Microscopy [SEM]).

Bucco-pharyngeal apparatus with antero-ventral mouth (Fig. 1b, d). Buccal ring with ten lamellae on its external margin. Buccal armature composed of: an anterior band of small teeth; a posterior line of conical teeth; three dorsal and three ventral transversal crests, the medio-ventral crest reduced to two or four mucrones in some smaller specimens (Fig. 1c), the latero-ventral crests shorter than the latero-dorsal ones. Short and straight stylet supports with distal flat enlargement, inserted at the 73.8–79.4% of the buccal tube. Typically-shaped stylet furca, with spherical condyles supported by short branches provided with small apophyses. Buccal tube ending with a thick cuticular ring within the pharynx (Fig. 1d). In the pharynx: pear-shaped pharyngeal apophyses; three grain-shaped (in lateral view) macroplacoids and an evident drop-shaped microplacoid. In frontal view, first macroplacoid triangular, second and third rectangular with rounded corners, and third with a deep distal constriction (Fig. 1b); length sequence 3 > 2 > 1.

Double-claws of Mesobiotus type (Fig. 1e–i) with evident accessory points on the main branch. All claws similar in shape, external claws slightly larger than internal. Claws increasing in size from the first to the fourth, claws of hind legs clearly the largest. Smooth lunules under all claws, larger under claws of the hind legs (the anterior lunules clearly larger with respect to the posterior).

Spherical eggs free laid, ornamented with processes in shape of large and short cones or mammillated with tips of different lengths (generally short), sometimes terminating in a tuft of filaments (Fig. 2a–c). Process wall formed by two sides (an internal and an external), interspersed with trabecular structures forming irregular meshes (Fig. 2b), in shape of bubble-like structures in the longer process tips (Fig. 2c). Base of the processes with a crown of irregular and small thickenings: smaller thickenings in shape of large dots, the larger ones triangular-shaped (Fig. 2b). Filaments of the process tips mostly short, elongated in abnormal processes. Processes in numbers of 11–14 on the circumference. Egg surface between the processes smooth or sparsely dotted. Egg with embryos found.

Differential diagnosis

According to the taxonomic key of Mesobiotus species^32,33, M. emiliae sp. nov. is different from any other described species of this genus. Mesobiotus emiliae sp. nov. belongs to the harmsworthi group of species, and within this group Mesobiotus insuetus (Pilato, Sabella & Lisi, 2014)³⁴, Mesobiotus lusitanicus (Maucci & Durante Pasa, 1984)³⁵, Mesobiotus occultatus Kaczmarek, Zawierucha, Buda, Stec, Gawlak, Michalczyk & Roszkowska, 2018³⁶, Mesobiotus patiens (Pilato, Binda, Napolitano & Moncada, 2000)³⁷, Mesobiotus pseudoblocki Roszkowska, Stec, Ciobanu & Kaczmarek, 2016³⁸, and Mesobiotus snaresensis (Horning, Schuster & Grigarick, 1978)³⁹ share with Mesobiotus emiliae sp. nov. the following characteristics: smooth cuticle; buccal armature with both anterior and posterior rows of teeth visible with LM, and without accessory teeth between posterior row and transversal crests; smooth lunules in the claws of the hind legs; eggs with processes in shape of cone or hemisphere with elongated tip or inverted funnel surrounded by a basal crown of dots or digitations, and with smooth or wrinkled surface between them.

Mesobiotus emiliae sp. nov. differs from:

M. insuetus by: the presence of granulation on legs IV, the shorter first macroplacoid (pt 15.4–16.8 in M. insuetus; pt 7.9–12.7 in M. emiliae), the shorter macroplacoids row (pt 46.2–48.9 in M. insuetus; pt 35.2–46.0 in M. emiliae), the different morphology of the hind claws (i.e. not with secondary branches diverging distally and forming a right angle with the primary branches as in M. insuetus), the shorter primary branch of all claws (e.g., pt of the primary branch of the claws II, 31.9–36.0 in M. insuetus; 20.4–25.4 in M. emiliae), and the processes of the egg with larger meshes on the surface (with LM, as small separated dots in M. insuetus and bubbles in contact to each other in M. emiliae);

