Tardigrades of the genus Halobiotus (Eutardigrada, Isohypsibioidea) inhabiting Arctic seas – new taxonomic and biological data

Tardigrades of the genus Halobiotus (Eutardigrada, Isohypsibioidea) inhabiting Arctic seas – new taxonomic and biological data | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tardigrades of the genus Halobiotus (Eutardigrada, Isohypsibioidea) inhabiting Arctic seas – new taxonomic and biological data Denis V. TUMANOV, Elena A. GOLIKOVA, Olga V. KNYAZEVA, Alexandra Yu. TSVETKOVA This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8224833/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In this paper we present the results of taxonomic and ecological investigation of several populations of marine tardigrades of the genus Halobiotus inhabiting the seas of Russian Arctic. We studied representatives of the genus Halobiotus collected from three localities of the White Sea, from the Barents Sea and from the Laptev Sea. Morphological analysis was performed using the methods of light and scanning electron microscopy, and supplemented with statistical analysis of the morphometric data. For the comparative genetic analysis, we obtained data on mitochondrial COI gene and on 18S rRNA, 28rRNA and ITS-2 sequences. Our study revealed the presence of two species, clearly differentiated both morphologically and genetically in the fauna of Arctic seas of Russia. Comparison of our specimens with the available type material of the previously described Halobiotus species and the obtained gene sequences with those deposited in GenBank made it possible to attribute the White Sea and the Barents Sea populations to the species Halobiotus crispae , while the Laptev Sea population was recognized as belonging to Halobiotus arcturulius . We provide here emended morphological descriptions for both species based on the in-depth study of the obtained material together with the type specimens of both species. In our opinion, the genus Halobiotus currently includes only two adequately described species, other species of this genus should be considered nomina inquirendae. We provide a new diagnosis for the family Halobiotidae based on the new data, and specify taxonomic position of the species Isohypsibius occultus as Grevenius occultus comb. nov. We also provide new biological data for the Halobiotus spp. populations from the Barents Sea and the Laptev Sea. Animal Science Marine and Freshwater Biology Taxonomy morphology morphometry ecology tardigrades Halobiotidae White Sea Laptev Sea Arctic fauna Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Introduction Tardigrades are a group of microscopic invertebrates, inhabiting all types of waterbodies from ocean depths to ephemeral micro reservoirs in hygroscopic terrestrial substrates, like moss cushions (Nelson et al. 2018). Most of the marine tardigrade species belong to the taxa currently classified as the class Heterotardigrada – specifically, the order Arthrotardigrada and the family Echiniscoididae within the order Echiniscoidea (Nelson et al. 2018). Beside these primarily marine groups, several species of the class Eutardigrada can be found in marine environments. Some of them belong to the freshwater taxa that occasionally occur in marine sediments, likely due to the accidental transportation from their natural habitats by currents (e.g. the genera Borealibius Pilato, Guidetti, Rebecchi, Lisi, Hansen & Bertolani, 2006, Murrayon Bertolani & Pilato, 1988 and Thulinius Bertolani, 2003; Biserov 1998; Kaczmarek et al. 2015). Only one group within the class Eutardigrada is permanently associated with marine environments – the genus Halobiotus Kristensen, 1982 comprising the monotypic family Halobiotidae Gąsiorek, Stec, Morek & Michalczyk, 2019. Descriptions of the first two species of this genus— Halobiotus stenostomus (Richters, 1908) and H. appelloefi (Richters, 1908)—were published alongside in the publication by Richters (1908). Both species were described from the Baltic Sea. Like many species descriptions of the early period of the tardigrade systematics they are very brief and incomplete, and do not meet modern requirements for the taxonomic descriptions. It is very likely that H. stenostomus is a cyclomorphic stage characteristic of this genus (Kristensen 1982). Moreover, other authors (Marcus 1936 and Thulin 1928) described several forms of marine Eutardigrada from the Baltic Sea under the names H. stenostomus and H. appelloefi , making the situation even more complicated. Another species ( Halobiotus geddesi (Hallas, 1971)) was described by Hallas (1971) on the basis of the material from Kattegat and Norwegian Sea. Later Petelina & Tchesunov (1983) synonymized this species with H. appelloefi . Kristensen (1982) published the first modern description of the Halobiotus species. In his work, he described a new species from Greenland ( Halobiotus crispae Kristensen, 1982), instituted a new genus Halobiotus for the stenostomus / appelloefi / geddesi / crispae species complex and described a life cycle with “pseudosimplex” cyclomorphic stage. A year later Crisp & Kristensen (1983) described another species of the genus – Halobiotus arcturulius Crisp & Kristensen, 1983, from Greenland as well. Pilato & Binda (1996) revised the type material of H. crispae and amended the diagnosis of the genus. They pointed out the absence of the ventral lamina in the buccal-pharyngeal apparatus and described apophyses for the insertion of the stylet muscles (AISM) as hook-like and symmetrical in respect of the frontal plane. Addition of the molecular genetic methods to the tools for the tardigrade phylogeny studies led to significant changes in the taxonomy of this group. An isolated position of the forms morphologically close to the genus Isohypsibius Thulin, 1928 was revealed, and they were allocated to the independent superfamily Isohypsibioidea Sands, McInnes, Marley, Goodall-Copestake, Convey & Linse, 2008. A later integrative revision of this taxon (Gasiorek et al. 2019) confirmed the separate position of the genus Halobiotus within the superfamily, and a new family Halobiotidae was established. Later, these conclusions were supported by the results of other works devoted to the phylogeny of the superfamily Isohypsibioidea (Mioduchowska et al. 2021; Tumanov 2022; Tumanov et al. 2025). It should be noted that, though the descriptions of the two latter species of the genus Halobiotus are accurate and supported by high-quality photographs acquired using both the light microscopy (LM) and scanning electron microscopy (SEM), they do not completely meet the standards of the taxonomic descriptions accepted in the systematics of the Eutardigrada nowadays. Morphometric data are provided for a small number of specimens and the set of measurements differs from the currently accepted for the Eutardigrada descriptions. Global distribution of the genus Halobiotus is still poorly understood. Most of the records are limited to the cold Arctic and Subarctic waters (Kaczmarek et al. 2015), but there are a few reports from the Black Sea (Romania and Ukraine; Rudescu 1964; Kharkevych 2013). Taxonomic status of these records is unclear due to the limited data on the specimens’ morphology and complete absence of molecular data. Four species of the genus Halobiotus were previously reported from Russian seas: 1. Halobiotus appelloefi was recorded by Petelina & Tchesunov (1983) from the Kandalaksha Bay (White Sea). Tardigrades were found in periphyton on the littoral and sublittoral macrophytes, on the pier and in the silted sand near the macrophytes’ bases. The authors, having analyzed the literature data available at the time and the morphometric data they had obtained, showed that the description of H. geddesi as a separate species is poorly justified. They also noted an unclear taxonomic situation in the appelloefi/stenostomus complex (Kristensen’s work (1982) on cyclomorphosis was not known to them yet) and pointed out the importance of studying the limits of variability of morphometric features of tardigrades for the reliability of taxonomic studies. 2. In their study of head sensory structures in genus Halobiotus , Biserova & Kuznetsova (2011) identified specimens collected from intertidal filamentous algae in the Kandalaksha Bay (White Sea) as H. stenostomus , which they accepted as the senior synonym of H. appelloefi . At the same time, they noted that the studied material had a number of features characteristic of the species H. crispae , therefore the exact species identification of the White Sea Halobiotus requires clarification. In addition, Biserov (1991) indicated H. stenostomus for the White Sea (without specifying a locality). 3. In the most recent publication (Biserova et al. 2025) specimens from subtidal sandy sediments in the Kandalaksha Bay (White Sea) were identified using the methods of the DNA barcoding and the analyzed sequences of the nuclear and mitochondrial markers (18S rRNA, 28S rRNA, ITS-2 and COI) were proved to be conspecific with H. crispae , but no detailed morphological data were provided. 4. Halobiotus arcturulius was reported to inhabit silty-sand tidal flats in the Kara (Baydaratskaya Bay) and the Laptev (near the mouth of the Kurkum/Urya River) Seas (Biserov 1998). Furthermore, several specimens presumably assigned to this species were found in a freshwater lake on the Yuzhny Island of the Novaya Zemlya archipelago (Biserov 1999). To address the uncertain taxonomy and incomplete descriptions of the tardigrade genus Halobiotus in Russian seas and to amend the existing taxonomic descriptions of the species belonging to the genus, we conducted an integrative morphological and molecular genetic analysis of populations from three Russian Arctic seas. This study aimed to determine the species affiliations of these populations and to generate a comprehensive dataset for reliable species identification. Furthermore, to elucidate the ecology of these species, we quantified the distribution of two Halobiotus species across two Arctic tidal flats. Materials and methods Sampling Material was collected from three localities of the White Sea Kandalaksha Bay (Fettah island, Gorelyj island, and Matrenin island), from the Barents Sea and the Laptev Sea (see detailed information below in the Results section). The White Sea samples were obtained by the following procedure. Sampled bottom material was washed through two sieves (with 1 mm and 29 µm mesh respectively) (Tumanov 2018 ). Contents of the fine sieve from Fettah island were fixed with 96% ethanol. We also examined the content of the fine sieve from locations Fettah island and Matrenin island under a stereo microscope and isolated animals using a glass pipette. These specimens were fixed in RNA later ™ Stabilization Solution (Thermo Fisher Scientific Inc.). Under a stereo microscope specimens from Gorelyj island were manually picked from the tubes of polychaete Dipolydora quadrilobata (Jacobi, 1883) and mounted on the permanent slides without fixation. Quantitative data on tardigrade distribution across the intertidal zone were obtained from two locations: the Barents Sea and the Laptev Sea. Sediment samples from the Barents Sea coast were collected on 22 July 2019 from silty-sand tidal flats near the Mishukovo village (Kola Bay). The sampling transect included four stations with two stations located on the tidal flat and two stations – on the adjacent salt marsh. Sediment temperature and pore water salinity were measured at each station (for details see Golikova et al. 2024 ). Station 3 was primarily vegetated by Puccinellia phryganodes , while Agrostis straminea dominated at Station 4. At each station, three replicate samples of 10 cm 3 each were collected from the top 1 cm sediment layer, yielding a total of 12 samples. These were immediately preserved in 80% ethanol with Rose Bengal dye (2 g/L). After four weeks of staining, samples were washed with tap water and sieved simultaneously through 0.5 mm (removing plant debris) and 0.125 mm meshes, with the > 0.125 mm fraction retained for the analysis. The Laptev Sea specimens were collected on 17 September 2017 across an intertidal transect on Bolshoy Begichev Island in the Khatanga River estuary. Following a similar design to the Barents Sea study, the transect comprised four stations spaced 2–3 m apart and extended from a tidal flat with silty sand substrate (stations 1–2) to an upper intertidal zone colonized by facultative halophytic vegetation (hereafter referred to as salt marsh; stations 3–4) (Fig. 1 ). Station 3 was covered with a dense turf of Puccinellia phryganodes , while Station 4 was dominated by a mix of Dupontia psilosantha and Arctophyla fulva . Plant specimens had marks of geese grazing. No temperature or salinity measurements were taken. At each station, three replicate sediment samples (20 cm 3 each) were collected using a 20-cm 2 scoop, sampling the sediment top 1 cm of with 5–30 cm spacing between replicates. In total, 12 samples were obtained. Samples were fixed in 70% ethanol with Rose Bengal dye (2 g/L). After one month of preservation, samples were washed with tap water through nested 0.5 mm and 0.125 mm sieves simultaneously to remove plant debris, retaining > 0.125 mm fraction for analysis. Tardigrades were counted wet in a Petri dish under a LeicaM205C stereomicroscope prior to morphological and molecular analysis. The counts were standardized to 10 cm 3 . Microscopy and imaging For light microscopy (LM) investigation tardigrades were mounted on slides in Hoyer’s medium. Permanent slides were examined under a Leica DM2500 microscope equipped with phase contrast (PhC) and differential interference contrast (DIC). Photographs were taken using a Nikon DS-Fi3 digital camera with NIS software. For scanning electron microscopy (SEM), ethanol fixed specimens were transferred to acetone, critical-point dried in CO 2 , mounted on stubs and coated with gold. Tescan MIRA3 LMU Scanning Electron Microscope (Tescan, Brno, Czech Republic) and Hitachi TM-1000 (Hitachi, Japan) were used for observations. Morphometrics and terminology The sample size for morphometrics was chosen following the recommendations of Stec et al. ( 2016 ). All dimensions are given in micrometers (µm). Structures were measured only if their orientation was suitable. Body length was measured from the anterior end of the body to the posterior end, excluding the hind legs. The bucco-pharyngeal tube was measured from the anterior margin of the stylet sheaths to the caudal end of the buccal tube, excluding the buccal apophyses. Terminology for structures of the bucco-pharyngeal apparatus and claws follows Pilato & Binda ( 2010 ). Elements of the buccal apparatus were measured according to Kaczmarek & Michalczyk ( 2017 ). Claws were measured according to Beasley et al. ( 2008 ), with the additional measurement of the total claw length (according to Pilato et al. 2002 ) to ensure compatibility with older publications. We calculated the pt index, which is the percentage ratio between the length of a structure and the length of the buccal tube (Pilato 1981 ), and presented it in italics. Average values are given with standard deviation (SD). Morphometric data were handled using ver. 1.6 of the ‘Parachela’ template, which is available from the Tardigrada Register (Michalczyk & Kaczmarek 2013 ), with the addition of the total length of the claws. Statistical analysis For the statistical analyses we chose the measurements accessible for the most specimens we analyzed. Namely, we used stylet supports insertion point, internal and external buccal tube width, length of the macroplacoids 1–3, length of the macroplacoid row for the buccal-pharyngeal apparatus and length of the base, the primary and the secondary branches for the posterior claws of the hind legs. To avoid the effect of the absolute body size of the specimens we used the pt index (Pilato 1981 ) in the analyses. Statistical analyses were performed using ‘R’ (R Core Team 2017) in RStudio environment (RStudio Team 2017). Ordination of morphometry data was performed on the distance matrix calculated with Euclidean distances. To visualize the multidimensional distance matrix, we ran non-metric multidimensional scaling (Borcard et al. 2011 ) of individuals in ‘vegan’ package (Oksanen et al. 2017) and plotted the two-dimensional outcome with ‘ggplot2’ package (Wickham 2009 ). A stress value was calculated as well and is shown on the graph. A Permutational Multivariate Analysis of Variance (PERMANOVA) was conducted to evaluate morphological differences between the Laptev Sea and the White Sea (Fettah