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R Core Team R: A Language and Environment for Statistical Computing Version 3.6.0 (R Foundation for Statistical Computing, 2019). https://www.R-project.org/ More

Systematic palaeontology
Tetrapoda Jaekel, 1909 fide Sues20
Family undesignated
Termonerpeton makrydactylus gen. et sp. nov. (Fig. 1)Fig. 1: Termonerpeton makrydactylus gen. et sp. nov. holotype UMZC 2019.1.a Specimen photograph. b Interpretive drawing. Scale bars 10 mm. Abbreviations: acet acetabulum, fem femur, fib fibula, ha haemal arch, ic intercentrum, l left, na neural arch, phal phalanx, piliac p post-iliac process, plc pleurocentrum, r right, sac rib sacral rib, tib tibia.Full size image

EtymologyGenus: from τέρμωυ (térmon) meaning boundary and ερπετό (erpetó) meaning ‘crawler’, referring to the field boundary walls near the East Kirkton quarry where the late Stan Wood initially discovered fossils from the East Kirkton Limestone and from where the type specimen may have been collected; species: from μακρύς (makrýs) meaning ‘elongate’ and δάχτυλο (dáchtylo; more precisely, δάχτυλο ποδιού, dáchtylo podioú) meaning ‘toe’, referring to the very long pedal digit IV.HolotypeUniversity of Cambridge Museum of Zoology (UMZC) 2019.1. A partial tetrapod postcranium, preserving both pelves, a femur, fibula, tibia, and an almost complete but disarticulated pes. Closely associated with the appendicular elements are dorsally open hoop-shaped centra, a few neural and haemal arches, curved ribs, and a section of articulated gastralia.Locality and horizonEast Kirkton quarry, near Bathgate, Scotland, UK. East Kirkton Limestone, Bathgate Hills Volcanic Formation. Exact horizon is unknown. Brigantian, Viséan, early Carboniferous (=Mississippian)21.Differential diagnosisPossible autapomorphies: ilium with drawn-out, flat, blade-like dorsal process; very large, stout, and elongate metatarsal IV, greatly exceeding the length of metatarsals III and V (~30% or more). Possible tetrapod synapomorphies among post-Devonian taxa: distinct interepipodial space between tibia and fibula; well-ossified tarsus comprising tibiale, fibulare, intermedium, four centralia, and five distal tarsals. Possible amniote synapomorphies, but often showing reversed polarity in several stem- and crown amniote taxa: presumed pedal phalangeal formula 23454; robust and long pedal digit IV; enlarged intermedium and fibulare, together occupying more than half of proximal moiety of tarsus; curved ribs. Characters of uncertain polarity (also present in Caerorhachis): elongate, slender, and posterodorsally oblique post-iliac process; short puboischiadic plate with almost vertical anterior margin; stout femur with poorly pronounced waisting along the shaft, longer than puboischiadic plate; hoop-shaped centra.Attributed specimenNational Museums Scotland (NMS) G.1992.22.1. An articulated, partially complete, large tetrapod pes, preserving a nearly complete array of tarsals, all metatarsals, and the proximal phalanges of digits I–III. Unit 82, East Kirkton Limestone, East Kirkton quarry, near Bathgate, Scotland, UK.