M. lusitanicus by: the presence of granulation on legs IV, the length sequence of macroplacoids (3 > 1 > 2 in M. lusitanicus), the more evident microplacoid (pt 4.2 in M. lusitanicus; pt 7.3–12.3 in M. emiliae), and the shape of egg processes (i.e. not in shape of hemispheres terminating with a cap-like structure or with a fringed cones as in M. lusitanicus);

M. occultatus by: the absence of granulation on the legs I-III (with LM), the length sequence of macroplacoids (1 ≥ 3 > 2 in M. occultatus), the smaller eggs (full diameter 97.4–126.6 µm in M. occultatus and 62.4–76.5 µm in M. emiliae), the shape of the egg processes with a base/height ratio (74–106% in M. occultatus and 144–164% in M. emiliae), the distance between the egg processes (mean 2.6 µm, 0.6 SD in M. occultatus and mean 1.1 µm, 0.4 SD in M. emiliae);

M. patiens by: the absence of granulation on the legs I-III (with LM), the length sequence of macroplacoids (1 > 3 > 2 in M. patiens), the smaller eggs (full diameter 90.5–100.0 µm in M. patiens and 62.4–76.5 µm in M. emiliae), absence of slender tips in the egg processes;

M. pseudoblocki by: the presence of granulation on legs IV, the length sequence of macroplacoids (1 > 3 > 2 in M. pseudoblocki), the smaller anterior claw of the hind legs (pt 27.5–33.5 in the primary branch and 19.8–27.4 in the secondary branch in M. pseudoblocki and pt 27.5–33.5 in the primary branch and 19.8–27.4 in the secondary branch in M. emiliae), the closer processes on the egg surface (mean 2.8 µm, 0.6 SD in M. pseudoblocki and mean 1.1 µm, 0.4 SD in M. emiliae), the processes of the egg not in shape of sharpened narrow cones, the processes base/height ratio (47–70% in M. pseudoblocki and 144–164% in M. emiliae);

M. snaresensis by: the presence of granulation on hind legs, the more evident microplacoid (pt 4.2–7.3 in M. snaresensis; pt 7.3–12.3 in M. emiliae), the processes of the egg not terminating with a sharp or bifid tips, and the absence of pseudoareolation on the egg surface between the processes.

Molecular characterization

The analyses of the molecular markers were not possible due to the lack of alive specimens: the genomic material extracted from dead specimens gave no amplicons.

Etymology

We dedicate this species to Emilia Lonis, the beloved hundred-years-old grandmother of the coauthor Massa E., one of the last living workers that with their hand-on work for the reclamation of the “Piana di Terralba” (Sardinia, Italy; now site of the “Natura 2000” network) have contributed to the eradication of malaria in the island saving thousands of life.

Xerobiotus gretae sp. nov.

ZooBank: lsid:zoobank.org:act:E5265E82-86E9-4069-9B3B-B0F11D43DE79.

Type locality

Sånnarna, (Kristianstad, Skane, Sweden). Moss on ground (55.928056 N, 14.252694 E), collected on June 10th, 2014. Sample SVC15 (C4341 in the Bertolani’s Collection). An animal of this species was also found within a Saxifraga sp. (SVC19; Supplementary Table S2).

Type repositories

The holotype (SVC15 s2m), 51 paratypes, and an egg (SVC15 s2, s3) are at HKR, 50 paratypes (SVC15 s5) are in the collection of the SMNH, and 29 paratypes and an egg (C4341 s1, s4) are in the Bertolani’s Collection of Unimore. Five paratypes recovered from the old slide C4341 s1 (for the extraction and mounting protocol, see: Methods section) together with four paratypes and an egg freshly extracted were mounted on stubs for SEM observation.