island) populations. This analysis was performed using the ‘adonis’ function in the ‘vegan’ package for R. These specific populations were selected as they were the only ones with an adequate sample size and corresponding molecular data required for a robust comparison. Genotyping DNA was extracted from 22 individual specimens using QuickExtractTM DNA Extraction Solution (Lucigen Corporation, USA, see complete protocol description in Tumanov 2020 ). Four genes were sequenced: a small ribosome subunit (18S rRNA) gene, a large ribosome subunit (28S rRNA) gene, internal transcribed spacer (ITS-2) and the cytochrome oxidase subunit I (COI) gene. PCR reactions included 5 µL template DNA, 1 µL of each primer, 1 µL DNTP, 5 µL Taq Buffer (10×) (− Mg), 4 µL 25 mM MgCl 2 and 0.2 µL Taq DNA Polymerase (Thermo Scientific™) in a final volume of 50 µL. The lists of primers and PCR programs are provided in Table 1. The PCR products were visualized in 1.5% agarose gel stained with ethidium bromide. All amplicons were sequenced directly using the ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the help of an ABI Prism 310 Genetic Analyser. Sequences were assembled using ChromasPro software (Technelysium, USA). To check for the presence of stop codons and therefore of pseudogenes, the COI sequences were translated into amino acids using the invertebrate mitochondrial code implemented in MEGA11 (Tamura et al. 2021 ). Unadjusted pairwise distances were calculated using MEGA11 (Tamura et al. 2021 ) with the treatment of gaps/missing data set to ‘pairwise deletion’. Institutional acronyms Specimens from the following institutions and collections were examined (the curator is given in parenthesis). SPbU = St. Petersburg State University, Russia, Faculty of Biology, Department of Invertebrate Zoology (Denis Tumanov). NHMD = Natural History Museum of Denmark (Martin Vinther Sørensen) ZMUC = Zoological Museum, University of Copenhagen (Martin Vinther Sørensen). Results Taxonomic account Phylum Tardigrada Doyère, 1840 Class Eutardigrada Richters, 1926 Order Parachela Schuster, Nelson, Grigarick & Christenberry, 1980 Superfamily Isohypsibioidea Sands, McInnes, Marley, Goodall-Copestake, Convey & Linse, 2008 Family Halobiotidae Gąsiorek, Stec, Morek & Michalczyk, 2019 Genus Halobiotus Kristensen, 1982 Halobiotus crispae Kristensen, 1982 Material examined WHITE SEA – Kandalaksha Bay, Fettah island • 65 specs; 66.334° N, 33.652° E; 15. Aug. 2022; D.V. Tumanov leg.; below the water’s edge at low tide, Ascophyllum sp. overgrown by sponges and hydroids; GenBank nos: PX248699, PX248700, PX248701, PQ070004 (18S), PQ070031 (28S), PX260918, PX260919 (ITS-2), PX248704 (COI); SPbU 301(1–15) • 22 specs; same data as for preceding; SEM stubs SPbU Tar_57, SPbU Tar_60. – Kandalaksha Bay, vicinity of Luvenga village • 25 specs; Gorelyj island, Ilistaja Bay; 67.095° N, 32.679° E; 1992; V.M. Khaitov leg.; in tubes of Dipolydora quadrilobata ; SPbU 327(1–5). – Kandalaksha Bay, Chupa Bay • 1 spec.; Matrenin island; 66.310° N, 33.630° E; 13 Jun. 2021; V.R. Khabibulina leg.; intertidal colonies of hydroids; GenBank nos: PX248698 (18S), PX260917 (ITS-2), PX248703 (COI); SpbU 278(1). BARENTS SEA • 21 specs; vicinity of Mishukovo village, 69.041° N, 33.028° E; 22 Jul. 2019; D.A. Mikhailov leg.; silty-sand tidal flats,; SPbU 262(1–5). Additional material Baffin Bay • 14 specs, paratypes; Nipissat Bay, Disko Island, West Greenland; 69°26′ N, 54°12′ W; depth 0.5 m; 18 May 1979; R.M. Kristensen leg.; subtidal on the brown alga Sphacelaria arctica ; ZMUC TAR-253 • 12 specs, paratypes; same data as for preceding; 03 Jan. 1979; R.M. Kristensen leg.; subtidal on the brown alga Sphacelaria arctica ; ZMUC TAR-255. Øresund • 1 specs; Helsingør, Denmark, S for Båkerne; labelled as Isohypsibius appelloefi ; ZMUC TAR-181. Øresund • 1 specs; Helsingør, Denmark; depth 28 m; 17 Apr. 1980; R.M. Kristensen leg.; mud, labelled as Isohypsibius appelloefi ; ZMUC TAR-182. Skagerrak • 7 specs; Tjarno, Sweden; depth 17–20 m; 23 Oct. 1987; R.M. Kristensen leg.; on Zostera , labelled as Halobiotus stenostomus ; NHMD-633704–633707. Photographs of H. geddesi type material were provided by Prof. Roberto Bertolani (University of Modena and Reggio Emilia). Amended morphological description Based on the material from the White Sea (Figs. 2 – 5 , 7 A–C) and the type material (Figs. 6 , 7 D). Active stage. Medium-sized animals (body length 273–555 µm, other morphometrics provided in Table 2). Body elongated, with the greatest width at the level of legs III (Figs. 2 A–C, 6 A, 7 A). Fresh specimens uncoloured or whitish with greenish gut content, transparent after fixation in Hoyer’s medium. Some specimens with intensive purple body colouring, which persists in the mounted animals. Eyes present in living specimens, dissolving after mounting in in Hoyer’s medium. Dorsal cuticle with sculpture. The degree of development of the cuticular sculpture varies significantly – from well-defined (Figs. 2 D–F, 6 B) to practically indistinguishable. The cuticle sculpture in both the type material and the White Sea specimens is not “reticular”, as indicated in the original description, but is a system of irregular winding folds, which only in rare cases looks like “reticular” sculpture at low magnifications of the microscope (Fig. 2 D). External cephalic sense organs include two pairs of sensory areas, which in fixed specimens are visible as flat plates, distinctly demarcated (in both LM and SEM) with a circular groove. The anterior pair was designated by Kristensen ( 1982 ) as “cephalic papillae” (Fig. 3 B–D) corresponds to the “antero-lateral sensory fields” (Walz 1978 ) or “frontal lobes” (Gąsiorek et al. 2019 ) of other eutardigrades. The posterodorsal pair designated by Kristensen ( 1982 ) as “temporalia” (Fig. 3 A, C, white arrowheads) corresponds to the “elliptical organs” (Pilato & Binda 2010 ) or “posterolateral sensory fields” (Gross et al. 2021 ; Kihm et al. 2023 ) of other eutardigrades. Additionally, in SEM a third pair of very poorly demarcated ventrolateral sensory fields (according to Gross et al. 2021 ) is sometimes visible (Fig. 3 B, D). No pore-like structures, typical for the head sensory areas of eutardigrades (see Tumanov et al. 2025 ) are visible on the surface of the sense organs of both pairs. Mouth opens on the top of the well-developed retractable mouth cone, lacks peribuccal lamellae with continuous membrane being present only and is surrounded by six peribuccal lobes (Fig. 3 E, F). Each lobe often with a clear depression in the apical part (Fig. 3 F, black arrowheads) Bucco-pharyngeal apparatus is of Halobiotus type (Fig. 3 G). In SEM, the oral cavity armature (OCA) consists of three rows of teeth (Fig. 3 H). First (anterior) row consists of a band of thin longitudinally elongated teeth located on the anterior ring of the buccal cavity right behind the peribuccal membrane. Second row consists of a wide band of strong conical teeth situated right behind the ring fold of the oral cavity. Third row comprises two strong transverse ridges (one dorsal and one ventral) consisted of partially fused large teeth. Sometimes the most lateral teeth seem to be separated from the ridges, being positioned in line with them. In LM only the ridges of the third row are always well-visible (Fig. 3 I, J, K, black arrowheads), teeth of the second row can be visible, but poorly distinguishable, in largest specimens (Fig. 3 I, white arrowheads). The dorsal and ventral apophyses for the insertion of the stylet muscles (AISMs) are both hook-like, but differ from each other (Figs. 3 K, 4 A, 6 D). The dorsal apophyses are better developed than the ventral and their apical part usually forms a three-pointed structure, being observed from the dorsal side, with apices directed backwards (Fig. 4 C). The points are often partially fused, forming asymmetrical structures (Fig. 4 D). The ventral apophyses are smaller than the dorsal and with lesser developed hook-like structure (sometimes nearly indistinguishable), without a three-pointed structure possessing two oblique latero-caudal processes. Caudally to the apophyses an elongated zone of the thickened buccal tube wall is present both dorsally and ventrally, the dorsal thickening is usually better developed than the ventral (Figs. 4 A, black arrowheads, 6D, white arrowheads). These thickenings are thin and can resemble ventral lamina when observed in dorso-ventral projection (Figs. 3 G, 4 A, C, black arrowheads)). The buccal tube is rigid, its caudal part slightly bends ventrally (Figs. 4 A, 6 C). The buccal tube structure evidently different in the anterior and the posterior parts with the transition zone located slightly caudally to the stylet supports insertion point. Anterior part of the buccal tube has “double” walls (Fig. 4 B, black arrowheads), and its surface is usually covered with numerous oblique folds (Figs. 4 C, 6 C, white arrowheads). Posterior part has solid walls, without such folds (Fig. 4 B). The stylet furca has a typical shape. Pharyngeal bulb is subspherical, with well-developed apophyses, thin oblique cuticular bars, and three elongated macroplacoids, microplacoids absent (Figs. 4 E, F, 6 E, F). The first macroplacoid is longer than the second and is usually saddle-shaped with a pronounced constriction on its outer side in the middle region (Figs. 4 E, 6 E, black arrowheads). Rarely, in large specimens the outer side of macroplacoids can be ragged (Figs. 4 F, 6 E, white arrowheads). The second macroplacoid is the shortest and is often is saddle-shaped (Fig. 4 F, black arrowhead), but sometimes have no median groove. The third macroplacoid is the longest with a deep preterminal constriction, separating its caudal part. This element is usually bent in the caudal direction and elongated transversely (with its width exceeding its length). Anterior part on the third macroplacoid is usually saddle-shaped, similar to the first macroplacoids (Fig. 4 F, black arrowhead). Legs are relatively long; outer surface of legs I–III often has an area of better developed cuticular sculpture, visible in SEM only (Fig. 5 A, black arrowhead). All legs possess large claws, which increase in size from legs I to legs IV (Fig. 2 A–C). Claws are of the Isohypsibius type, with thin stalk and expanded bases (Figs. 5 B, C, F, G, 6 G, I). Both main and secondary claw branches with thick walls and a large internal cavity (Figs. 5 C, G, 6 G, I). Claws are very flexible, especially in the zone of connection between the main branch and the other part of the claw, where the claw walls are evidently thinned. All claws have accessory points (Fig. 5 B); free apexes of accessory points of claws on legs I–III are often lateral to the main branch and poorly discernible under LM. All claws with smooth lunules (Figs. 5 C–E, G, 6 G–I). Lunules of internal (anterior) claws are small and often difficult to observe (Fig. 5 D, 6 H, black arrowhead). On legs I–III they are not connected with a cuticular bar, which present below the inner claw bases (Figs. 2 A, 5 C, D, 6 G, H). On legs IV below the claw bases a horseshoe-like zone of thickened cuticle is usually present in large specimens, connected with the muscle attachment zone (Fig. 5 E). Smooth eggs laid in exuvium. Pseudosimplex stage Animals in the pseudosimplex stage clearly differs from the active stage specimens in the configuration of the buccal-pharyngeal apparatus (Fig. 7 ). Buccal tube is much thinner (external diameter up to 2 µm), flexible, walls of the anterior part retain the “double” structure, evident oblique folds are also present in the anterior part of the buccal tube (Fig. 7 B, D). AISMs are hook-like, but much less developed and of nearly equal size and configuration (Fig. 7 C). Pharyngeal bulb elongated, without developed placoids, with thin usually continuous lining only (Fig. 7 B, D). Rarely some of these continuous cuticular lines are interrupted in the anterior part. Claws of the animals in the pseudosimplex stage in our material do not differ in their morphology from the active stage. DNA sequences We obtained sequences for 18S rRNA marker from five specimens, sequence for 28S rRNA marker from a single specimen, sequences for ITS-2 marker from three specimens, and sequences for COI marker from two specimens. All markers were represented by single haplotype. Halobiotus arcturulius Crisp & Kristensen, 1983 Material examined LAPTEV SEA • 111 specs; Bolshoy Begichev island in the mouth of the Khatanga River, 74.365° N, 112.04° E; 17 Sep. 2017; D.A. Mikhailov leg.; silty-sand tidal flats and salt marsh covered with Puccinellia phryganodes , Dupontia psilosantha и Arctophyla fulva ; GenBank nos: MZ050455 (18S), MZ050452 (28S), MZ078284 (ITS-2); SPbU 260(1–25) • 41 specs; same data as for preceding; SEM stubs SPbU Tar_28, SPbU Tar_32. Additional material GREENLAND SEA • 11 specs, paratypes; Mesters Vig, Kong Oscars Fjord, Central East Greenland; 6 Aug. 1974; M. Crisp and R.M. Kristensen leg.; sandy arctic beach; ZMUC TAR-192, ZMUC TAR-194, ZMUC TAR-195, ZMUC TAR-197. NOVAYA ZEMLYA • 1 spec.; Yuzhny Island, freshwater lake; 1995(?); V.I. Biserov leg.; labelled as Halobiotus arcturulius cfr., R.M. Kristensen det.; ZMUC TAR-773. Amended morphological description Based on the material from the Laptev Sea (Figs. 8 – 10 ), the type material (Fig. 11 A–D), and the specimen from a freshwater lake on the Yuzhny Island of the Novaya Zemlya archipelago (Fig. 11 E–H)). Large animals (body length 473–693 µm, other morphometrics provided in Table 3). Body elongated, with the greatest width at the level of legs III (Figs. 8 A–C, 11 A, E). Fresh specimens uncoloured or whitish, transparent after fixation in Hoyer’s medium. Eyes present, not dissolving after mounting in in Hoyer’s medium (Fig. 8 A, black arrowheads). Cuticle without developed sculpture, only slight unevenness can be visible in SEM (Fig. 8 A–E). External cephalic sense organs visible in SEM only and comprise poorly demarcated anterolateral fields (without pore-like structures) (Fig. 8 E, black arrowheads) and mediodorsal cephalic pore (Fig. 8 E, white arrowhead). “Temporalia” (or “elliptical organs”) not visible (Fig. 8 D). Mouth opens on the top of the retractable mouth cone, lacks peribuccal lamellae with continuous membrane being present only and is surrounded by six peribuccal lobes (Fig. 8 F). Bucco-pharyngeal apparatus is of the Halobiotus type (Figs. 9 A, 11 B, F). In SEM, the oral cavity armature (OCA) consists of three rows of teeth (Fig. 9 B, C). First (anterior) row consists of a band of small teeth located on the anterior ring of the buccal cavity right behind the peribuccal membrane (Fig. 9 B). Second row consists of a wide band of strong conical teeth situated just behind the ring fold of the oral cavity, teeth in the caudal zone of the band can be enlarged (Fig. 9 B). Third row comprises two strong transverse ridges (one dorsal and one ventral) consisted of partially fused large teeth (Fig. 9 C). Sometimes the most lateral teeth seem to be separated from the ridges, being positioned in line with them. In LM only the teeth of the second (Fig. 9 D, E, white arrowheads) and the third rows (Fig. 9 D, E, black arrowheads) are visible. The dorsal and ventral apophyses for the insertion of the stylet muscles (AISMs) are ridge-like, without hook-like structure, slightly different from each other (Fig. 9 F, 11 B, black arrowheads). The dorsal apophyses are better developed than the ventral, with step-like caudal end without hook-like appendage. The ventral apophyses are smaller than the dorsal in the form of simple ridge slightly indented in the middle. The dorsal apophyses do not form clear three-pointed structure, being observed from the dorsal side (Figs. 9 G, 11 G). Caudally to the apophyses an elongated zone of the thickened buccal tube wall is present both dorsally and ventrally (Fig. 9 F, white arrowheads), the dorsal thickening is usually better developed than the ventral (Fig. 9 G, 11 F, G, black arrowheads). The buccal tube is rigid; its caudal part slightly bends ventrally (Fig. 11 B). The buccal tube structure evidently different in the anterior and the posterior parts with the transition zone located slightly caudally to the stylet supports insertion point. Anterior part of the buccal tube has “double” walls (Fig. 9 G, white arrowheads), and its surface is usually covered with numerous oblique folds (Fig. 11 F, G). Posterior part has solid walls, without such folds. The stylet furca has a typical shape. Pharyngeal bulb is subspherical, with well-developed apophyses, thin oblique cuticular bars, and three elongated macroplacoids, microplacoids