Specimen description
Appendicular skeletonMost of the description is based upon the holotype. Both pelves are preserved, one mainly as a natural mould. The puboichiadic plates are short and deep, with an almost vertical anterior margin to the pubis (Fig. 1). In one, the surface of the puboischiadic plate is strongly convex, in the other it is strongly concave. The concave plate may belong to the left pelvis, with the concavity indicating the acetabulum. Both iliac processes of the presumed right ilium are overlain by a neural arch and part of the femur and cannot be seen. The presumed left ilium shows a long, posteriorly pointing post-iliac process that extends as far backward as the posterior edge of the ischium. It retains the proximal, stump-like portion of a dorsal iliac process, continued distally in natural mould as a mediolaterally flattened and blade-like structure. Both processes sit above a short iliac neck. The dorsal iliac process is proportionally longer than in other tetrapods and its knife-like appearance is unique. The angle between the two processes is much more acute than in most other tetrapods, and the nearest comparison is with the divided iliac process of the microsaurs Tuditanus and Ricnodon22 which, however, could merely represent a bifid post-iliac process. Two gaps in ossification are taken as evidence of an ilio-ischiadic suture half-way down the posterior margin on the left pelvis and an ilio-pubic suture halfway down the anterior margin of the right pelvis (Fig. 1). There is no evidence of a puboischiadic suture, although a shallow depression along the ventral margin of the left puboischiadic plate probably marks the junction between pubis and ischium. The complete left puboischiadic plate is 20 mm deep behind the ilium and 30 mm long, with the pubis contributing about one-third of its length and the ischium the remaining two thirds. The anterior margin of the pubis is almost vertical. The dorsal margin of the ischium is shallowly convex for half its length before extending posteroventrally to meet the upturned posterior extremity of the ischium’s ventral margin. There is no evidence as to the angle at which the two pelvic plates met at the symphysis, which would affect the position of the acetabulum relative to the substrate, and thus the effective resting posture of the hindlimb.The left femur is at least 39 mm in length, and longer than the puboischiadic plate. The entire bone is crushed, and its distal end lies partly beneath one of the pelvic halves and a neural arch so that its features cannot easily be made out. A possible intercondylar groove may be present distally, and the extensor surface of its proximal extremity appears to show a subcentral depression. The femur itself is robust with little waisting at mid-shaft. A small internal trochanter lies near its proximal end. The left fibula is approximately 26 mm long along its lateral margin. Its proximal end is narrow and grooved. Its broad and strongly flared distal end suggests a broad contact with the tarsus. The medial turn of the distal end indicates a large interepipodial space. The left tibia is about 20 mm long, slender, and shallowly waisted at mid-shaft. It is not clear which end is proximal and which distal, although probably the proximal is the broader. The tibia is probably more than half the length of the femur. Based upon the femur and tibia lengths, and omitting the ankle and pes, the above figures indicate a total stylopod-zeugopod length of about 65 mm, assuming a fully extended limb.Most of the morphology of the left pes is preserved, showing many well-ossified tarsal bones (Fig. 2). Several of these, including possible distal tarsals II and III lie more or less in anatomical continuity relative to metatarsals II and III, respectively. Other tarsal elements, including possible fibulare, tibiale, centralia, and distal tarsals, are illustrated in Fig. 2. Metatarsal IV lies in anatomical position relative to metatarsals II and III and, at 7 mm in length, it is significantly larger than the latter. The presumed first phalanx of pedal digit IV lies close to metatarsal IV, at an angle of nearly 90° to the latter. It is long and slender, indicating an unusually elongate fourth pedal digit.Fig. 2: Termonerpeton makrydactylus gen. et sp. nov. left hindlimb of UMZC 2019.1.a Specimen photograph, showing close-up view of hindlimb skeleton, b Interpretive drawing, with centralia, distal tarsals, and metatarsals indicated by red, blue, and black Roman numerals, respectively, c Interpretive drawing with dashed lines connecting elements of individual digits, d Reconstruction of left tibia, fibula and pes. Scale bars 10 mm. Abbreviations: interm intermedium, tib tibialia.Full size imageAn array of about 12 phalanges is preserved. They are all disrupted but occur in proximity to one another and, like the first phalanx of pedal digit IV, also mainly lie