Description

Body whitish or pale green, 177.5–438.3 µm in length (Figs. 3a, 4a,f). Orange eye-spots present in mounted specimens. Very small scattered pores (about 0.5 µm in diameter) in the dorso-lateral cuticle (Figs. 3b, 4b,c). Very small single granules, distributed almost regularly, present on the entire cuticle (only visible with SEM; Fig. 4c). Legs of the first pair smaller than those of the second and third pairs. The area of the leg cuticle surrounding the claws with a swelling (forming a garter-like structure; Fig. 4a, d–f). These swellings appearing covered with microdigitations and few minute scattered granules (with SEM; Fig. 4d).

Bucco-pharyngeal apparatus with antero-ventral mouth (Fig. 3c,d). Buccal ring with ten peribuccal lamellae (Fig. 4g). Buccal tube of Macrobiotus type, curved in the first half, and ending with a thick cuticular ring within the pharynx (Fig. 3c,d). Ventral lamina with an antero-ventral thickening (Fig. 3d). Buccal armature (Figs. 3c,e, 4g) composed of: an anterior band of small teeth at the base of the peribuccal lamellae (with SEM); a thin posterior band of small teeth not always visible with LM, but clearly visible with SEM; three dorsal and three ventral transversal crests, medio-ventral crest appearing split in two or three mucrones in some specimens (with LM). Lateral cribrose areas posterior to the transversal crests visible with SEM (Fig. 4g). Stylet support, inserted at 77.0–81.2% of buccal tube, in shape of an elongated sigma with a distal flat enlargement (Fig. 3c). Typically-shaped stylet furcae, with oval condyles supported by short branches provided with rounded apophyses (Fig. 3f). In the pharynx: large and triangular pharyngeal apophyses overlapping the first macroplacoid; two rod-shaped macroplacoids (in lateral view; Fig. 3d), and evident drop-shaped microplacoid. In frontal view (Fig. 3g), the first macroplacoid in shape of a drop with a medial slight constriction longer than the second, the second rectangular with rounded corners and with a small terminal slight constriction.

Double-claws I-III different from claws IV (Figs. 3h,i, 4d,e): claws I–III of Xerobiotus type (without lunules), claws IV with a longer common tract, small and short claw branches, and pale lunules (more sclerified proximally than distally) sometimes visible (Fig. 3i). Internal and external claws of the same leg similar in shape, external (or posterior, in claw IV) claw slightly larger than the internal (or anterior, in claw IV). Proximal portion of the basal part of all claws with a small enlargement, larger in claw IV (Figs. 3h,i, 4d). Claws increasing in length from the first to the third pair. Primary branch of all claws with short accessory points (larger in claws IV). Cuticular bars under the base of the claw IV thick and with ragged margin (Fig. 3i): cuticular bar under the posterior claw wider and stretched toward the anterior claw, cuticular bar under anterior claw developed toward the front of the body (Fig. 3i).

Spherical eggs laid free (Fig. 2d,h), ornamented with processes in shape of inverted goblets with straight or concave cross section (according to Kaczmarek et al.³⁵; Fig. 2e–g,i,j). Processes base surrounded by a crown of dots (Fig. 2g); terminal disc slightly concave and divided by a septum from the trunk (Fig. 2e,f). The edge of the terminal disks indented, the indentations appearing like tapered tip (with LM; Fig. 2g) and like elongated processes ornamented with granules (with SEM; Fig. 2i); in several processes indentation-like structures (provided with granular ornamentation) occurring also in the upper surfaces of the disk (Fig. 2j). Wrinkled egg surface between the processes (Fig. 2g, i) and scattered with dot-like pores (with SEM; Fig. 2i). Egg with an embryo found.

Differential diagnosis

Xerobiotus gretae sp. nov. differs from all other Xerobiotus species by having pores on the cuticle visible with LM, an enlargement in the basal part of the claws, and cuticular bars under the claws IV.