absent (Figs. 9 H, I, 11 C, H). The first macroplacoid is longer than the second and is usually ragged with numerous small protrusions on its outer side. The second macroplacoid is the shortest. The third macroplacoid is the longest with a very strong preterminal constriction, its caudal part is nearly completely detached from the anterior part. This element is usually slightly bent in the caudal direction; its width is usually equal to its length. Anterior part on the third macroplacoid and the second macroplacoid are usually ragged on their outer side, similar to the first macroplacoid. Legs are relatively long, all legs possess large claws, which increase in size from legs I to legs IV (Fig. 8 A, C). Claws are of the Isohypsibius type, with thin stalk and expanded bases (Fig. 10 A–F). Both main and secondary claw branches with thick walls and a large internal cavity (Fig. 10 A, B, F). Claws are very flexible, especially in the zone of connection between the main branch and the other part of the claw, where the claw walls are evidently thinned. All claws have accessory points; free apexes of accessory points of claws on legs I–III are often lateral to the main branch and poorly discernible under LM. All claws with smooth lunules (Fig. 10 A, B, F). Lunules of internal (anterior) claws are small and often difficult to observe. On legs I–III they are not connected with a cuticular bar, which present below the inner claw bases (Figs. 10 A, F, 11 D, black arrowheads). Cuticular bars on legs I–III are less pronounced than in H. crispae , often poorly visible on legs I–II (Fig. 11 D). Weak sclerotization causes evident unevenness of the bars’ margins, described as “toothed” margin by Crisp & Kristensen ( 1983 ) (Figs. 10 F, 11 D). On legs IV very poorly developed diffuse zone of the thickened cuticle is sometimes visible below the claw bases, never forms horseshoe-like structure (Fig. 10 G, black arrowheads). In one specimen abnormal internal claw of the leg III bearing additional secondary branch was observed (Fig. 10 E). Smooth eggs laid in exuvium. No specimens in the cyclomorphic stages were observed. DNA sequences We obtained sequences from a single specimen for 18S rRNA, 28S rRNA, and ITS-2 markers. Numerous attempts to obtain COI sequence were unsuccessful. Statistical analysis Permutational Multivariate Analysis of Variance showed that the Laptev Sea and the White Sea populations significantly diverge in their morphometric features used for the analysis, and the geographical location contributes prominently to the observed differences (r 2 = 0.76, p = 0.001). Consistently, on the ordination plot the individuals from different populations form two separate groups without overlapping (Fig. 12 ). Ecological data Quantitative data on tardigrade distribution were obtained only from the Barents and Laptev Sea coasts. In both regions, the tardigrade assemblages were monospecific. The Barents Sea intertidal zone was exclusively populated by H. crispae , while H. arcturulius was found only on the Laptev Sea coast. A total of 21 specimens of H. crispae were documented from the 10 cm 3 samples transect in the Barents Sea. In contrast, the abundance of H. arcturulius in the Laptev Sea was markedly higher, with a total of 1,586 individuals collected along a transect of 20 cm 3 samples, which equivalent to 793 individuals per standardized 10 cm 3 volume. Thus, population density differed substantially between the two species. The density of H. crispae was sparse, with a low average value of 2 individuals per 10 cm 3 , ranged in 1–9 ind./10 cm 3 . In contrast, a considerably higher mean density of 66 ind./10 cm 3 (range: 3-212 ind./10 cm 3 ) was observed in H. arcturulius (Table 4). In both locations, the highest tardigrade densities were observed at the lowest tidal flat station (Station 1). At this station, the mean density of H. crispae was 3.7 ind./10 cm 3 at the Mishukovo (Barents Sea), and the density of H. arcturulius reached 131.3 ind./10 cm 3 at the Bolshoy Begichev Island (Laptev Sea) (Table 4). Exuviae with eggs were found on both coasts, indicating reproductive activity of tardigrades. On the Barents Sea intertidal zone, we found egg-bearing exuviae of H. crispae in mid-July and only at the lowest tidal flat station. We did not count these exuviae. In the Laptev Sea, exuviae of H. arcturulius were found in mid-September, mainly in the upper part of the tidal flat (station 2), and once at the lower salt marsh station (station 3) (Table 4). The sediment samples from both sites also contained typical meiofaunal organisms, including copepods, nematodes, oligochaetes, chironomids, halacarid mites, and foraminiferans. Discussion Comparison with the original descriptions Halobiotus crispae Animals from the White Sea and Barents Sea populations match the original description of H. crispae in such key details as the presence of pronounced cuticular sculpture on the body surface and well-developed cephalic sensory appendages. It should be noted that the sensory areas are not protruding dome-shaped papillae, as in Kristensen’s ( 1982 ) description and drawings, but are visible as clearly demarcated flat plates. The length ratio of the pharyngeal placoids and the size characteristics of the claws in our material also match the original description. Our material, however, also exhibits certain features that diverge from the description: The cuticular sculpture varies significantly in development, from well-defined to practically indistinguishable. These data correlate well with the results of Danish researchers, who initially attributed the material found near the island of Ærø (Baltic Sea) to H. stenostomus and later identified it as H. crispae based on the results of molecular genetic analysis (Jørgensen & Kristensen 2004 ; Møbjerg et al. 2007 ). These observations were recently supported by Biserova et al. ( 2025 ) who noted the smooth cuticle surface of the specimens from the White Sea identified as H. crispae based on the results of molecular genetic analysis. As mentioned above, the sculpture in the White Sea specimens differs from the “reticular” type in the original description. Moreover, the SEM photographs, provided in the description (Kristensen 1982 , Figs. 29–32) correspond well to our material, showing a system of irregular winding folds (Figs. 2 F, 6 B). The claws of pairs I of legs are somewhat shorter than indicated by Kristensen ( 1982 ) for the type material (external claw 43 µm, internal claw 31 µm in the type material and external claws 33.7–37.6 µm, internal claws 17.5–26.4 µm in our specimens). For the claws of the fourth pair such differences are still present, but much lesser pronounced, affecting the anterior claws only (37 µm in the type material and 22.5–33.0 µm in our specimens). However, the measurement technique is not specified in the original description, the specimens were embedded in a different medium (glycerol or polyvinyl-lactophenol), and the long and thin claws of Halobiotus bend very easily, making their accurate measurement a significant problem. In this regard, these differences do not seem significant to us. Moreover, the evident variability if the claw length was noted in the original description. It was not possible to carry out new measurements on the type material provided to us due to the unfortunate orientation of the specimens in the preparation. The outer lunulae of the claws of pairs I–III of legs are described by Kristensen ( 1982 ) as “weakly serrated”. We were unable to detect serration on these elements either in the type material or in the White Sea specimens (Figs. 5 C, 6 G). The noticeable unevenness sometimes present of the lunulae edges was probably taken by the author of the description for the presence of denticles. Kristensen also described the cuticular bar at the base of the inner claws of pairs I–III of legs as part of a large lunula. A repeated examination of the type material revealed the presence of a small typical lunula on the inner claws (Fig. 6 H), often folded under the base of the claw and therefore unnoticeable. The White Sea specimens have the same claw structure (Fig. 5 D). The cuticular bar is not part of the claw lunula, but is homologous to other similar structures that are widespread within eutardigrades. For all populations with significant number of specimens examined (two populations from the White Sea and one from the Barents Sea), the presence of a cyclomorphic pseudosimplex stage characteristic of H. crispae was shown (Fig. 7 ). Halobiotus arcturulius Animals from the Laptev Sea populations match the original description of Halobiotus arcturulius (Crisp & Kristensen 1983 ) and reinvestigated type material (Figs. 8 – 10 , 11 A–D). Authors of the description gave no formal differential diagnosis for the species, but all discriminative traits mentioned by them (i.e., larger body length, smooth cuticle, less pronounced AISM’s) are present in our material and confirm the presence of this species in the Laptev Sea. As in the case of the type population of this species no cyclomorphic stages were observed. Investigated specimen of H. arcturulius collected by Biserov from a small lake on Yuzhny Island of the Novaya Zemlya archipelago (slide number: ZMUC TAR-773) also conforms the original description and material from the Laptev Sea in all observed characters (Fig. 11 E–H). Genetic data on the White Sea material Molecular genetic analysis also revealed a high similarity between our material and H. crispae. According to the mitochondrial marker COI, sampled populations from the White Sea (Fettah Island and Matrenin Island) were identical to each other and completely identical to the Danish specimens (Vellerup Vig); in addition, they were extremely close to samples from the White Sea published by Biserova et al. ( 2025 ) (Olenevsky Island, p -distance 0,17%) and from the type locality (Greenland, Nipisat) and another Danish location (Ærø) ( p -distance 0.86% and 0.85%, respectively). This result is slightly in contrary with the statement of Biserova et al. ( 2025 ) that “cox1 gene sequences of the White Sea H. crispae are 100% identical with H. crispae isolates from GenBank database”, but the differences are still minor enough to consider all sequences conspecific. Median Joining haplotype network for the COI gene is presented in Fig. 13 A. Based on the nuclear ITS-2 marker, White Sea samples from both populations were identical to each other and completely matched those from Greenland and two Danish localities. Surprisingly, sample from the other White Sea population (Olenevsky Island; Biserova et al. 2025 ) was significantly different from all other H. crispae populations. P -distance for the ITS-2 marker of this population is 8.49%, which exceed even the distance for the same marker of H. arcturulius. In our opinion such difference is likely to be a result of amplification or sequencing artifact. Surely, such phenomenon needs careful investigation of the additional material from the same locality. Median Joining haplotype network for the ITS-2 marker is presented in Fig. 13 B. Conservative marker 18S rRNA shows very low variability within all studied populations (do not exceed 0.10%). There are no data on the homological regions of the 28S rRNA marker of H. crispae from outside the White Sea in GenBank. The 28S rRNA marker shows relatively high variability ( p -distance is 3.20%) between our results and sequences obtained by Biserova et al. ( 2025 ). We consider these results as requiring additional investigation. Complete results of the p -distances calculations are presented in Supplementary file SM.01. Comparative morphometry of H. crispae and H. arcturulius Direct comparison of the morphometric traits of Halobiotus populations from the White Sea and the Laptev Sea revealed their similarity (see Tables 2 and 3). Meanings of the pt index interleave or close for most of the measurements. The most constant differences were observed in the length of the first macroplacoids ( pt value is 8.3–15.0 in H. crispae and 15.3–21.5 in H. arcturulius ), macroplacoid row length ( pt value is 38.9–52.8 in H. crispae and 56.0–72.2 in H. arcturulius ), and internal (anterior) claws secondary branch length of all legs (e.g., for the anterior claws of the hind legs secondary branch pt value is 34.6–44.5 in H. crispae and 48.3–58.0 in H. arcturulius ). The results of the performed statistical analyses (Fig. 12 ) additionally support our conclusion of presence of two distinct species of the genus Halobiotus in the fauna of Russia. Taxonomic composition of the genus Halobiotus in North European seas. It can be confidently concluded that in the fauna of the White Sea only one species of the genus Halobiotus is reliably found, namely H. crispae . Considering that trustworthy morphological identification of the species of this complex is nearly impossible, only molecular taxonomy methods can answer the question of whether the Barents Sea population belongs to the same species or represents a separate taxon. But taking into account its morphological identity with the studied White Sea populations and presence of the typical cyclomorphic stages we can assume that in the Barents Sea the same species H. crispae is present. Our investigation of several specimens identified as H. appelloefi from Sweden (Tjärnö; slides NHMD-633704–633707) and Denmark (Helsingør; slide ZMUC TAR-181) from collection of Zoological Museum, University of Copenhagen revealed presence of the cuticular sculpture at least in some specimens (Fig. 14 A, B), which indicates that these populations should be also considered as belonging to species H. crispae. Taking into account that specimens of H. crispae can possess nearly completely smooth cuticle with sculpture invisible both in LM and SEM (Jørgensen & Kristensen 2004 ; Møbjerg et al. 2007 ; Biserova et al. 2025 ; our data), which is the main character discriminating H. crispae from the species of H. appelloefi/stenostomus complex, and considering the incomplete and outdated descriptions of H. appelloefi and H. stenostomus both these species should be considered as nomina inquirendae . All records of these species should be considered doubtful, waiting for the confirmation by the methods of modern taxonomy. In concordance with Petelina & Tchesunov ( 1983 ) we do not accept H. geddesi as a valid species. Taking into account the presence of the cuticular sculpture well visible in the photographs of the type specimen (Fig. 14 C–F; kindly provided by R. Bertolani) this species is likely a junior synonym of H. crispae . Until the new material is collected and genetically characterized from the type locality of H. geddesi this species should be considered nomen inquirendum as proposed by Gąsiorek et al. ( 2019 ). We therefore conclude that the genus Halobiotus currently comprises only two adequately described species, which are reliably distinguished using both morphological and molecular methods. Notes on the oral cavity armature in Isohypsibioidea In the latest revision of the Isohypsibioidea morphology and phylogeny Gąsiorek et al. ( 2019 ) stated that all genera of this clade have no more than two rows of teeth on the OCA. In their opinion this fundamental difference with the Macrobiotoidea clade which typically have three rows of teeth on OCA makes it difficult to homologize these structures between the clades. Our observations revealed presence of the three rows of teeth on the OCA of both investigated Halobiotus species. In each case the OCA comprises: 1) the anteriormost row of minute teeth, located on the anterior ring of the oral cavity immediately below the peribuccal cuticular membrane, 2) the second row, consisted of larger conical teeth, located on and behind the ring fold of the oral cavity, and 3) the third row, consisted of large partially fused teeth, which forms strong mediodorsal and medioventral transverse ridges in the caudal part of the oral cavity. This configuration perfectly coincides with the typical OCA configuration within Macrobiotoidea, and in our opinion those three rows of teeth are evidently homologous within Isohypsibioidea, Eohypsibioidea and Macrobiotoidea. It is interesting to note that Halobiotus is not the only taxon within Isohypsibioidea possessing strong mediodorsal and medioventral transverse ridges in the caudal part of the oral cavity. Such structures are also present in the OCA of species, currently attributed to the genus Grevenius Gąsiorek, Stec, Morek & Michalczyk, 2019 , more precisely, to the group of species similar to Grevenius monoicus (Bertolani, 1981). This group incorporates