at right angles to metatarsals III and IV. An additional, acutely angled pointed ungual phalanx, possibly associated with digit II, is also visible. A further two phalanges have been displaced and rest along the anterior edge of the left pelvis. In total, we were, therefore, able to identify 15 elements. The preservation of the pes suggests it was strongly flexed either at death or from tissue shrinkage thereafter. An isolated metatarsal, presumably from the other, missing foot, lies some distance away near the edge of the block. Together, the pedal elements suggest a relatively large foot.A second specimen, NMS G.1992.22.1 (Fig. 3), is represented by an isolated pes. It may belong to Termonerpeton, although it is from a much larger individual. It shows five metatarsals of which the fourth is much longer and more robust than the other four and about twice as long as that of the holotype, while metatarsal V is the smallest. There are three phalanges, plus five distal tarsals. A D-shaped element closely associated with three centralia could be either a fibulare, a displaced intermedium, or centrale IV.Fig. 3: Termonerpeton makrydactylus gen. et sp. nov partial pes, attributed specimen NMS G.1992.22.1.a Specimen photograph, b with centralia, distal tarsals, and metatarsals indicated by red, blue, and black Roman numerals, respectively. Scale bars 10 mm.Full size imageAxial skeletonWhere visible, neural arches have short neural spines and prominent zygapophyses, but their shape is hard to assess as none is well preserved. The element overlying part of the right pelvis and the femur is 7 mm high in total. Numerous dorsally open, hoop-shaped centra about 5 mm in diameter are visible, as well as a few small, oval, shallowly curved elements (Fig. 1). Without further evidence, it is uncertain which of these elements are intercentra and which pleurocentra, though we assume that the larger elements are pleurocentra. The preserved ribs are slender and curved, and include trunk ribs, a possible presacral rib, a possible sacral rib, and a possible postsacral rib. This is long but more or less straight. A bone situated among a cluster of centra, somewhat distant from the other tarsal bones, was originally interpreted by us as a possible fibulare, similar to the fibulare in Proterogyrinus23. However, it might also be interpreted as a sacral rib. If so, its morphology is unique. It is short and widens distally into a fan-shaped structure but does not appear to have a bifid proximal end, unlike the sacral rib in Proterogyrinus23. Three haemal arches are present, one still attached to its half-hoop centrum, a second slightly longer, and a third very short and presumably from a more posterior region of the tail.ComparisonsThe exceptional preservation of tetrapods from the East Kirkton Limestone provides a unique opportunity to study portions of the skeletal anatomy that are otherwise poorly preserved or absent among Mississippian tetrapods. In particular, hindlimbs with a complete or near-complete array of tarsal elements and digits are notably rare. The unusual construction of the pes of Termonerpeton prompted us to examine the hindlimb morphology of six other East Kirkton tetrapods (Fig. 4a–g) alongside a selection of additional, mostly Carboniferous taxa (Fig. 4h–n). We focus on epipodials, tarsi, phalangeal formulae and digit length and proportions. To facilitate visual inspection of these elements, all hindlimbs are drawn to a common tibial length, except for the stem diapsid Petrolacosaurus, in which the epipodials are greatly elongate.Fig. 4: Comparison of the left tibia, fibula, tarsus, and digits of early tetrapods.a Balanerpeton after 2, b Eucritta after 12, c Eldeceeon after 6, d Silvanerpeton after 4, e Westlothiana after 7, f Kirktonecta original, see 15 (the grey area marks the estimated position and extent of the tarsus), g Termonerpeton, h Pederpes after 24, i, Greererpeton after 27, j Caerorhachis after 31, k Archeria after 30, l Hylonomus after 28, m Tuditanus after 22, n Petrolacosaurus after 29. Drawn to the same tibial length apart from n. Scale bars 10 mm.Full size imageIn terms of pes size relative to the tibia, the East Kirkton taxa Balanerpeton, Eucritta, and Silvanerpeton (Fig. 4a, b, d) are similarly proportioned. In contrast, Eldeceeon and Westlothiana (Fig. 4c, e) exhibit somewhat larger pedes. Kirktonecta has proportionally the largest pedes of all (Fig. 4f). Termonerpeton (Fig. 4g) has a pes of similar size to the first three taxa except that digit IV is relatively much longer than in any of the others, with an exceptionally large metatarsal IV. In all those taxa in which digit IV is fully preserved, it is the longest, especially in Eldeceeon and Kirktonecta, but in none does it approach in size and proportions that of Termonerpeton. The illustrated limbs also differ from one another in the degree of ossification of the tarsal