Moreover, Xerobiotus gretae sp. nov. differs from:

Xerobiotus xerophilus (Dastych, 1978)^41,42 by: the presence of a posterior band of teeth and the dorsal transversal crests not fused in the buccal armature, the shape of the egg processes (flattened hemispherical processes in X. xerophilus), and the egg surface lacking reticulation;

Xerobiotus euxinus Pilato, Kiosya, Lisi, Inshina & Biserov, 2011⁴³ by: the dorsal transversal crests not fused in the buccal armature, and the presence of cuticular bars under the claws of the hind legs;

Xerobiotus pseudohufelandi (Iharos, 1966)⁴⁴ by: the presence of a posterior band of teeth, shorter common tract in the claws I-III (pt 11.07–11.99 in X. pseudohufelandi; pt 9.19–9.91 in X. gretae), and the egg surface lacking reticulation.

Molecular characterization

It was not possible to extract genetic material from the specimens recollected from the permanent slides (C4341 A–E). The analyses of the molecular markers amplified from four specimens (C4341 G–L; GenBank accession number: MW581665-8, cox1; MW588431-3, ITS2; MW588438-41, 28S; MW588434-7, 18S; Supplementary Table S5) revealed single haplotypes for ITS2, 18S, and 28S genes, and three haplotypes for cox1 gene (highest p-distance = 0.4%; Supplementary Table S3).

Xerobiotus gretae sp. nov., in comparison to the more similar GenBank sequences which belong to Xerobiotus sp. collected in South Africa (Cape of Good Hope, Western Cape)⁴⁵, differs for p-distances of 1.6–2.6% for cox1 (796 bp), 1.0% for ITS2 (452 bp), and 0.0% for 18S (870 bp). Xerobiotus pseudohufelandi, collected in Italy (Monte Calvario)⁴⁶, differs from X. gretae sp. nov. for p-distance of 16.9–17.8% for cox1, and 0.0–0.1% for 18S, Xerobiotus sp., collected in Poland (Błedowska Desert)⁴⁵, differs from X. gretae sp. nov. for p-distances of 17.8–18.1% for cox1, 5.1% for ITS2, and 0.1–0.2% for 18S (Supplementary Table S3).

Etymology

We dedicate this species to the climate activist Greta Thunberg, for her brave and insightful efforts to open the eyes of the world leaders about the need for action against climate change. The achievements of Greta Thunberg give us hope that the challenges of changing the unsustainable path of human societies may still be possible, just like the tiny tardigrades are able to overcome seemingly impossible environmental challenges. But we have to act now!

Itaquascon magnussoni sp. nov.

ZooBank: lsid:zoobank.org:act:254843BC-60F7-4B8E-A94D-8783214F3399.

Type locality

Näsby Fält (Kristianstad, Skåne, Sweden), along a trail to Araslövssjön Lake. Moss on bark of Alnus sp. (56.059328 N, 14.136678 E), 2 m up on the tree, collected on June 10th, 2014. Sample SVC32 (C4344 in the Bertolani’s Collection). The species was also found in two other localities (SVC3, 27; Supplementary Table S2).

Type repositories

The holotype (SVC32 s4c) and 13 paratypes are at HKR, four paratypes (SVC32 s8) are in the collection of the SMNH, and eight paratypes (C4344 s12) in the Bertolani’s Collection of Unimore.

Description

Body whitish, 135.9–509.3 µm in length (Fig. 5a). Eye-spots absent in mounted specimens. Cuticle smooth. Bucco-pharyngeal apparatus of Itaquascon type (Fig. 5b). Rigid and straight buccal tube, clearly longer than the apophyses for the insertion of the stylet muscles [AISM]. AISM symmetrical and flat ridge-shaped. Buccal tube followed by a pharyngeal tube almost of the same length (pharyngeal tube pt 91.6–113.8). Flexible pharyngeal tube formed by a rope-shaped thickening organized in a geometrical repeated pattern resembling an alternating hexagonal “wire meshes” (Fig. 5b,e,f); the “wire meshes” pattern begins in a more anterior position dorsally and ventrally than laterally (Fig. 5e,f). Very thin stylet supports present but hardly detectable and inserted on the pharyngeal tube in its anterior portion (Fig. 5b,e,f). Small stylet furca with short branches ending in drop-shaped condyles. Stylet coat more sclerotized in its proximal and distal portions than in its middle part. Pharyngeal tube ending within the pharynx with three small triangular apophyses (Fig. 5b). In the pharynx, only a single long, straight, and weakly thickened bar present (Fig. 5b).