species with rugose cuticular sculpture, three wide massive macroplacoids and OCA with strong dorsal and ventral toothed ridges (Bertolani 1988 ) (Fig. 14 G). Three species can be currently attributed to this morphological group: G. monoicus , Grevenius baicalensis (Ramazzotti, 1966), and Grevenius ladogensis (Tumanov, 2003). The fourth species with the same set of characters was described by Pilato et al. ( 2019 ) as Isohypsibius occultus Pilato, D’Urso, Sabella & Lisi, 2019 simultaneously with the publication of the Isohypsibioidea revision by Gąsiorek et al. ( 2019 ), where all freshwater species of the former genus Isohypsibius were transferred into the newly instituted genus Grevenius . Taking into account the morphological similarity of Is. occultus and G. monoicus and the aquatic lifestyle of the former species we suppose to move this species formally into the genus Grevenius as Grevenius occultus (Pilato, D’Urso, Sabella & Lisi, 2019 ) comb. nov. But in our opinion taxonomic position of the monoicus morphogroup within the genus Grevenius should be considered as tentative and questionable, because of the divergent configuration of the buccal apparatus, which strongly resembles the genus Halobiotus , and the evidently polyphyletic nature of the genus Grevenius itself (see Tumanov 2022 ; Tumanov et al. 2025 ). This problem should be solved in future using the methods of molecular phylogenetic analysis. Amended diagnosis of the family Halobiotidae The current diagnosis of the family Halobiotidae was given by Gąsiorek et al. ( 2019 ). It was formulated as follows: “ Marine eutardigrades with six peribuccal lobes equipped with chemosensory organs. Two large, dome-shaped cephalic papillae present. Mouth opening surrounded by the peribuccal lamina. No ventral lamina on the buccal tube. AISM symmetrical, divided into the anterior semilunar hook and the posterior slight thickening. Claws with pseudolunulae ”. Some of these statements need to be corrected or excluded. First, the representatives of the family are not exclusively marine – H. arcturulius was registered in the freshwater lake (Biserov 1999). Second, cephalic sense organs are not dome-shaped, but rather flat plates. They are definitely marked in H. crispae (see Fig. 3 A–C and Fig. 4 C in Gąsiorek et al. 2019 ), being nearly completely invisible in H. arcturulius. Third, AISM are symmetrical and hook-like only in H. crispae , while in H. arcturulius they are evidently asymmetrical and ridge-like. Fourth, presence of lunulae (pseudolunulae according to Gąsiorek et al. 2019 ) in our opinion should be excluded from the diagnosis, because this character can hardly be accepted as family-level, being rather variable in nearly all Eutardigrada families. On the other hand, some characters seem to be unique for the family Halobiotidae. These are the presence of the “double wall” in the anterior part of the buccal tube, presence of three rows of teeth in the OCA, and presence of three massive macroplacoids with detached caudal part of the third macroplacoid. Here we propose an amended diagnosis of the family Halobiotidae: Isohypsibioidea, six peribuccal lobes equipped with chemosensory organs, mouth opening surrounded by the peribuccal lamina, no ventral lamina on the buccal tube, anterior part of the buccal tube with “double wall”, three rows of teeth in the OCA, third row in the shape of massive transverse ridges dorsally and ventrally, three massive macroplacoids with detached caudal part of the third macroplacoid, long claws with flexible main branches. Ecology of Halobiotus crispae and H. arcturulius on Arctic tidal flats Tardigrade assemblages sampled quantitatively along two high-latitude intertidal transects were both monospecific with exclusively H. crispae for the Barents Sea and H. arcturulius for the Laptev Sea coast. Our findings are consistent with previous records of these species in high latitudes. Indeed, monospecific assemblages of H. crispae have been documented in intertidal and shallow subtidal environments across the Holarctic. These include locations in Greenland, Spitsbergen, Arctic Canada, Norway, Alaska, Sweden, Denmark, and the White Sea (Kristensen 1982 ; Møbjerg et al. 2007 ; Smykla et al. 2011 ; Halberg et al. 2013 ; Biserova et al. 2025 ). Similarly, monospecific assemblages of H. arcturulius have been reported from intertidal zone of Greenland, Spitsbergen, the Kara and Laptev Seas, and tidal flats of Alaska (Feder & Paul 1980 as Hypsibius appelloefi ; Crisp & Kristensen 1983 ; Mokievsky 1992 ; Biserov 1998 ). All findings of H. arcturulius , including our own, were located in estuarine areas or near freshwater streams, suggesting a specific habitat preference. The prevalence of monospecific assemblages is often associated with harsh environments, such as the intertidal zone, where stress is caused by the regular fluctuation of environmental factors’ impact (e.g., Kemp et al. 2017 ). At high latitudes, this stress is amplified by extreme climates, which can favor the dominance of highly adapted stress-tolerant species. Our data revealed a stark contrast in the population densities of the two species. Densities of H. arcturulius in the Laptev Sea intertidal were more than an order of magnitude higher than those of H. crispae in the Barents Sea tidal flat. The Laptev Sea population of H. arcturulius surpassed the maximal densities previously described for this species in other regions, such as Greenland (5.8–11.6 ind./10 cm 3 ; Crisp & Kristensen 1983 ) and Spitsbergen (maximum: 40 ind./10 cm 3 , mean density per transect 9 ind./10 cm 3 ; Mokievsky 1992 ), suggesting the local conditions in the Laptev Sea are more favorable for H. arcturulius . In contrast, the Barents Sea site was sparsely populated by H. crispae , although comparisons with other regions are hindered by the scarcity of quantitative data and inconsistent methodologies. A study from Vellerup Vig, Denmark, recorded high population densities of up to 1,300 individuals per sample but omitted sample volumes, which prevents direct comparisons (Halberg et al. 2013 ). Another study conducted in Spitsbergen reported 268 specimens of H. crispae found in a transect of ten 10 cm 3 samples, with no reported densities per sample (Smykla et al. 2011 ). The resulting average density was 26.8 ind./10 cm 3 , which is more than 10 times higher than the average density of this species found in the Barents Sea. The lower abundance in our study may be related to a less preferred biotope and/or may reflect the uneven distribution of this species, which has been previously reported for H. arcturulius (Crisp & Kristensen 1983 ; Mokievsky 1992 ) and which is typical for Arctic meiobenthos (Szymelfenig et al. 1995 ; Weslawski et al. 1999 ; Urban-Malinga et al. 2005 ; Urban-Malinga 2014 ). The highest mean tardigrade densities were observed at the lowest tidal stations on the transect in both seas. Specimens of H. crispae were restricted to lower elevations and absent from the upper, vegetated zones, whereas H. arcturulius displayed a broader range, extending to the upper salt marsh. Mokievsky ( 1992 ) also reported peak densities of H. arcturulius at the low intertidal of Spitsbergen with its sparse abundance on the other stations. The preference of both species for lower tidal elevations is likely due to more frequent and prolonged submersion during tidal cycles, which provides more favorable and stable environmental conditions. Such pattern of vertical zonation of benthic organisms is typical for Arctic intertidal communities (Thyrring et al. 2021 ). The presence of exuviae with eggs confirms that the sampling periods captured the reproductive phase of H. crispae in the Barents Sea (mid-July) and H. arcturulius in the Laptev Sea (mid-September). These findings align with reports of abundant exuviae with eggs of H. arcturulius in eastern Greenland from mid-July to mid-August (Crisp & Kristensen 1983 ) and in western Greenland from June to August (Møbjerg et al. 2007 ). These records suggest that reproduction in Arctic populations occurs from mid-summer to early autumn. In contrast, temperate-latitude populations of H. crispae reproduce from mid-January to mid-April (Møbjerg et al. 2007 ; Halberg et al. 2013 ). This latitudinal pattern of reproductive timing likely reflects an adaptive response to regional climatic conditions, particularly the short Arctic summer preceding the seasonal freezing of intertidal sediments, which suppresses tardigrade activity. Our study revealed contrasting densities of two tardigrade species in the Arctic intertidal zone: low densities of H. crispae in the Barents Sea and high densities of H. arcturulius in the Laptev Sea. The high abundance of H. arcturulius suggests that, despite the highly stressed high-latitude intertidal environment of the Laptev Sea, there are likely to be favorable local conditions that support high population densities. However, the factors determining the distribution and abundance of both studied species, as well as marine tardigrades in general, remain unclear and require further study. Declarations Supplementary materials SM.01. Uncorrected genetic p -distances between the genes’ sequences of the studied specimens and sequences of the genus Halobiotus available in GenBank. Acknowledgements Samples from the Barents Sea intertidal were obtained in 2019 during the coastal expedition under the RFBR project 18-54-20001 (SPbU, PI Andrey Granovitch). Laptev Sea samples were collected during a cruise aboard the R/V Akademik Mstislav Keldysh in 2017 operated by the P.P. Shirshov Institute of Oceanology RAS. We gratefully acknowledge these research opportunities. We are also grateful to Dmitrii Mikhailov for collecting samples from the Bolshoy Begichev Island (Laptev Sea), Valeria Khabibulina (St Petersburg State University) for collecting samples from the Matrenin Island (White Sea), and to Luidmila A. Segrienko (Petrozavodsk University) and Vladislav V. Petrovsky (Komarov Botanical Institute RAS) for salt marsh plants identification. We would like to thank Reinhardt Møbjerg Kristensen and Martin Vinther Sørensen (Zoological Museum, University of Copenhagen) for the loan of the patatypes of Halobiotus crispae and H. arcturulius and additional specimens of the genus Halobiotus . We also want to thank Roberto Bertolani (University of Modena and Reggio Emilia, Italy) for providing the photographs of the type specimen of H. geddesi . Specimens of Grevenius cf. monoicus were provided by Elena Chertoprud (A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences). This study was carried out with the use of the equipment of the Core Facilities Centre “Centre for Molecular and Cell Technologies” of St Petersburg State University ( https://researchpark.spbu.ru/index.php/en/biomed-eng ) and “Taxon” Research Resource Center of the Zoological Institute of the Russian Academy of Sciences ( http://www.ckp-rf.ru/ckp/3038/ ). The study was supported by the Russian Science Foundation, grant No. 25-74-20033 “Evolutionary transformations of nanostructural elements of the Ecdysozoa cuticle using the example of the integumentary structures of tardigrades (Tardigrada)”. References Astrin JJ, Stüben PE (2008) Phylogeny in cryptic weevils: molecules, morphology and new genera of western Palaearctic Cryptorhynchinae (Coleoptera: Curculionidae). Invertebrate Syst 22:503–522 Beasley CW, Kaczmarek Ł, Michalczyk Ł (2008) Doryphoribius mexicanus , a new species of Tardigrada (Eutardigrada: Hypsibiidae) from Mexico (North America). Proceedings of the Biological Society of Washington 121 (1): 34–40. https://doi.org/10.2988/07-30.1 Bertolani R (1988) Tardigradi delle acque dolci, con riferimento ai corsi d'acqua della Lunigiana e della Garfagnana. Bollettino del Museo di Storia Naturale della Lunigiana 6–7:133–138 Biserov VI (1991) An annotated list of Tardigrada from European Russia. 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Zoomorphologie 89(1):1–19 Weslawski JM, Szymelfenig M, Zajaczkowski M, Keck A (1999) Influence of salinity and suspended matter on benthos of an Arctic tidal flat. ICES J Mar Sci 56(Supplement):S194–S202 Wickham H (2009) ggplot2: Elegant Graphics for Data Analysis. Springer-, New York Tables Tables are available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files ESM1.xlsx Uncorrected genetic p -distances between the genes’ sequences of the studied specimens and sequences of the genus Halobiotus available in GenBank. Table1.docx Table4.docx Table2.docx Table3.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8224833","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":551926799,"identity":"9a24da09-2602-423f-bc5d-3051fd9298ff","order_by":0,"name":"Denis V. 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Stations locations (1–4) are identified by both the placed sample jars and corresponding red numerals added to the image. Stations span an elevation gradient from the lower tidal flat to a slightly elevated vegetated salt marsh. Numbers of replicates are shown in brackets.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/5adaf47b102434116a5ce999.jpg"},{"id":98429661,"identity":"9c8a2a8a-219a-461e-be8b-6b80e6f93f9f","added_by":"auto","created_at":"2025-12-17 16:43:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3409504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHalobiotus crispae, \u003c/em\u003eWhite Sea population, total view and cuticular sculpture. \u003cstrong\u003eA.\u003c/strong\u003e Dorso-ventral view, PhC. \u003cstrong\u003eB.\u003c/strong\u003e Lateral view in SEM. \u003cstrong\u003eC.\u003c/strong\u003e Ventral view in SEM. \u003cstrong\u003eD.\u003c/strong\u003e Sculpture of the dorsal body surface, PhC. \u003cstrong\u003eE.\u003c/strong\u003e Sculpture of the dorsal body surface, DIC. \u003cstrong\u003eF.\u003c/strong\u003eSculpture of the dorsal body surface in SEM. Scale bars A–C = 100 µm; D, E = 10 µm; F = 5 µm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/ce83815122de1e5000ad7d3d.png"},{"id":98427040,"identity":"a4347360-841b-4d93-a3d1-0d337d4bcaeb","added_by":"auto","created_at":"2025-12-17 16:39:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3288885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHalobiotus crispae, \u003c/em\u003eWhite Sea population, cephalic sensory structures and buccal-pharyngeal apparatus. \u003cstrong\u003eA.\u003c/strong\u003eDorsal surface of the cephalic body region, PhC, white arrowheads indicate “temporalia”. \u003cstrong\u003eB.\u003c/strong\u003e Ventral surface of the cephalic body region, PhC, black arrowheads indicate “cephalic papillae”, white arrowheads indicate “ventrolateral sensory fields”. \u003cstrong\u003eC.\u003c/strong\u003e Lateral view of the cephalic body region, SEM, black arrowhead indicates “cephalic papilla”, white arrowhead indicates “temporalia”. \u003cstrong\u003eD.\u003c/strong\u003e Frontal view of the cephalic body region, SEM, white arrowheads indicate “cephalic papillae”, black arrowheads indicate “ventrolateral sensory fields”. \u003cstrong\u003eE. \u003c/strong\u003eProtruded mouth cone, SEM. \u003cstrong\u003eF.\u003c/strong\u003e Mouth opening with peribuccal lobes, SEM, black arrowheads indicate depressions in the apical part of each lobe. \u003cstrong\u003eG.\u003c/strong\u003e Total dorso-ventral view of the bucco-pharyngeal apparatus, PhC, black arrowhead indicates the zone of the thickened buccal tube wall. \u003cstrong\u003eH.\u003c/strong\u003e Mouth opening with oral cavity armature, SEM. \u003cstrong\u003eI.\u003c/strong\u003e Oral cavity armature, dorsal, DIC, white arrowhead indicates the teeth of the second raw, black arrowhead indicates the teeth of the third row. \u003cstrong\u003eJ.\u003c/strong\u003e Oral cavity armature, ventral, DIC, black arrowhead indicates the teeth of the third row. \u003cstrong\u003eJ.\u003c/strong\u003e Anterior part of the buccal tube, saggital optical section, DIC, white arrowheads indicate the dorsal and ventral apophyses for the insertion of the stylet muscles (AISM), black arrowheads indicate the teeth of the third row. Scale bars A, B, C, D, G = 20 µm; I, J, K = 10 µm; E, F = 5 µm; H = 2 µm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/dd898342eb4beb246cf51712.png"},{"id":98428564,"identity":"d2ad58ff-c934-4351-b3e3-b62ed9305ccc","added_by":"auto","created_at":"2025-12-17 16:42:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1715791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHalobiotus crispae, \u003c/em\u003eWhite Sea population, buccal-pharyngeal apparatus. \u003cstrong\u003eA.