bones. Most taxa except Eucritta have some indication of ossified tarsal elements, and some of them, including Balanerpeton and Silvanerpeton, show a complete or almost complete tarsal set. Kirktonecta does have an ossified tarsus, but specimen preservation does not allow us to identify individual elements. The phalangeal count, where known, also varies: 22343 in Balanerpeton2; 223?? in Eucritta12; 23455 in Silvanerpeton4; 23454 in Eldeceeon6, Kirktonecta15, Termonerpeton, and Westlothiana7.In addition, we compared the pedes of East Kirkton tetrapods with those of seven other taxa (Fig. 4h–n): one earlier, Pederpes24; one almost contemporary, Caerorhachis25; four later Carboniferous, Greererpeton26, Hylonomus27, Tuditanus22, and Petrolacosaurus28; and one early Permian, Archeria29. Of these, Greererpeton has relatively the smallest pes. In most, digit IV is the longest, though in Pederpes and Caerorhachis it is incomplete. The pes of Caerorhachis was originally restored with only three phalanges in digit IV30. This is probably incorrect and would be unusual in Carboniferous tetrapods. The pes of the anthracosaur Archeria was originally reconstructed with digit V as the longest29, but again this is unusual among later Carboniferous and early Permian tetrapods and we suspect that digits IV and V have been transposed, and Romer himself expressed doubt about this reconstruction29. In either case, the phalangeal formula of Archeria is similar to that of the East Kirkton anthracosaur Silvanerpeton, as 23455.Among Carboniferous tetrapods, temnospondyls such as Balanerpeton and colosteids such as Greererpeton show a digit IV that is somewhat longer than the others, but metatarsal IV is very similar in length and breadth to the adjacent metatarsals. In anthracosaurs, digit IV is the longest, but again metatarsal IV is not significantly broader than adjacent metatarsals. This is also the case in the early amniote Hylonomus and the microsaur Tuditanus. Among the taxa illustrated here, Termonerpeton shows a strikingly similar pes to that of the Late Pennsylvanian araeoscelidian diapsid Petrolacosaurus (Fig. 4n). In both, metatarsal IV is significantly longer and stouter than others and forms part of a similarly long digit IV. In early amniotes, an elongate digit IV coupled with an elongate metatarsal IV is a common occurrence in other taxa, such as protothyridids (e.g. Anthracodromeus31), basal araeoscelidians (e.g. Spinoaequalis32), younginids (e.g. Youngina33), saurians33, and basal synapsids (e.g. Heleosaurus34,35,36), among others.Based upon available evidence, an elongate digit IV is likely to be the plesiomorphic condition for crown amniotes, being present in Hylonomus, Paleothyris, and Petrolacosaurus (Fig. 4l, n), and shortening of this digit certainly represents a derived feature. In later crown amniotes, the conditions vary, with larger, heavier-bodied tetrapods such as dicynodonts and diadectids having generally shorter toes and adopting a more clearly plantigrade posture. An elongate metatarsal IV and associated digit, however, are not universal among Palaeozoic amniotes, and modifications of these conditions occur repeatedly across clades. For instance, in the eureptile captorhinid Eocaptorhinus, digit IV is also the longest, but the length of metatarsal IV does not greatly exceed that of other metatarsals37. The same is true of some early Permian clades, including seymouriamorphs (e.g. Seymouria38; Discosauriscus39), and diadectids (e.g. Diadectes40), although in the diadectomorph Orobates digit III is a little longer than digit IV41. Among synapsids, dicynodonts such as Diictodon42 and caseids43, to name a few, have five pedal digits of approximately uniform length.We further point out that, while digit IV attains a certain degree of elongation in other early tetrapod groups, such as temnospondyls, in none of them do the relative proportions of this digit (where known) compare to those of several stem and crown amniotes (Fig. 4).Phylogenetic relationshipsThe results of various phylogenetic analyses lend some support to the interpretation of Termonerpeton as a stem amniote, despite its uncertain placement in the unweighted character parsimony analysis (Fig. 5a). In the latter analysis, Termonerpeton appears in a polytomous node alongside baphetids (Eucritta; Baphetes; Megalocephalus), temnospondyls (Balanerpeton; Dendrysekos), the anthracosauroids Eldeceeon and Silvanerpeton, and the problematic Caerorhachis. In all other analyses—implied weights, reweighted characters, and Bayesian—Termonerpeton is placed on the amniote stem group, albeit in different positions, among a diverse array of ‘reptiliomorph’ clades and grades. In the implied weights analysis (Fig. 5b), Termonerpeton, Silvanerpeton, and Eldeceeon form a monophyletic group branching crownward of chroniosaurs plus anthracosaurs and anti-crownward of paraphyletic gephyrostegids. In