Double-claws of Hypsibius type (Fig. 5c,d), internal (anterior, in the claw IV) and external (posterior, in the claw IV) claws of the same legs different both in shape and size. Claws increasing in length from the first to the fourth legs. Basal part of all claws long, with enlarged base. Main branch of external claw (posterior, in the claw IV) long, quite straight, and poorly sclerotized throughout its length, with evident accessory points; its proximal part placed on a cuticular digit and connected with the secondary branch with a pair of filaments spanning from the tip of the branch (Fig. 5c). Main branch of internal (anterior, in the claw IV) claw shorter and more curved than the external, with evident accessory points. Thin and hardly detectable pseudolunules present under all claws (Fig. 5c,d). Straight cuticular bars (Fig. 5c), similar in size, with ragged margins on the internal side of the legs I-III, extending from the internal claw base to the anterior side of the leg, weakly visible only on the first pair of legs.

Eggs unknown.

Differential diagnosis

Itaquascon magnussoni sp. nov. differs from all other Itaquascon species by having the stylet support inserted on the flexible pharyngeal tube. Considering the presence of the thickening within the pharynx, the most similar species of I. magnussoni sp. nov. are Itaquascon placophorum Maucci, 1973⁴⁷ and Itaquascon simplex (Mihelčič, 1971)⁴⁸ (considered nomen dubium by Ramazzotti et al.⁴⁹, thus not considered in this diagnosis).

Itaquascon magnussoni sp. nov. differs from I. placophorum by: the longer buccal tube with respect to the bucco-pharyngeal tube (buccal tube 16–17% of the bucco-pharyngeal tube in the holotype of I. placophorum and 48.0–48.4% in I. magnussoni), the longer thickening in the pharynx (calculated pt 31.3 in the holotype of I. placophorum; pt 44.8–63.5 in I. magnussoni), the claws with pseudolunules and evident accessory points on the main branch.

Molecular characterization

The analyses of the molecular markers were not possible due to the lack of alive specimens: the genomic material extracted from dead specimens gave no amplicons.

Etymology

The species name is to honor Sven-Erik Magnusson, a sustainability visionary and leading person behind the development of Kristianstads Vattenrike Biosphere Reserve, and the first Coordinator of the Biosphere Reserve.

Thulinius gustavi sp. nov.

ZooBank: lsid:zoobank.org:act:149B5B73-580F-4BBE-BCB0-BCFD53E54EB5.

Type locality

Araslövssjön Lake, Näsby Fält (Skåne, Sweden). Upper layer of freshwater sediments in the bottom of the shore of the lake (56.059050 N, 14.135425 E), sample SVC31 (C4343 in Bertolani’s Collection).

Type repositories

The holotype (SVC31 s3b) and nine paratypes (SVC31 s2, s4, s5; SVC31b s2, s6, s8) are at HKR, one paratype (SVC31 s4) is in the collection of the SMNH, and one paratype (C4343 s1) in the Bertolani’s Collection of the Unimore. Two paratypes were mounted on stubs for SEM observation.

Description

Body whitish, 231.0–346.0 µm in length (Fig. 6a,j). Eye-spots present. Dorsal cuticle sculptured with large tubercles, with polygonal base, that gradually increase in size from the head to the posterior side of the body (Fig. 6b,k).