\u003c/strong\u003e Lateral view of the buccal tube, PhC, black arrowheads indicate zone of the thickened buccal tube wall. \u003cstrong\u003eB.\u003c/strong\u003e Optical section of the buccal tube, DIC, black arrowhead indicates the transition between the “double” buccal tube wall of the anterior part of the buccal tube and the solid caudal part. \u003cstrong\u003eC.\u003c/strong\u003e Dorsal view of the buccal tube anterior part with three-pointed structure of the dorsal AISM, DIC, white arrowhead indicates zone of the thickened buccal tube wall, white arrowhead indicates numerous oblique folds of the buccal tube wall. \u003cstrong\u003eD.\u003c/strong\u003e Dorsal view of the buccal tube anterior part with asymmetrical three-pointed structure of the dorsal AISM, DIC. \u003cstrong\u003eE. \u003c/strong\u003eVentral row of macroplacoids, DIC, black arrowhead indicates saddle-shaped margin of the first macroplacoid. \u003cstrong\u003eF.\u003c/strong\u003e Dorso-lateral rows of macroplacoids of the same specimen, DIC, black arrowheads indicate saddle-shaped margins of the second and third macroplacoids, white arrowhead indicates ragged margin of the first macroplacoid. Scale bars = 10 µm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/daad75eda5b3e9828645d89b.png"},{"id":98073203,"identity":"3187fb44-0e71-4eee-948f-cb6f26a54387","added_by":"auto","created_at":"2025-12-12 13:21:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1758121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHalobiotus crispae, \u003c/em\u003eWhite Sea population, legs and claws. \u003cstrong\u003eA.\u003c/strong\u003e Outer surface of leg II, SEM, black arrowhead indicates the area of better developed cuticular sculpture. \u003cstrong\u003eB.\u003c/strong\u003e Claws of leg II, SEM, white arrowhead indicates cuticular bar below the inner claw base. \u003cstrong\u003eC.\u003c/strong\u003e Claws of leg III, PhC, black arrowhead indicates smooth lunule margin, white arrowhead indicates cuticular bar below the inner claw base. \u003cstrong\u003eD.\u003c/strong\u003e Smooth lunule at the base of the inner claw of leg II, PhC. \u003cstrong\u003eE.\u003c/strong\u003e Ventral surface of the leg IV, PhC, black arrowheads indicate horseshoe-like zone of thickened cuticle, white arrowhead indicates muscle attachment zone. \u003cstrong\u003eF.\u003c/strong\u003e Claws of leg IV, SEM. \u003cstrong\u003eG.\u003c/strong\u003e Claws of leg IV, PhC. Scale bars: A–C, E–G = 10 µm; D = 5 µm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/f8234793bcedf2a120878d57.png"},{"id":98428955,"identity":"b76a869e-6916-4f2d-a655-fee1838d9189","added_by":"auto","created_at":"2025-12-17 16:42:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2740846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHalobiotus crispae, \u003c/em\u003eparatype specimens, slides ZMUC TAR-253 and ZMUC TAR-255. \u003cstrong\u003eA.\u003c/strong\u003e Lateral view, PhC. \u003cstrong\u003eB.\u003c/strong\u003e Sculpture of the dorsal body surface, PhC. \u003cstrong\u003eC.\u003c/strong\u003e Buccal tube, PhC, black arrowhead indicates numerous oblique folds of the buccal tube wall. \u003cstrong\u003eD.\u003c/strong\u003e Anterior part of the buccal tube, saggital optical section, PhC, black arrowheads indicate the dorsal and ventral apophyses for the insertion of the stylet muscles (AISM), white arrowheads indicate zone of the thickened buccal tube wall. \u003cstrong\u003eE.\u003c/strong\u003e Macroplacoids, DIC, black arrowheads indicate saddle-shaped margins of macroplacoids, white arrowheads indicate ragged margins of macroplacoids. \u003cstrong\u003eF.\u003c/strong\u003e Macroplacoids, PhC. \u003cstrong\u003eG.\u003c/strong\u003e Claws of leg II, PhC. \u003cstrong\u003eH.\u003c/strong\u003e Smooth lunule at the base of the inner claw of leg III, PhC. \u003cstrong\u003eI.\u003c/strong\u003e Claws of leg IV, PhC. Scale bars: A = 100 µm; G, I = 20 µm; B–F, H = 10 µm.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/ed332c004fcfb6953b0ebd4f.png"},{"id":98073205,"identity":"564b5b41-6e3c-4063-b982-f6b6411c0ba0","added_by":"auto","created_at":"2025-12-12 13:21:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1145259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHalobiotus crispae, \u003c/em\u003epseudosimplex stage. \u003cstrong\u003eA.\u003c/strong\u003e Total view, White Sea population specimen, PhC. \u003cstrong\u003eB.\u003c/strong\u003e Dorso-ventral view of the buccal-pharyngeal apparatus, White Sea population specimen, PhC. \u003cstrong\u003eC.\u003c/strong\u003e Lateral view of the buccal tube anterior part, White Sea population specimen, PhC, black arrowheads indicate dorsal and ventral apophyses for the insertion of the stylet muscles. \u003cstrong\u003eD.\u003c/strong\u003eLateral view of the buccal-pharyngeal apparatus, paratype specimen, slide ZMUC TAR-255, PhC. Scale bars: A = 100 µm; B–D = 10 µm.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/32824e52cfa75643c52888c1.png"},{"id":98429398,"identity":"60a08f8b-c5cd-4f21-8add-89f597fccdff","added_by":"auto","created_at":"2025-12-17 16:43:19","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3037649,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHalobiotus arcturulius, \u003c/em\u003eLaptev Sea population, total view and cephalic sensory organs. \u003cstrong\u003eA.\u003c/strong\u003e Dorso-ventral view, PhC, black arrowheads indicate eyes. \u003cstrong\u003eB.\u003c/strong\u003eDorsal view in SEM. \u003cstrong\u003eC.\u003c/strong\u003e Lateral view in SEM. \u003cstrong\u003eD.\u003c/strong\u003e Dorsal surface of the cephalic body region showing the absence of the “temporalia”, PhC. \u003cstrong\u003eE.\u003c/strong\u003e Frontal view of the cephalic body region, SEM, black arrowheads indicate anterolateral sensory fields, white arrowhead indicates mediodorsal cephalic pore. \u003cstrong\u003eF.\u003c/strong\u003e Mouth opening with peribuccal lobes, SEM. Scale bars A–C = 100 µm; D, E = 20 µm; F = 5 µm.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/2f8fc24239c4dc965dcba837.png"},{"id":98073201,"identity":"9176d317-e6a9-434e-b769-abbfc5ae5cef","added_by":"auto","created_at":"2025-12-12 13:21:51","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2840904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHalobiotus arcturulius, \u003c/em\u003eLaptev Sea population, buccal-pharyngeal apparatus. \u003cstrong\u003eA.\u003c/strong\u003e Total dorso-ventral view of the bucco-pharyngeal apparatus, PhC. \u003cstrong\u003eB.\u003c/strong\u003eMouth opening with the first and the second rows of the oral cavity armature visible, SEM. \u003cstrong\u003eC.\u003c/strong\u003eMouth opening with the second and the third rows of the oral cavity armature visible, SEM. \u003cstrong\u003eD.\u003c/strong\u003e Oral cavity armature, dorsal, DIC, white arrowhead indicates the teeth of the second raw, black arrowhead indicates the teeth of the third row. \u003cstrong\u003eE. \u003c/strong\u003eOral cavity armature, ventral, DIC, white arrowhead indicates the teeth of the second raw, black arrowhead indicates the teeth of the third row. \u003cstrong\u003eF.\u003c/strong\u003e Anterior part of the buccal tube, saggital optical section, PhC, black arrowheads indicate the dorsal and ventral apophyses for the insertion of the stylet muscles (AISM), white arrowheads indicate zones of the thickened buccal tube wall,black arrows indicate the teeth of the third row. \u003cstrong\u003eG.\u003c/strong\u003e Optical section of the buccal tube, DIC, white arrowhead indicates the transition between the “double” buccal tube wall of the anterior part of the buccal tube and the solid caudal part, black arrowhead indicates dorsal zone of the thickened buccal tube wall. \u003cstrong\u003eH. \u003c/strong\u003eMacroplacoid rows, PhC. \u003cstrong\u003eI. \u003c/strong\u003eMacroplacoid rows, DIC. Scale bars: A = 20 µm; D–I = 10 µm; B, C = 2 µm.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/f8c53c61f7024b2a14e606c7.png"},{"id":98429698,"identity":"a4e883af-3603-464d-ba92-f474315abb83","added_by":"auto","created_at":"2025-12-17 16:44:01","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2456406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHalobiotus arcturulius, \u003c/em\u003eLaptev Sea population, legs and claws. \u003cstrong\u003eA.\u003c/strong\u003e Claws of leg III, PhC, black arrowhead indicates cuticular bar below the inner claw base. \u003cstrong\u003eB.\u003c/strong\u003e Claws of leg IV, PhC. \u003cstrong\u003eC.\u003c/strong\u003e Claws of leg II, SEM, black arrowhead indicates cuticular bar below the inner claw base. \u003cstrong\u003eD.\u003c/strong\u003e Claws of leg IV, SEM. \u003cstrong\u003eE.\u003c/strong\u003e Abnormal inner claw of leg III, SEM, black arrowhead indicates cuticular bar below the inner claw base. \u003cstrong\u003eF.\u003c/strong\u003e Inner claw of leg III, PhC, black arrowhead indicates cuticular bar below the inner claw base. \u003cstrong\u003eG.\u003c/strong\u003e Ventral surface of the leg IV, PhC, black arrowheads indicate diffuse zones of thickened cuticle. Scale bars: A–F = 10 µm; G = 20 µm.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/72339df924079c6f865e0e5d.png"},{"id":98428483,"identity":"4722bcbf-2bbb-482a-923a-0e1b93d05ff2","added_by":"auto","created_at":"2025-12-17 16:42:03","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":2848533,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHalobiotus arcturulius. \u003c/em\u003eA–D – paratype specimens, slides ZMUC TAR-194, ZMUC TAR-194. E–H – from the collection of V.I. Biserov, slide ZMUC TAR-773. \u003cstrong\u003eA.\u003c/strong\u003e Dorso-ventral view, PhC. \u003cstrong\u003eB.\u003c/strong\u003e Lateral view of the buccal-pharyngeal apparatus, PhC, black arrowhead indicates the dorsal apophysis for the insertion of the stylet muscles (AISM). \u003cstrong\u003eC.\u003c/strong\u003e Macroplacoid rows, PhC. \u003cstrong\u003eD.\u003c/strong\u003e Claws of leg I, PhC, black arrowhead indicates cuticular bar below the inner claw base. \u003cstrong\u003eE.\u003c/strong\u003e Dorso-ventral view, PhC. \u003cstrong\u003eF.\u003c/strong\u003e Dorso-ventral view of the buccal-pharyngeal apparatus, PhC, black arrowhead indicates the zone of the thickened buccal tube wall. \u003cstrong\u003eG.\u003c/strong\u003e Dorsal view of the buccal tube, DIC, black arrowhead indicates the zone of the thickened buccal tube wall. \u003cstrong\u003eH.\u003c/strong\u003e Macroplacoid rows, DIC. Scale bars: A, E = 100 µm; B, F = 20 µm; C, D, G, H = 10 µm.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/cde667f97f07a94c4eb1bb80.png"},{"id":98073209,"identity":"e944c1fc-1b76-46ea-9db3-855f51614602","added_by":"auto","created_at":"2025-12-12 13:21:51","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1797385,"visible":true,"origin":"","legend":"\u003cp\u003enMDS plot of the specimens ordination based on the Pilato index.\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/86297faaea64ba28d53d8944.png"},{"id":98073207,"identity":"b74df603-a978-4c6c-b10b-ab9ac869e689","added_by":"auto","created_at":"2025-12-12 13:21:51","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":2333798,"visible":true,"origin":"","legend":"\u003cp\u003eMedian Joining haplotype networks for the genus \u003cem\u003eHalobiotus\u003c/em\u003e. \u003cstrong\u003eA.\u003c/strong\u003e COI marker. \u003cstrong\u003eB.\u003c/strong\u003eITS-2 marker. Black circles represent putative haplotypes required to join the detected haplotypes. Transverse striae at connecting lines indicate mutations between the haplotypes.\u003c/p\u003e","description":"","filename":"Figure13.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/7dbe969dd1fadf000dfaa2b5.png"},{"id":98073213,"identity":"c86ff635-0ef1-40b8-ad31-9a655c7b8ce8","added_by":"auto","created_at":"2025-12-12 13:21:51","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":2484148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA, B.\u003c/strong\u003e Cuticular sculpture of specimens previously identified as \u003cem\u003eH. appelloefi.\u003c/em\u003e \u003cstrong\u003eC–F.\u003c/strong\u003eMorphology of the type series specimen of \u003cem\u003eH. geddesi\u003c/em\u003e. \u003cstrong\u003eG.\u003c/strong\u003e \u003cem\u003eGrevenius\u003c/em\u003e cf. \u003cem\u003emonoicus\u003c/em\u003e. \u003cstrong\u003eA.\u003c/strong\u003e Specimen from Tjarno, Sweden, slide number NHMD-633705, PhC. \u003cstrong\u003eB.\u003c/strong\u003e Specimen from Helsingør, Denmark, slide number ZMUC TAR-181, PhC. \u003cstrong\u003eC.\u003c/strong\u003e Dorsal cuticular sculpture of \u003cem\u003eH. crispae\u003c/em\u003e-type, black arrowhead indicates the zone of the sculptured cuticle, PhC. \u003cstrong\u003eD.\u003c/strong\u003e Buccal-pharyngeal apparatus, PhC. \u003cstrong\u003eE.\u003c/strong\u003e Claws of leg III, PhC. \u003cstrong\u003eF.\u003c/strong\u003e Claws of leg IV, PhC. \u003cstrong\u003eG.\u003c/strong\u003e Mouth opening with oral cavity armature, SEM. Scale bars A–F = 10 µm; G = 2 µm. Photographs C–F by R. Bertolani.\u003c/p\u003e","description":"","filename":"Figure14.png","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/ee8c2a7b7b7e28473280cf9a.png"},{"id":100421671,"identity":"2f151a9a-74fc-4a05-bc9b-40949c51f4df","added_by":"auto","created_at":"2026-01-16 13:41:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":35187341,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/c9873bd7-7af0-4d86-96d3-07de138d46f6.pdf"},{"id":98073198,"identity":"645301f2-a5b1-435c-9574-9746af524fa6","added_by":"auto","created_at":"2025-12-12 13:21:51","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14072,"visible":true,"origin":"","legend":"\u003cp\u003eUncorrected genetic \u003cem\u003ep\u003c/em\u003e-distances between the genes’ sequences of the studied specimens and sequences of the genus \u003cem\u003eHalobiotus \u003c/em\u003eavailable in GenBank.\u003c/p\u003e","description":"","filename":"ESM1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/f3efd47ed4b223cdb01bc06f.xlsx"},{"id":98073190,"identity":"d621f518-002d-41e6-9f2a-61e5bdcddeb9","added_by":"auto","created_at":"2025-12-12 13:21:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12829,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/45092d5c34236db3d3d29717.docx"},{"id":98073192,"identity":"bdd7ea6a-d306-4051-8659-9a5582201edf","added_by":"auto","created_at":"2025-12-12 13:21:51","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16641,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.docx","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/65e75d81c55b4f4224e2c920.docx"},{"id":98073196,"identity":"c2738573-1ad8-4097-a973-f8d964a9cd98","added_by":"auto","created_at":"2025-12-12 13:21:51","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":24106,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/e098d08b1365ff762e52d093.docx"},{"id":98428959,"identity":"3d84091f-567b-4463-91ae-042fab412014","added_by":"auto","created_at":"2025-12-17 16:42:37","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":24139,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8224833/v1/cd88b9dc4a3ac6c19adebad1.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003eTardigrades of the genus Halobiotus (Eutardigrada, Isohypsibioidea) inhabiting Arctic seas – new taxonomic and biological data\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTardigrades are a group of microscopic invertebrates, inhabiting all types of waterbodies from ocean depths to ephemeral micro reservoirs in hygroscopic terrestrial substrates, like moss cushions (Nelson et al. 2018). Most of the marine tardigrade species belong to the taxa currently classified as the class Heterotardigrada \u0026ndash; specifically, the order Arthrotardigrada and the family Echiniscoididae within the order Echiniscoidea (Nelson et al. 2018). Beside these primarily marine groups, several species of the class Eutardigrada can be found in marine environments. Some of them belong to the freshwater taxa that occasionally occur in marine sediments, likely due to the accidental transportation from their natural habitats by currents (e.g. the genera \u003cem\u003eBorealibius\u003c/em\u003e Pilato, Guidetti, Rebecchi, Lisi, Hansen \u0026amp; Bertolani, 2006, \u003cem\u003eMurrayon\u003c/em\u003e Bertolani \u0026amp; Pilato, 1988 and \u003cem\u003eThulinius\u003c/em\u003e Bertolani, 2003; Biserov 1998; Kaczmarek et al. 2015). Only one group within the class Eutardigrada is permanently associated with marine environments \u0026ndash; the genus \u003cem\u003eHalobiotus\u003c/em\u003e Kristensen, 1982 comprising the monotypic family Halobiotidae Gąsiorek, Stec, Morek \u0026amp; Michalczyk, 2019.