the reweighted analysis (Fig. 5c), Termonerpeton and Caerorhachis appear as successive sister taxa, in that order, to monophyletic anthracosaurs. In the Bayesian analysis (Fig. 5d), the amniote total group receives moderate support with a credibility value (c.v.) of 76 with Caerorhachis as the most plesiomorphic stem amniote. Crownward of Caerorhachis is a polytomy with low support (c.v. = 59) that subtends Termonerpeton, a clade consisting of Eldeceeon plus Silvanerpeton, a clade of anthracosaurs, and a clade that includes all remaining taxa. In crownward succession, these taxa include chroniosaurs, gephyrostegids, seymouriamorphs, Solenodonsaurus, and Westlothiana as successive sister groups to a strongly supported (c.v. = 100) clade containing diadectomorphs, synapsids, and eureptiles. Although eureptile monophyly is not retrieved, strong support (c.v. = 100) is given to the branch subtending diadectomorphs plus synapsids44.Fig. 5: Results of phylogenetic analyses.a Strict consensus of 120 shortest trees from unweighted analysis (tree length = 1286 steps, ensemble consistency index C.I. = 0.2738 without uninformative characters, ensemble retention index R.I. = 0.5768), b Single tree from implied weights analysis (tree length = 1298 steps, Goloboff fit = −202.59266, C.I. = 0.2712, R.I. = 0.5713), c Single tree from reweighted analysis (tree length = 212,68965 steps, C.I. = 0.4755, R.I. = 0.774), d Bayesian topology with branches reporting credibility values.Full size image More

Species used in the experimentsMilnesium inceptum32 (Fig. 1A, a picture taken using Olympus BX41 Phase Contrast light Microscope associated with Olympus SC50 digital camera) is an obligatory predatory species with the body length ranging from 326 to 848 μm. It feeds on rotifers, nematodes and other tardigrades and lays smooth eggs in exuviae. To stay active, M. inceptum needs a thin water film around its body14. The species inhabits places exposed to shorter and longer periods of drying i.e. frequently drying mosses growing on cement walls32. Till now it was reported in Poland, Germany, Japan, Switzerland and Bulgaria32. At the same time, it is a perfect organism for our research because (1) it is large and easy to observe, (2) it tolerates frequent periods of entering and leaving anhydrobiosis, (3) it easily creates a tun stage. Milnesium inceptum for experimental purposes were acquired from a moss sample from a cement wall in Poznań, Poland (52°24′15″N, 16°53′18″E). The extraction of tardigrades was conducted under stereomicroscope (Olympus SZ51) using standard methods33. Then specimens, further used in our experiments, have been cultured based on protocol proposed by Roszkowska et al.34. Only fully active, adult specimens were selected for the experiments.Figure 1Model animals used in experiments: (A) Milnesium inceptum; insert shows tardigrade in the tun state; (B) Cepaea nemoralis in its natural environment; (C) a tardigrade that appeared on moss surface during in vivo observation of rehydrated moss cushion (red arrow). Figures were assembled in Corel Photo-Paint 2017 (http://www.corel.com).Full size imageCepaea nemoralis35 (Fig. 1B, a picture taken using Motorola g(9), Camera version 7.3.63.53-whitney) is a stylommatophoran European land snail species, which is widespread and common throughout the continent36. The average maximum shell diameter is 20 to 22 mm37. It feeds on plant materials available, yet has a strong preference for dead and senescent herbs38. C. nemoralis occurs in variable habitats (frequently in synanthropic ones) such as forests, meadows, gardens, near shrubs or dunes36.The period of its activity falls on the growing season; it usually comes out of the shell and crawls when the air humidity reaches 70% or more, independently from solar radiation and air temperature28. The species is a good model for our study due to its: (1) large size compared to tardigrades, and (2) co-occurrence with M. inceptum in natural environments. Individuals of C. nemoralis were harvested from anthropogenic environment: gardens adjacent to detached houses (52°25′28″N, 16°46′52″E). Snails were collected from plants, cement walls and ground surfaces. After collection, all C. nemoralis specimens were washed-up and placed in 30 L (480 × 360 × 252 mm) transparent plastic box with mesh covering for ventilation. Soil and rocks were placed in the box allowing to maintain a moist shelter for snails, and a sepia was used as a source of a calcium. Animals were fed with lettuce, cabbage and nettle twice a week and sprinkled with water to stimulate their activity. Box containing snails was kept in a rearing room, at 17 °C in 12:12 photoperiod. Snails were kept in the box for 1.5 months prior to the experiments. For the experiments we used only adult animals. The snails were checked under Olympus SZX7 stereomicroscope prior to the experiment to ensure they were free of