Bucco-pharyngeal apparatus with antero-ventral mouth opening (Fig. 6c,n). Buccal tube straight. Twelve peribuccal lamellae present. Buccal armature formed by a posterior line of small round teeth, followed by a line of large rounded teeth in the position of the transversal crests (Fig. 6d,e). Two bigger rounded teeth present ventrally within the second line in correspondence of the stylet sheaths (Fig. 6d). Ventral and dorsal AISM crest-shaped and symmetrical with respect to the frontal plane (Fig. 6n). Long and straight stylet supports with a distal flat enlargement. Typically-shaped stylet furca, with long branches provided with large apophyses (Fig. 6f). In the pharynx, large pharyngeal apophyses overlapping the first macroplacoid (Fig. 6n); three rod-shaped (in lateral view) macroplacoids arranged in a curved line; first and second almost fuse together, third spaced from the second (Fig. 6g). In frontal view, the first macroplacoid in shape of a triangle, second in shape of a rectangle with rounded corners, and the third almond-shaped and slightly constricted in the middle; length sequence 3 > 1 > 2.

Double-claws of Isohypsibius type (Fig. 6h,i,l,m) increasing in length from the first to the fourth pair of legs, external claw slightly longer than the internal. Basal portions of all claws short and slender, enlarged in their proximal portion. Primary branch of all claws with thin and short accessory points (never reaching the end of the branch and not always visible) and divided from the rest of the claw by a basal septum, with a dorsal knob-like thickening (Fig. 6h,i). Pseudolunules hardly detectable and small on external claws of legs I–III (Fig. 6h,l), larger on posterior claw of legs IV (Fig. 6i,m).

Smooth oval eggs laid in exuvium (an exuvium with four eggs was found).

Differential diagnosis

Thulinius gustavi sp. nov. differs from all other Thulinius species by having a dorso-lateral ornamented cuticle with tubercles. Considering the presence of pseudolunules only under the external (posterior, in claws IV) claws, the most similar species are Thulinius romanoi bertolani, bartels, Guidetti, Cesari & Nelson, 2014⁵⁰ and Thulinius saltarsus (Schuster, Toftner & Grigarick, 1978)⁵¹.

T. gustavi sp. nov. differs from:

T. romanoi by: the absence of ornamented cuticle in ventral side, the presence of eye-spots, the more narrow buccal tube (pt 21.9 and 13.4 in the holotype and a paratype of T. romanoi; pt 9.1–10.7 in T. gustavi), the stylet support inserted more posteriorly (pt 62.9 in the holotype of T. romanoi; pt 70.2–73.1 in T. gustavi), the macroplacoid length sequence (1 > 3 > 2 in T. romanoi), the shorter accessory points that never reach the end of the branch, and the presence of evident pseudolunules in claw IV;

T. saltarsus by: the dorso-lateral ornamented cuticle, presence of eye-spots, the macroplacoid length sequence (1 > 3 > 2 in T. saltarsus).

Molecular characterization

The analyses of the molecular markers were not possible due to the lack of alive specimens: the genomic material extracted from dead specimens gave no amplicons.

Etymology

The species name has been chosen in honor of Gustav Thulin (1889–1945), the first internationally recognized Swedish tardigradologist, who made important contributions to the knowledge of the Swedish tardigrade fauna and to modern taxonomy and phylogenetics of tardigrades.

Faunistic results

The analysis of the 34 samples collected (33 terrestrial and one freshwater; see “Methods”) in the five sampled areas within the KVBR revealed the presence of 33 morphospecies belonging to 20 genera (Table 3) of Eutardigrada (18 genera and 29 species of Parachela, and one genus and two species of Apochela), and Heterotardigrada (one genus, two species). The identification of the morphospecies was carried out with morphological and morphometric approaches.

The highest densities of tardigrades (ind/g) were found in a lichen and a moss (Table 3, Supplementary Table S4). However, tardigrade densities in both lichens (five samples) and mosses (13 samples) were highly variable, ranging from 2.7 to 75.4 ind/g (mean: 27.6 ind/g, SD: 29.5, N = 5) in lichens, and from 0.2 to 66.4 ind/g (mean: 11.1 ind/g, SD: 17.9, N = 13) in mosses. In contrast, leaf litter (two samples) and soil with grass (three samples) were less abundant in animals and had a more homogeneous density: 0.1–1.9 ind/g (mean: 0.9 ind/g, SD: 0.68, N = 5; Table 3, Supplementary Table S4).