\u003c/p\u003e\n\u003cp\u003eDescriptions of the first two species of this genus\u0026mdash;\u003cem\u003eHalobiotus stenostomus\u003c/em\u003e (Richters, 1908) and \u003cem\u003eH. appelloefi\u003c/em\u003e (Richters, 1908)\u0026mdash;were published alongside in the publication by Richters (1908). Both species were described from the Baltic Sea. Like many species descriptions of the early period of the tardigrade systematics they are very brief and incomplete, and do not meet modern requirements for the taxonomic descriptions. It is very likely that \u003cem\u003eH. stenostomus\u003c/em\u003e is a cyclomorphic stage characteristic of this genus (Kristensen 1982). Moreover, other authors (Marcus 1936 and Thulin 1928) described several forms of marine Eutardigrada from the Baltic Sea under the names \u003cem\u003eH. stenostomus\u003c/em\u003e and \u003cem\u003eH. appelloefi\u003c/em\u003e, making the situation even more complicated. Another species (\u003cem\u003eHalobiotus geddesi\u003c/em\u003e (Hallas, 1971)) was described by Hallas (1971) on the basis of the material from Kattegat and Norwegian Sea. Later Petelina \u0026amp; Tchesunov (1983) synonymized this species with \u003cem\u003eH. appelloefi\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eKristensen (1982) published the first modern description of the \u003cem\u003eHalobiotus\u003c/em\u003e species. In his work, he described a new species from Greenland (\u003cem\u003eHalobiotus crispae\u003c/em\u003e Kristensen, 1982), instituted a new genus \u003cem\u003eHalobiotus\u003c/em\u003e for the \u003cem\u003estenostomus / appelloefi / geddesi / crispae\u003c/em\u003e species complex and described a life cycle with \u0026ldquo;pseudosimplex\u0026rdquo; cyclomorphic stage. A year later Crisp \u0026amp; Kristensen (1983) described another species of the genus \u0026ndash; \u003cem\u003eHalobiotus arcturulius\u003c/em\u003e Crisp \u0026amp; Kristensen, 1983, from Greenland as well.\u003c/p\u003e\n\u003cp\u003ePilato \u0026amp; Binda (1996) revised the type material of \u003cem\u003eH. crispae\u003c/em\u003e and amended the diagnosis of the genus. They pointed out the absence of the ventral lamina in the buccal-pharyngeal apparatus and described apophyses for the insertion of the stylet muscles (AISM) as hook-like and symmetrical in respect of the frontal plane.\u003c/p\u003e\n\u003cp\u003eAddition of the molecular genetic methods to the tools for the tardigrade phylogeny studies led to significant changes in the taxonomy of this group. An isolated position of the forms morphologically close to the genus \u003cem\u003eIsohypsibius\u003c/em\u003e Thulin, 1928 was revealed, and they were allocated to the independent superfamily Isohypsibioidea Sands, McInnes, Marley, Goodall-Copestake, Convey \u0026amp; Linse, 2008. A later integrative revision of this taxon (Gasiorek \u003cem\u003eet al.\u003c/em\u003e 2019) confirmed the separate position of the genus \u003cem\u003eHalobiotus\u003c/em\u003e within the superfamily, and a new family Halobiotidae was established. Later, these conclusions were supported by the results of other works devoted to the phylogeny of the superfamily Isohypsibioidea (Mioduchowska et al. 2021; Tumanov 2022; Tumanov et al. 2025).\u003c/p\u003e\n\u003cp\u003eIt should be noted that, though the descriptions of the two latter species of the genus \u003cem\u003eHalobiotus\u003c/em\u003e are accurate and supported by high-quality photographs acquired using both the light microscopy (LM) and scanning electron microscopy (SEM), they do not completely meet the standards of the taxonomic descriptions accepted in the systematics of the Eutardigrada nowadays. Morphometric data are provided for a small number of specimens and the set of measurements differs from the currently accepted for the Eutardigrada descriptions.\u003c/p\u003e\n\u003cp\u003eGlobal distribution of the genus \u003cem\u003eHalobiotus\u003c/em\u003e is still poorly understood. Most of the records are limited to the cold Arctic and Subarctic waters (Kaczmarek et al. 2015), but there are a few reports from the Black Sea (Romania and Ukraine; Rudescu 1964; Kharkevych 2013). Taxonomic status of these records is unclear due to the limited data on the specimens\u0026rsquo; morphology and complete absence of molecular data.\u003c/p\u003e\n\u003cp\u003eFour species of the genus \u003cem\u003eHalobiotus\u003c/em\u003e were previously reported from Russian seas:\u003c/p\u003e\n\u003cp\u003e1. \u003cem\u003eHalobiotus appelloefi\u003c/em\u003e was recorded by Petelina \u0026amp; Tchesunov (1983) from the Kandalaksha Bay (White Sea). Tardigrades were found in periphyton on the littoral and sublittoral macrophytes, on the pier and in the silted sand near the macrophytes\u0026rsquo; bases. The authors, having analyzed the literature data available at the time and the morphometric data they had obtained, showed that the description of \u003cem\u003eH. geddesi\u003c/em\u003e as a separate species is poorly justified. They also noted an unclear taxonomic situation in the \u003cem\u003eappelloefi/stenostomus\u003c/em\u003e complex (Kristensen\u0026rsquo;s work (1982) on cyclomorphosis was not known to them yet) and pointed out the importance of studying the limits of variability of morphometric features of tardigrades for the reliability of taxonomic studies.\u003c/p\u003e\n\u003cp\u003e2. In their study of head sensory structures in genus \u003cem\u003eHalobiotus\u003c/em\u003e, Biserova \u0026amp; Kuznetsova (2011) identified specimens collected from intertidal filamentous algae in the Kandalaksha Bay (White Sea) as \u003cem\u003eH. stenostomus\u003c/em\u003e, which they accepted as the senior synonym of \u003cem\u003eH. appelloefi\u003c/em\u003e. At the same time, they noted that the studied material had a number of features characteristic of the species \u003cem\u003eH. crispae\u003c/em\u003e, therefore the exact species identification of the White Sea \u003cem\u003eHalobiotus\u003c/em\u003e requires clarification. In addition, Biserov (1991) indicated \u003cem\u003eH. stenostomus\u003c/em\u003e for the White Sea (without specifying a locality).\u003c/p\u003e\n\u003cp\u003e3. In the most recent publication (Biserova et al. 2025) specimens from subtidal sandy sediments in the Kandalaksha Bay (White Sea) were identified using the methods of the DNA barcoding and the analyzed sequences of the nuclear and mitochondrial markers (18S rRNA, 28S rRNA, ITS-2 and COI) were proved to be conspecific with \u003cem\u003eH. crispae\u003c/em\u003e, but no detailed morphological data were provided.\u003c/p\u003e\n\u003cp\u003e4. \u003cem\u003eHalobiotus arcturulius\u003c/em\u003e was reported to inhabit silty-sand tidal flats in the Kara (Baydaratskaya Bay) and the Laptev (near the mouth of the Kurkum/Urya River) Seas (Biserov 1998). Furthermore, several specimens presumably assigned to this species were found in a freshwater lake on the Yuzhny Island of the Novaya Zemlya archipelago (Biserov 1999).\u003c/p\u003e\n\u003cp\u003eTo address the uncertain taxonomy and incomplete descriptions of the tardigrade genus \u003cem\u003eHalobiotus\u003c/em\u003e in Russian seas and to amend the existing taxonomic descriptions of the species belonging to the genus, we conducted an integrative morphological and molecular genetic analysis of populations from three Russian Arctic seas. This study aimed to determine the species affiliations of these populations and to generate a comprehensive dataset for reliable species identification. Furthermore, to elucidate the ecology of these species, we quantified the distribution of two \u003cem\u003eHalobiotus\u003c/em\u003e species across two Arctic tidal flats.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSampling\u003c/h2\u003e\u003cp\u003eMaterial was collected from three localities of the White Sea Kandalaksha Bay (Fettah island, Gorelyj island, and Matrenin island), from the Barents Sea and the Laptev Sea (see detailed information below in the Results section).\u003c/p\u003e\u003cp\u003eThe White Sea samples were obtained by the following procedure. Sampled bottom material was washed through two sieves (with 1 mm and 29 \u0026micro;m mesh respectively) (Tumanov \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Contents of the fine sieve from Fettah island were fixed with 96% ethanol. We also examined the content of the fine sieve from locations Fettah island and Matrenin island under a stereo microscope and isolated animals using a glass pipette. These specimens were fixed in RNA\u003cem\u003elater\u003c/em\u003e\u0026trade; Stabilization Solution (Thermo Fisher Scientific Inc.). Under a stereo microscope specimens from Gorelyj island were manually picked from the tubes of polychaete \u003cem\u003eDipolydora quadrilobata\u003c/em\u003e (Jacobi, 1883) and mounted on the permanent slides without fixation.\u003c/p\u003e\u003cp\u003eQuantitative data on tardigrade distribution across the intertidal zone were obtained from two locations: the Barents Sea and the Laptev Sea.\u003c/p\u003e\u003cp\u003eSediment samples from the Barents Sea coast were collected on 22 July 2019 from silty-sand tidal flats near the Mishukovo village (Kola Bay). The sampling transect included four stations with two stations located on the tidal flat and two stations \u0026ndash; on the adjacent salt marsh. Sediment temperature and pore water salinity were measured at each station (for details see Golikova et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Station 3 was primarily vegetated by \u003cem\u003ePuccinellia phryganodes\u003c/em\u003e, while \u003cem\u003eAgrostis straminea\u003c/em\u003e dominated at Station 4. At each station, three replicate samples of 10 cm\u003csup\u003e3\u003c/sup\u003e each were collected from the top 1 cm sediment layer, yielding a total of 12 samples. These were immediately preserved in 80% ethanol with Rose Bengal dye (2 g/L). After four weeks of staining, samples were washed with tap water and sieved simultaneously through 0.5 mm (removing plant debris) and 0.125 mm meshes, with the \u0026gt;\u0026thinsp;0.125 mm fraction retained for the analysis.\u003c/p\u003e\u003cp\u003eThe Laptev Sea specimens were collected on 17 September 2017 across an intertidal transect on Bolshoy Begichev Island in the Khatanga River estuary. Following a similar design to the Barents Sea study, the transect comprised four stations spaced 2\u0026ndash;3 m apart and extended from a tidal flat with silty sand substrate (stations 1\u0026ndash;2) to an upper intertidal zone colonized by facultative halophytic vegetation (hereafter referred to as salt marsh; stations 3\u0026ndash;4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Station 3 was covered with a dense turf of \u003cem\u003ePuccinellia phryganodes\u003c/em\u003e, while Station 4 was dominated by a mix of \u003cem\u003eDupontia psilosantha\u003c/em\u003e and \u003cem\u003eArctophyla fulva\u003c/em\u003e. Plant specimens had marks of geese grazing. No temperature or salinity measurements were taken. At each station, three replicate sediment samples (20 cm\u003csup\u003e3\u003c/sup\u003e each) were collected using a 20-cm\u003csup\u003e2\u003c/sup\u003e scoop, sampling the sediment top 1 cm of with 5\u0026ndash;30 cm spacing between replicates. In total, 12 samples were obtained. Samples were fixed in 70% ethanol with Rose Bengal dye (2 g/L). After one month of preservation, samples were washed with tap water through nested 0.5 mm and 0.125 mm sieves simultaneously to remove plant debris, retaining\u0026thinsp;\u0026gt;\u0026thinsp;0.125 mm fraction for analysis. Tardigrades were counted wet in a Petri dish under a LeicaM205C stereomicroscope prior to morphological and molecular analysis. The counts were standardized to 10 cm\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMicroscopy and imaging\u003c/h3\u003e\n\u003cp\u003eFor light microscopy (LM) investigation tardigrades were mounted on slides in Hoyer\u0026rsquo;s medium. Permanent slides were examined under a Leica DM2500 microscope equipped with phase contrast (PhC) and differential interference contrast (DIC). Photographs were taken using a Nikon DS-Fi3 digital camera with NIS software. For scanning electron microscopy (SEM), ethanol fixed specimens were transferred to acetone, critical-point dried in CO\u003csub\u003e2\u003c/sub\u003e, mounted on stubs and coated with gold. Tescan MIRA3 LMU Scanning Electron Microscope (Tescan, Brno, Czech Republic) and Hitachi TM-1000 (Hitachi, Japan) were used for observations.\u003c/p\u003e\n\u003ch3\u003eMorphometrics and terminology\u003c/h3\u003e\n\u003cp\u003eThe sample size for morphometrics was chosen following the recommendations of Stec et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). All dimensions are given in micrometers (\u0026micro;m). Structures were measured only if their orientation was suitable. Body length was measured from the anterior end of the body to the posterior end, excluding the hind legs. The bucco-pharyngeal tube was measured from the anterior margin of the stylet sheaths to the caudal end of the buccal tube, excluding the buccal apophyses. Terminology for structures of the bucco-pharyngeal apparatus and claws follows Pilato \u0026amp; Binda (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Elements of the buccal apparatus were measured according to Kaczmarek \u0026amp; Michalczyk (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Claws were measured according to Beasley et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), with the additional measurement of the total claw length (according to Pilato et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) to ensure compatibility with older publications. We calculated the \u003cem\u003ept\u003c/em\u003e index, which is the percentage ratio between the length of a structure and the length of the buccal tube (Pilato \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1981\u003c/span\u003e), and presented it in italics. Average values are given with standard deviation (SD). Morphometric data were handled using ver. 1.6 of the \u0026lsquo;Parachela\u0026rsquo; template, which is available from the Tardigrada Register (Michalczyk \u0026amp; Kaczmarek \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), with the addition of the total length of the claws.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eFor the statistical analyses we chose the measurements accessible for the most specimens we analyzed. Namely, we used stylet supports insertion point, internal and external buccal tube width, length of the macroplacoids 1\u0026ndash;3, length of the macroplacoid row for the buccal-pharyngeal apparatus and length of the base, the primary and the secondary branches for the posterior claws of the hind legs. To avoid the effect of the absolute body size of the specimens we used the \u003cem\u003ept\u003c/em\u003e index (Pilato \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) in the analyses.\u003c/p\u003e\u003cp\u003eStatistical analyses were performed using \u0026lsquo;R\u0026rsquo; (R Core Team 2017) in RStudio environment (RStudio Team 2017). Ordination of morphometry data was performed on the distance matrix calculated with Euclidean distances. To visualize the multidimensional distance matrix, we ran non-metric multidimensional scaling (Borcard et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) of individuals in \u0026lsquo;vegan\u0026rsquo; package (Oksanen \u003cem\u003eet al.\u003c/em\u003e 2017) and plotted the two-dimensional outcome with \u0026lsquo;ggplot2\u0026rsquo; package (Wickham \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). A stress value was calculated as well and is shown on the graph.\u003c/p\u003e\u003cp\u003eA Permutational Multivariate Analysis of Variance (PERMANOVA) was conducted to evaluate morphological differences between the Laptev Sea and the White Sea (Fettah island) populations. This analysis was performed using the \u0026lsquo;adonis\u0026rsquo; function in the \u0026lsquo;vegan\u0026rsquo; package for R. These specific populations were selected as they were the only ones with an adequate sample size and corresponding molecular data required for a robust comparison.