tardigrades.Pilot studiesDoes the tardigrades’ distribution within a moss cushion enable tardigrade-snail contact?To check whether tardigrades may come into a close encounter with the snail in the natural environment (which would be impossible if the tardigrades were only present in the lower layers of the moss), we investigated the distribution of water bears within moss cushions. The observations were performed for 6 samples of dried moss cushions (ca. 1 cm high and 3 cm in diameter). The moss containing M. inceptum specimens, was collected from a concrete wall in Poznań, Poland (52°24′15″N, 16°53′18″E), the same from which tardigrades were initially collected for the culturing purposes. Three moss cushions were rehydrated, and left for 3 h followed by further observation to check whether tardigrades may actively move across the moss cushion. On the remaining three moss samples, a horizontal cut was made through the center of the moss cushion to check in which layer tardigrade tuns are present while the moss remains dry. The extraction of tardigrades from separated layers was conducted under stereomicroscope (Olympus SZ51) using standard methods33.Within the dry moss cushions tardigrades were present in both the upper and lower moss layers. We did not observe any difference in the number of individuals of M. inceptum that would be dependent on the moss layer. A total of 353 tardigrades were extracted from one moss cushion (dry weight of moss = 0.332 g), what gives the density of tardigrades per 1 g of dry moss sample equal to 1063 specimens. The observation of rehydrated moss cushions conducted in vivo using Olympus SZX16 stereomicroscope associated with Olympus DP74 digital camera and cellSens software revealed that single active tardigrades may also appear on the moss surface (Fig. 1C, red arrow). Therefore, observed in the pilot studies tardigrades distribution within the moss cushion enables tardigrade-snail contact.Is it possible for a tardigrade to take a snail ride?The initial observations were carried out for snails and tardigrades to check whenever a tardigrade may be transferred by a snail. In total, 10 snails and 20 active tardigrades were used. Two variants of Petri dishes (ø 90 mm) were prepared: (1) with smooth and (2) scratched bottom, to avoid and allow tardigrade attachment to the bottom of the dish, respectively. We repeated the observation five times per option. For each single observation we used one snail and two tardigrades.Snails and tardigrades were split equally between the pilot’s experimental options (in total 5 snails and 10 tardigrades per option). We checked whether tardigrades may be transferred by snails by putting tardigrades in the drop of water in the center of a Petri dish and releasing an active snail to crawl through the drop. In total, in the case of the smooth-bottom option, three tardigrades glued to the snail’s body within which two were moved to a distance up to a few centimeters. The third one fixed to a snail’s leg and had a potential to be transferred to a greater distance. In the case of the dishes with the scratched bottom, we did not notice any transfer. Tardigrades were attached tightly to the dishes’ bottom and remained unmoved after the snail had passed through them. Therefore, the observation in the pilot study confirmed that tardigrades may stick to snails’ body and be transferred by a gastropod at least when the substratum (bottom of the dish) is smooth.Experimental design
Experiment 1. Do snails have a significant effect on tardigrade dispersion that depends on the substrate type?As the laboratory environment offers limited possibilities to reflect natural conditions, we aimed to create an environment similar to the natural one by eliminating as many artificial elements as possible and, at the same time, enabling observation and data collection. To imitate a natural microhabitat of water bears we used a piece of moss as a substrate. Moss is a natural shelter and a hunting space for these animals, and a gripping surface that prevents them from being easily carried away by a stream of water or wind. The moss Vesicularia dubyana39 used in the experiment was purchased in an aquarium shop and was derived from an in vitro culture. It was checked under Olympus SZX7 stereomicroscope prior to the experiment to ensure it was free of tardigrades. For experimental purposes we used plastic ventilated boxes with dimensions 950 mm × 950 mm × 600 mm, tightly closed with a plastic lid. The bottom of each box was scratched with sandpaper in order to (1) imitate a rough surface of a concrete wall to which mosses are attached in the natural environment; (2) allow tardigrade locomotion. At the same time, moss and (unfortunately) plastic elements are quite common surroundings of C. nemoralis frequently found in anthropogenic habitats36.Using transparent, non-toxic aquarium silicone, a square with a side