The species belonging to the family Macrobiotidae were the most represented, found in 23 samples. The 11 macrobiotid species belonged to five genera (Macrobiotus, Mesobiotus, Minibiotus, Paramacrobiotus, and Xerobiotus) and were found in 69.7% of the terrestrial substrates, with variable diversity (1–4 species per substrate) and variable density (0.1–22.4 ind/g; Table 3) within each sample. The genus Macrobiotus was the most represented among the Macrobiotidae and among all the genera identified in all samples (7 species distributed among 17 samples).

The sample SVC11 (C4340 in Bertolani’s Collection) was the richer in terms of overall density (66.4 ind/g; Table 3). Within this sample Macrobiotus polonicus Pilato, Kaczmarek, Michalczyk & Lisi, 2003⁵² and Macrobiotus wandae Kayastha, Berdi, Mioduchowska, Gawlak, Łukasiewicz, Gołdyn, & Kaczmarek, 2020⁵³ were initially morphologically identified, but the evidence of intraspecific variability for some characters led us to suspect the presence of cryptic species. The analyses were performed by genotyping the markers ITS2 and cox1. The analyses of the cox1 were unsuccessful, but the ITS2 sequences amplified from nine specimens (C4340 C–D, J–P; GenBank accession numbers: XXXX) were sufficient to reveal the presence of three species: Macrobiotus polonicus, Macrobiotus cf. polonicus, and Macrobiotus aff. wandae. Macrobiotus polonicus, already identified via morphology, was confirmed also by a very low p-distance of its sequences (0.00–0.01%; 587 bp) with respect to those already attributed to this species (Supplementary Table S3). In the population, eight males with spermatozoans within the gonad were found. One specimen previously identified as M. polonicus was revealed to belong to a cryptic taxon that we named M. cf. polonicus (p-distance 0.04–0.05% with respect to M. polonicus sequences; Supplementary Table S3). Macrobiotus cf. polonicus differs morphologically from M. polonicus by the presence of fine granules on the external side of all legs, for this species the egg morphology is unknown. Macrobiotus aff. wandae is probably a species new to science both for the ITS2 differences (p-distance 0.17–0.18% from the three available sequences of Macrobiotus wandae; Supplementary Table S3) and for the different shape of the egg having a more expanded distal disk on the processes. Since only one egg and few animals of this species were collected and the cox1 sequencing gave no result, further collection and analyses will needed before a possible new species description.

The most common morphospecies in the samples was Ramazzottius oberhaeuseri (Doyére, 1840)⁵⁴. It was retrieved from 39.4% (13 terrestrial samples) of the samples and from all the sampled areas except Balsberget, with a highly variable density: e.g., 70.8 ind/g in a lichen, 0.3 ind/g in a soil with grass, and 0.1 ind/g in a moss or in a leaf litter. Milnesium asiaticum Tumanov, 2006⁵⁵ was found in 33.3% (11 samples) of the terrestrial samples from all the sampled areas, but with low density (0.1–1.4 ind/g). Hypsibius convergens (Urbanowicz, 1925)⁵⁶ was found in 32.4% (11 samples) of both terrestrial and freshwater samples, and in all the sampled areas except the HKR campus, with a low density (0.1–6.2 ind/g). Macrobiotus persimilis Binda & Pilato, 1972⁵⁷ had a wide distribution, found in 30.3% (10 samples) of terrestrial samples, with low density (0.3–2.3 ind/g). All the other morphospecies have a more restricted distribution within the samples (Table 3).

Considering all samples, the species diversity within individual samples and between sampling areas was variable (number of morphospecies: 0–7; 12–19, respectively), but most of the samples (67.6%) had three to six morphospecies, and only within two samples (SVC5, 6; 0.5%) there were no tardigrades (Table 3).

Source: Ecology - nature.com