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenotyping\u003c/h3\u003e\n\u003cp\u003eDNA was extracted from 22 individual specimens using QuickExtractTM DNA Extraction Solution (Lucigen Corporation, USA, see complete protocol description in Tumanov \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Four genes were sequenced: a small ribosome subunit (18S rRNA) gene, a large ribosome subunit (28S rRNA) gene, internal transcribed spacer (ITS-2) and the cytochrome oxidase subunit I (COI) gene. PCR reactions included 5 \u0026micro;L template DNA, 1 \u0026micro;L of each primer, 1 \u0026micro;L DNTP, 5 \u0026micro;L Taq Buffer (10\u0026times;) (\u0026minus;\u0026thinsp;Mg), 4 \u0026micro;L 25 mM MgCl\u003csub\u003e2\u003c/sub\u003e and 0.2 \u0026micro;L Taq DNA Polymerase (Thermo Scientific\u0026trade;) in a final volume of 50 \u0026micro;L. The lists of primers and PCR programs are provided in Table\u0026nbsp;1. The PCR products were visualized in 1.5% agarose gel stained with ethidium bromide. All amplicons were sequenced directly using the ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the help of an ABI Prism 310 Genetic Analyser. Sequences were assembled using ChromasPro software (Technelysium, USA). To check for the presence of stop codons and therefore of pseudogenes, the COI sequences were translated into amino acids using the invertebrate mitochondrial code implemented in MEGA11 (Tamura et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Unadjusted pairwise distances were calculated using MEGA11 (Tamura et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) with the treatment of gaps/missing data set to \u0026lsquo;pairwise deletion\u0026rsquo;.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eInstitutional acronyms\u003c/h2\u003e\u003cp\u003eSpecimens from the following institutions and collections were examined (the curator is given in parenthesis).\u003c/p\u003e\u003cp\u003eSPbU\u0026thinsp;=\u0026thinsp;St. Petersburg State University, Russia, Faculty of Biology, Department of Invertebrate Zoology (Denis Tumanov).\u003c/p\u003e\u003cp\u003eNHMD\u0026thinsp;=\u0026thinsp;Natural History Museum of Denmark (Martin Vinther S\u0026oslash;rensen)\u003c/p\u003e\u003cp\u003eZMUC\u0026thinsp;=\u0026thinsp;Zoological Museum, University of Copenhagen (Martin Vinther S\u0026oslash;rensen).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eTaxonomic account\u003c/p\u003e\u003cp\u003ePhylum Tardigrada Doy\u0026egrave;re, 1840\u003c/p\u003e\u003cp\u003eClass Eutardigrada Richters, 1926\u003c/p\u003e\u003cp\u003eOrder Parachela Schuster, Nelson, Grigarick \u0026amp; Christenberry, 1980\u003c/p\u003e\u003cp\u003eSuperfamily Isohypsibioidea Sands, McInnes, Marley, Goodall-Copestake, Convey \u0026amp; Linse, 2008\u003c/p\u003e\u003cp\u003eFamily Halobiotidae Gąsiorek, Stec, Morek \u0026amp; Michalczyk, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e\u003c/p\u003e\u003cp\u003eGenus \u003cem\u003eHalobiotus\u003c/em\u003e Kristensen, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1982\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHalobiotus crispae\u003c/b\u003e Kristensen, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1982\u003c/span\u003e\u003c/p\u003e\n\u003ch3\u003eMaterial examined\u003c/h3\u003e\n\u003cp\u003eWHITE SEA \u0026ndash; \u003cb\u003eKandalaksha Bay, Fettah island\u003c/b\u003e \u0026bull; 65 specs; 66.334\u0026deg; N, 33.652\u0026deg; E; 15. Aug. 2022; D.V. Tumanov leg.; below the water\u0026rsquo;s edge at low tide, \u003cem\u003eAscophyllum\u003c/em\u003e sp. overgrown by sponges and hydroids; GenBank nos: PX248699, PX248700, PX248701, PQ070004 (18S), PQ070031 (28S), PX260918, PX260919 (ITS-2), PX248704 (COI); SPbU 301(1\u0026ndash;15) \u0026bull; 22 specs; same data as for preceding; SEM stubs SPbU Tar_57, SPbU Tar_60. \u0026ndash; \u003cb\u003eKandalaksha Bay, vicinity of Luvenga village\u003c/b\u003e \u0026bull; 25 specs; Gorelyj island, Ilistaja Bay; 67.095\u0026deg; N, 32.679\u0026deg; E; 1992; V.M. Khaitov leg.; in tubes of \u003cem\u003eDipolydora quadrilobata\u003c/em\u003e; SPbU 327(1\u0026ndash;5). \u0026ndash; \u003cb\u003eKandalaksha Bay, Chupa Bay\u003c/b\u003e \u0026bull; 1 spec.; Matrenin island; 66.310\u0026deg; N, 33.630\u0026deg; E; 13 Jun. 2021; V.R. Khabibulina leg.; intertidal colonies of hydroids; GenBank nos: PX248698 (18S), PX260917 (ITS-2), PX248703 (COI); SpbU 278(1).\u003c/p\u003e\u003cp\u003eBARENTS SEA \u0026bull; 21 specs; vicinity of Mishukovo village, 69.041\u0026deg; N, 33.028\u0026deg; E; 22 Jul. 2019; D.A. Mikhailov leg.; silty-sand tidal flats,; SPbU 262(1\u0026ndash;5).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAdditional material\u003c/h2\u003e\u003cp\u003eBaffin Bay \u0026bull; 14 specs, paratypes; Nipissat Bay, Disko Island, West Greenland; 69\u0026deg;26\u0026prime; N, 54\u0026deg;12\u0026prime; W; depth 0.5 m; 18 May 1979; R.M. Kristensen leg.; subtidal on the brown alga \u003cem\u003eSphacelaria arctica\u003c/em\u003e; ZMUC TAR-253 \u0026bull; 12 specs, paratypes; same data as for preceding; 03 Jan. 1979; R.M. Kristensen leg.; subtidal on the brown alga \u003cem\u003eSphacelaria arctica\u003c/em\u003e; ZMUC TAR-255.\u003c/p\u003e\u003cp\u003e\u0026Oslash;resund \u0026bull; 1 specs; Helsing\u0026oslash;r, Denmark, S for B\u0026aring;kerne; labelled as \u003cem\u003eIsohypsibius appelloefi\u003c/em\u003e; ZMUC TAR-181.\u003c/p\u003e\u003cp\u003e\u0026Oslash;resund \u0026bull; 1 specs; Helsing\u0026oslash;r, Denmark; depth 28 m; 17 Apr. 1980; R.M. Kristensen leg.; mud, labelled as \u003cem\u003eIsohypsibius appelloefi\u003c/em\u003e; ZMUC TAR-182.\u003c/p\u003e\u003cp\u003eSkagerrak \u0026bull; 7 specs; Tjarno, Sweden; depth 17\u0026ndash;20 m; 23 Oct. 1987; R.M. Kristensen leg.; on \u003cem\u003eZostera\u003c/em\u003e, labelled as \u003cem\u003eHalobiotus stenostomus\u003c/em\u003e; NHMD-633704\u0026ndash;633707.\u003c/p\u003e\u003cp\u003ePhotographs of \u003cem\u003eH. geddesi\u003c/em\u003e type material were provided by Prof. Roberto Bertolani (University of Modena and Reggio Emilia).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAmended morphological description\u003c/h2\u003e\u003cp\u003eBased on the material from the White Sea (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;C) and the type material (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eActive stage.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMedium-sized animals (body length 273\u0026ndash;555 \u0026micro;m, other morphometrics provided in Table\u0026nbsp;2). Body elongated, with the greatest width at the level of legs III (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Fresh specimens uncoloured or whitish with greenish gut content, transparent after fixation in Hoyer\u0026rsquo;s medium. Some specimens with intensive purple body colouring, which persists in the mounted animals. Eyes present in living specimens, dissolving after mounting in in Hoyer\u0026rsquo;s medium.\u003c/p\u003e\u003cp\u003eDorsal cuticle with sculpture. The degree of development of the cuticular sculpture varies significantly \u0026ndash; from well-defined (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;F, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) to practically indistinguishable. The cuticle sculpture in both the type material and the White Sea specimens is not \u0026ldquo;reticular\u0026rdquo;, as indicated in the original description, but is a system of irregular winding folds, which only in rare cases looks like \u0026ldquo;reticular\u0026rdquo; sculpture at low magnifications of the microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eExternal cephalic sense organs include two pairs of sensory areas, which in fixed specimens are visible as flat plates, distinctly demarcated (in both LM and SEM) with a circular groove. The anterior pair was designated by Kristensen (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) as \u0026ldquo;cephalic papillae\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;D) corresponds to the \u0026ldquo;antero-lateral sensory fields\u0026rdquo; (Walz \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1978\u003c/span\u003e) or \u0026ldquo;frontal lobes\u0026rdquo; (Gąsiorek et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) of other eutardigrades. The posterodorsal pair designated by Kristensen (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) as \u0026ldquo;temporalia\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C, white arrowheads) corresponds to the \u0026ldquo;elliptical organs\u0026rdquo; (Pilato \u0026amp; Binda \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) or \u0026ldquo;posterolateral sensory fields\u0026rdquo; (Gross et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kihm et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) of other eutardigrades. Additionally, in SEM a third pair of very poorly demarcated ventrolateral sensory fields (according to Gross et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) is sometimes visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D). No pore-like structures, typical for the head sensory areas of eutardigrades (see Tumanov et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) are visible on the surface of the sense organs of both pairs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMouth opens on the top of the well-developed retractable mouth cone, lacks peribuccal lamellae with continuous membrane being present only and is surrounded by six peribuccal lobes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). Each lobe often with a clear depression in the apical part (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, black arrowheads)\u003c/p\u003e\u003cp\u003eBucco-pharyngeal apparatus is of \u003cem\u003eHalobiotus\u003c/em\u003e type (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). In SEM, the oral cavity armature (OCA) consists of three rows of teeth (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). First (anterior) row consists of a band of thin longitudinally elongated teeth located on the anterior ring of the buccal cavity right behind the peribuccal membrane. Second row consists of a wide band of strong conical teeth situated right behind the ring fold of the oral cavity. Third row comprises two strong transverse ridges (one dorsal and one ventral) consisted of partially fused large teeth. Sometimes the most lateral teeth seem to be separated from the ridges, being positioned in line with them. In LM only the ridges of the third row are always well-visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J, K, black arrowheads), teeth of the second row can be visible, but poorly distinguishable, in largest specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, white arrowheads).\u003c/p\u003e\u003cp\u003eThe dorsal and ventral apophyses for the insertion of the stylet muscles (AISMs) are both hook-like, but differ from each other (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The dorsal apophyses are better developed than the ventral and their apical part usually forms a three-pointed structure, being observed from the dorsal side, with apices directed backwards (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The points are often partially fused, forming asymmetrical structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The ventral apophyses are smaller than the dorsal and with lesser developed hook-like structure (sometimes nearly indistinguishable), without a three-pointed structure possessing two oblique latero-caudal processes. Caudally to the apophyses an elongated zone of the thickened buccal tube wall is present both dorsally and ventrally, the dorsal thickening is usually better developed than the ventral (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, black arrowheads, 6D, white arrowheads). These thickenings are thin and can resemble ventral lamina when observed in dorso-ventral projection (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C, black arrowheads)).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe buccal tube is rigid, its caudal part slightly bends ventrally (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The buccal tube structure evidently different in the anterior and the posterior parts with the transition zone located slightly caudally to the stylet supports insertion point. Anterior part of the buccal tube has \u0026ldquo;double\u0026rdquo; walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, black arrowheads), and its surface is usually covered with numerous oblique folds (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, white arrowheads). Posterior part has solid walls, without such folds (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The stylet furca has a typical shape. Pharyngeal bulb is subspherical, with well-developed apophyses, thin oblique cuticular bars, and three elongated macroplacoids, microplacoids absent (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). The first macroplacoid is longer than the second and is usually saddle-shaped with a pronounced constriction on its outer side in the middle region (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, black arrowheads). Rarely, in large specimens the outer side of macroplacoids can be ragged (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, white arrowheads). The second macroplacoid is the shortest and is often is saddle-shaped (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, black arrowhead), but sometimes have no median groove. The third macroplacoid is the longest with a deep preterminal constriction, separating its caudal part. This element is usually bent in the caudal direction and elongated transversely (with its width exceeding its length). Anterior part on the third macroplacoid is usually saddle-shaped, similar to the first macroplacoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, black arrowhead).\u003c/p\u003e\u003cp\u003eLegs are relatively long; outer surface of legs I\u0026ndash;III often has an area of better developed cuticular sculpture, visible in SEM only (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, black arrowhead). All legs possess large claws, which increase in size from legs I to legs IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C). Claws are of the \u003cem\u003eIsohypsibius\u003c/em\u003e type, with thin stalk and expanded bases (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C, F, G, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, I). Both main and secondary claw branches with thick walls and a large internal cavity (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, G, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, I). Claws are very flexible, especially in the zone of connection between the main branch and the other part of the claw, where the claw walls are evidently thinned. All claws have accessory points (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB); free apexes of accessory points of claws on legs I\u0026ndash;III are often lateral to the main branch and poorly discernible under LM. All claws with smooth lunules (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;E, G, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eG\u0026ndash;I). Lunules of internal (anterior) claws are small and often difficult to observe (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, black arrowhead). On legs I\u0026ndash;III they are not connected with a cuticular bar, which present below the inner claw bases (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, H). On legs IV below the claw bases a horseshoe-like zone of thickened cuticle is usually present in large specimens, connected with the muscle attachment zone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eSmooth eggs laid in exuvium.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePseudosimplex stage\u003c/h2\u003e\u003cp\u003eAnimals in the pseudosimplex stage clearly differs from the active stage specimens in the configuration of the buccal-pharyngeal apparatus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Buccal tube is much thinner (external diameter up to 2 \u0026micro;m), flexible, walls of the anterior part retain the \u0026ldquo;double\u0026rdquo; structure, evident oblique folds are also present in the anterior part of the buccal tube (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, D). AISMs are hook-like, but much less developed and of nearly equal size and configuration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Pharyngeal bulb elongated, without developed placoids, with thin usually continuous lining only (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, D). Rarely some of these continuous cuticular lines are interrupted in the anterior part. Claws of the animals in the pseudosimplex stage in our material do not differ in their morphology from the active stage.