length of 3 cm and a height of 0.5 cm was mounted on the bottom of the box. Before starting the experiment, the tightness of the square silicone barrier was checked by pouring 2.5 ml of water inside and leaving the boxes for observation for 24 h. After this time, all silicone squares turned out to be impermeable to water.Boxes for each of the experimental option, namely: (A) control (further in the text referred as C), (B) tardigrades + snail (referred as TS), and (C) tardigrades + snail + moss (referred as TSM, see Fig. 2), were prepared in a following way: 2.5 ml of water was added to the scratched bottom of the box inside the silicone square and 7.5 ml to the area outside of the silicone square to enable survival and active locomotion of tardigrades on both sides of the silicone barrier. Then, 10 active individuals of M. inceptum taken from the culture were transferred to the center of the silicone square. It was repeated for 90 boxes (30 boxes per each C, TS and TSM option). Therefore we used 300 tardigrades per each experimental option which gives 900 tardigrades in total for all experimental options. In case of 30 boxes with TSM option, a piece of moss (ca. 2.5 cm in diameter) was added. It was situated in the center of the silicone square, just after the tardigrades were placed at the boxes in order to isolate tardigrades from the snail during the experiment.Figure 2Graphical representation of three designed experimental options of the experiment 1. (A) 10 tardigrades in the silicone square (control (C)); (B) 10 tardigrades in the silicone square and one snail placed in the box (tardigrades + snail (TS)); (C) 10 tardigrades in the silicone square, one snail placed in the box and additional piece of the moss added as a barrier between tardigrades and snail (tardigrades + snail + moss (TSM)). Figures were assembled in Corel Photo-Paint 2017 (http://www.corel.com).Full size imageFinally, in the boxes targeted for TS and TSM experimental options, one adult and active individual of C. nemoralis snail was placed in each box outside the silicone square. In total, 60 snails were used (30 individuals per experimental option).The boxes were then placed in the rearing room (17 °C, 80% of humidity, photoperiod 12:12) for 72 h. After this time, the number of tardigrades inside and outside the silicone square was counted (both: live and dead) separately for each box, using Olympus SZX7 stereomicroscope.Experiment 2. Effect of the snail’s mucus on tardigrade recovery to active life after anhydrobiosis
Milnesium inceptum anhydrobiosis protocolOnly fully active, adult specimens of medium body length were selected for the experiment. The animals were transferred to ø 3.5 cm vented Petri-dishes with bottom scratched by sandpaper to allow tardigrade locomotion. Five tardigrade individuals were placed to each Petri dish together with 450 µl of water and then dehydrated. In total, 16 Petri dishes with 5 tardigrades on each were prepared. Dehydration process lasted 72 h and was performed in the Q-Cell incubator (40–50% RH, 20 °C, darkness). After that time tardigrade tuns were kept under the abovementioned conditions for 7 days.Impact of the snail’s mucus on tardigrade tunsAfter 7 days of anhydrobiosis, one individual of C. nemoralis was transferred to each dish with tardigrade tuns and was left there for 1 min allowing the snail to actively crawl over the tuns. 30 min after the snail was removed from the dish, tardigrade tuns were observed under the Olympus SZX7 stereomicroscope for any animal movements. Then, all covered and vented dishes were left in the Q-Cell incubator overnight. After 24 h, the dried tuns were rehydrated by adding 3 ml of water to each Petri dish to check whether snail’s mucus affected mortality rates of tardigrades. After 3 and 24 h following rehydration tardigrade tuns were observed for any animal movements. Pictures of tuns were taken using Olympus SZ61 stereomicroscope associated with Olympus UC30 camera (Fig. 3). As reference data on the rehydration of the M. inceptum tuns free of the snail’s mucus, we used the data from Roszkowska et al.20 who tested anhydrobiosis survivability of above-mentioned species. Individuals used for the tuns preparation in the control option were collected from the same laboratory breeding stock, and prepared at the same laboratory conditions as those used in our experiments20.Figure 3Milnesium inceptum tuns: (A,B) before contact with snail mucus; (C,D) coated with wet snail mucus; (E,F) coated with dry snail mucus. Figures were assembled in Corel Photo-Paint 2017 (http://www.corel.com).Full size imageStatistical analysesThe number of tardigrades relocated in each experimental option (C, TS and TSM) was compared with a one-way ANOVA randomized version using RundomPro 3.14 software40. We used non-parametric methods because of the lack of normality. Differences were considered significant at p  More