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eDNA sequences\u003c/h2\u003e\u003cp\u003eWe obtained sequences for 18S rRNA marker from five specimens, sequence for 28S rRNA marker from a single specimen, sequences for ITS-2 marker from three specimens, and sequences for COI marker from two specimens. All markers were represented by single haplotype.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHalobiotus arcturulius\u003c/b\u003e Crisp \u0026amp; Kristensen, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMaterial examined\u003c/h2\u003e\u003cp\u003eLAPTEV SEA \u0026bull; 111 specs; Bolshoy Begichev island in the mouth of the Khatanga River, 74.365\u0026deg; N, 112.04\u0026deg; E; 17 Sep. 2017; D.A. Mikhailov leg.; silty-sand tidal flats and salt marsh covered with \u003cem\u003ePuccinellia phryganodes\u003c/em\u003e, \u003cem\u003eDupontia psilosantha\u003c/em\u003e и \u003cem\u003eArctophyla fulva\u003c/em\u003e; GenBank nos: MZ050455 (18S), MZ050452 (28S), MZ078284 (ITS-2); SPbU 260(1\u0026ndash;25) \u0026bull; 41 specs; same data as for preceding; SEM stubs SPbU Tar_28, SPbU Tar_32.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAdditional material\u003c/h2\u003e\u003cp\u003eGREENLAND SEA \u0026bull; 11 specs, paratypes; Mesters Vig, Kong Oscars Fjord, Central East Greenland; 6 Aug. 1974; M. Crisp and R.M. Kristensen leg.; sandy arctic beach; ZMUC TAR-192, ZMUC TAR-194, ZMUC TAR-195, ZMUC TAR-197.\u003c/p\u003e\u003cp\u003eNOVAYA ZEMLYA \u0026bull; 1 spec.; Yuzhny Island, freshwater lake; 1995(?); V.I. Biserov leg.; labelled as \u003cem\u003eHalobiotus arcturulius\u003c/em\u003e cfr., R.M. Kristensen det.; ZMUC TAR-773.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eAmended morphological description\u003c/h2\u003e\u003cp\u003eBased on the material from the Laptev Sea (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e), the type material (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eA\u0026ndash;D), and the specimen from a freshwater lake on the Yuzhny Island of the Novaya Zemlya archipelago (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eE\u0026ndash;H)).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLarge animals (body length 473\u0026ndash;693 \u0026micro;m, other morphometrics provided in Table\u0026nbsp;3). Body elongated, with the greatest width at the level of legs III (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;C, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eA, E). Fresh specimens uncoloured or whitish, transparent after fixation in Hoyer\u0026rsquo;s medium. Eyes present, not dissolving after mounting in in Hoyer\u0026rsquo;s medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, black arrowheads).\u003c/p\u003e\u003cp\u003eCuticle without developed sculpture, only slight unevenness can be visible in SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;E).\u003c/p\u003e\u003cp\u003eExternal cephalic sense organs visible in SEM only and comprise poorly demarcated anterolateral fields (without pore-like structures) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, black arrowheads) and mediodorsal cephalic pore (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, white arrowhead). \u0026ldquo;Temporalia\u0026rdquo; (or \u0026ldquo;elliptical organs\u0026rdquo;) not visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eMouth opens on the top of the retractable mouth cone, lacks peribuccal lamellae with continuous membrane being present only and is surrounded by six peribuccal lobes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eBucco-pharyngeal apparatus is of the \u003cem\u003eHalobiotus\u003c/em\u003e type (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eB, F). In SEM, the oral cavity armature (OCA) consists of three rows of teeth (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, C). First (anterior) row consists of a band of small teeth located on the anterior ring of the buccal cavity right behind the peribuccal membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Second row consists of a wide band of strong conical teeth situated just behind the ring fold of the oral cavity, teeth in the caudal zone of the band can be enlarged (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Third row comprises two strong transverse ridges (one dorsal and one ventral) consisted of partially fused large teeth (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). Sometimes the most lateral teeth seem to be separated from the ridges, being positioned in line with them. In LM only the teeth of the second (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eD, E, white arrowheads) and the third rows (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eD, E, black arrowheads) are visible.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe dorsal and ventral apophyses for the insertion of the stylet muscles (AISMs) are ridge-like, without hook-like structure, slightly different from each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eF, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eB, black arrowheads). The dorsal apophyses are better developed than the ventral, with step-like caudal end without hook-like appendage. The ventral apophyses are smaller than the dorsal in the form of simple ridge slightly indented in the middle. The dorsal apophyses do not form clear three-pointed structure, being observed from the dorsal side (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eG, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eG). Caudally to the apophyses an elongated zone of the thickened buccal tube wall is present both dorsally and ventrally (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eF, white arrowheads), the dorsal thickening is usually better developed than the ventral (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eG, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eF, G, black arrowheads).\u003c/p\u003e\u003cp\u003eThe buccal tube is rigid; its caudal part slightly bends ventrally (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eB). The buccal tube structure evidently different in the anterior and the posterior parts with the transition zone located slightly caudally to the stylet supports insertion point. Anterior part of the buccal tube has \u0026ldquo;double\u0026rdquo; walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eG, white arrowheads), and its surface is usually covered with numerous oblique folds (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eF, G). Posterior part has solid walls, without such folds. The stylet furca has a typical shape. Pharyngeal bulb is subspherical, with well-developed apophyses, thin oblique cuticular bars, and three elongated macroplacoids, microplacoids absent (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e9\u003c/span\u003eH, I, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eC, H). The first macroplacoid is longer than the second and is usually ragged with numerous small protrusions on its outer side. The second macroplacoid is the shortest. The third macroplacoid is the longest with a very strong preterminal constriction, its caudal part is nearly completely detached from the anterior part. This element is usually slightly bent in the caudal direction; its width is usually equal to its length. Anterior part on the third macroplacoid and the second macroplacoid are usually ragged on their outer side, similar to the first macroplacoid.\u003c/p\u003e\u003cp\u003eLegs are relatively long, all legs possess large claws, which increase in size from legs I to legs IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, C). Claws are of the \u003cem\u003eIsohypsibius\u003c/em\u003e type, with thin stalk and expanded bases (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003eA\u0026ndash;F). Both main and secondary claw branches with thick walls and a large internal cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003eA, B, F). Claws are very flexible, especially in the zone of connection between the main branch and the other part of the claw, where the claw walls are evidently thinned. All claws have accessory points; free apexes of accessory points of claws on legs I\u0026ndash;III are often lateral to the main branch and poorly discernible under LM. All claws with smooth lunules (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003eA, B, F). Lunules of internal (anterior) claws are small and often difficult to observe. On legs I\u0026ndash;III they are not connected with a cuticular bar, which present below the inner claw bases (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003eA, F, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eD, black arrowheads). Cuticular bars on legs I\u0026ndash;III are less pronounced than in \u003cem\u003eH. crispae\u003c/em\u003e, often poorly visible on legs I\u0026ndash;II (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eD). Weak sclerotization causes evident unevenness of the bars\u0026rsquo; margins, described as \u0026ldquo;toothed\u0026rdquo; margin by Crisp \u0026amp; Kristensen (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003eF, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eD). On legs IV very poorly developed diffuse zone of the thickened cuticle is sometimes visible below the claw bases, never forms horseshoe-like structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003eG, black arrowheads). In one specimen abnormal internal claw of the leg III bearing additional secondary branch was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eSmooth eggs laid in exuvium.\u003c/p\u003e\u003cp\u003eNo specimens in the cyclomorphic stages were observed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eDNA sequences\u003c/h2\u003e\u003cp\u003eWe obtained sequences from a single specimen for 18S rRNA, 28S rRNA, and ITS-2 markers. Numerous attempts to obtain COI sequence were unsuccessful.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003ePermutational Multivariate Analysis of Variance showed that the Laptev Sea and the White Sea populations significantly diverge in their morphometric features used for the analysis, and the geographical location contributes prominently to the observed differences (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.76, p\u0026thinsp;=\u0026thinsp;0.001). Consistently, on the ordination plot the individuals from different populations form two separate groups without overlapping (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eEcological data\u003c/h2\u003e\u003cp\u003eQuantitative data on tardigrade distribution were obtained only from the Barents and Laptev Sea coasts. In both regions, the tardigrade assemblages were monospecific. The Barents Sea intertidal zone was exclusively populated by \u003cem\u003eH. crispae\u003c/em\u003e, while \u003cem\u003eH. arcturulius\u003c/em\u003e was found only on the Laptev Sea coast.\u003c/p\u003e\u003cp\u003eA total of 21 specimens of \u003cem\u003eH. crispae\u003c/em\u003e were documented from the 10 cm\u003csup\u003e3\u003c/sup\u003e samples transect in the Barents Sea. In contrast, the abundance of \u003cem\u003eH. arcturulius\u003c/em\u003e in the Laptev Sea was markedly higher, with a total of 1,586 individuals collected along a transect of 20 cm\u003csup\u003e3\u003c/sup\u003e samples, which equivalent to 793 individuals per standardized 10 cm\u003csup\u003e3\u003c/sup\u003e volume. Thus, population density differed substantially between the two species. The density of \u003cem\u003eH. crispae\u003c/em\u003e was sparse, with a low average value of 2 individuals per 10 cm\u003csup\u003e3\u003c/sup\u003e, ranged in 1\u0026ndash;9 ind./10 cm\u003csup\u003e3\u003c/sup\u003e. In contrast, a considerably higher mean density of 66 ind./10 cm\u003csup\u003e3\u003c/sup\u003e (range: 3-212 ind./10 cm\u003csup\u003e3\u003c/sup\u003e) was observed in \u003cem\u003eH. arcturulius\u003c/em\u003e (Table\u0026nbsp;4).\u003c/p\u003e\u003cp\u003eIn both locations, the highest tardigrade densities were observed at the lowest tidal flat station (Station 1). At this station, the mean density of \u003cem\u003eH. crispae\u003c/em\u003e was 3.7 ind./10 cm\u003csup\u003e3\u003c/sup\u003e at the Mishukovo (Barents Sea), and the density of \u003cem\u003eH. arcturulius\u003c/em\u003e reached 131.3 ind./10 cm\u003csup\u003e3\u003c/sup\u003e at the Bolshoy Begichev Island (Laptev Sea) (Table\u0026nbsp;4).\u003c/p\u003e\u003cp\u003eExuviae with eggs were found on both coasts, indicating reproductive activity of tardigrades. On the Barents Sea intertidal zone, we found egg-bearing exuviae of \u003cem\u003eH. crispae\u003c/em\u003e in mid-July and only at the lowest tidal flat station. We did not count these exuviae. In the Laptev Sea, exuviae of \u003cem\u003eH. arcturulius\u003c/em\u003e were found in mid-September, mainly in the upper part of the tidal flat (station 2), and once at the lower salt marsh station (station 3) (Table\u0026nbsp;4).\u003c/p\u003e\u003cp\u003eThe sediment samples from both sites also contained typical meiofaunal organisms, including copepods, nematodes, oligochaetes, chironomids, halacarid mites, and foraminiferans.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eComparison with the original descriptions\u003c/h2\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eHalobiotus crispae\u003c/h2\u003e\u003cp\u003eAnimals from the White Sea and Barents Sea populations match the original description of \u003cem\u003eH. crispae\u003c/em\u003e in such key details as the presence of pronounced cuticular sculpture on the body surface and well-developed cephalic sensory appendages. It should be noted that the sensory areas are not protruding dome-shaped papillae, as in Kristensen\u0026rsquo;s (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) description and drawings, but are visible as clearly demarcated flat plates. The length ratio of the pharyngeal placoids and the size characteristics of the claws in our material also match the original description.\u003c/p\u003e\u003cp\u003eOur material, however, also exhibits certain features that diverge from the description:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe cuticular sculpture varies significantly in development, from well-defined to practically indistinguishable. These data correlate well with the results of Danish researchers, who initially attributed the material found near the island of \u0026AElig;r\u0026oslash; (Baltic Sea) to \u003cem\u003eH. stenostomus\u003c/em\u003e and later identified it as \u003cem\u003eH. crispae\u003c/em\u003e based on the results of molecular genetic analysis (J\u0026oslash;rgensen \u0026amp; Kristensen \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; M\u0026oslash;bjerg et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These observations were recently supported by Biserova et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) who noted the smooth cuticle surface of the specimens from the White Sea identified as \u003cem\u003eH. crispae\u003c/em\u003e based on the results of molecular genetic analysis.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eAs mentioned above, the sculpture in the White Sea specimens differs from the \u0026ldquo;reticular\u0026rdquo; type in the original description. Moreover, the SEM photographs, provided in the description (Kristensen \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1982\u003c/span\u003e, Figs.\u0026nbsp;29\u0026ndash;32) correspond well to our material, showing a system of irregular winding folds (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe claws of pairs I of legs are somewhat shorter than indicated by Kristensen (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) for the type material (external claw 43 \u0026micro;m, internal claw 31 \u0026micro;m in the type material and external claws 33.7\u0026ndash;37.6 \u0026micro;m, internal claws 17.5\u0026ndash;26.4 \u0026micro;m in our specimens). For the claws of the fourth pair such differences are still present, but much lesser pronounced, affecting the anterior claws only (37 \u0026micro;m in the type material and 22.5\u0026ndash;33.0 \u0026micro;m in our specimens). However, the measurement technique is not specified in the original description, the specimens were embedded in a different medium (glycerol or polyvinyl-lactophenol), and the long and thin claws of \u003cem\u003eHalobiotus\u003c/em\u003e bend very easily, making their accurate measurement a significant problem. In this regard, these differences do not seem significant to us. Moreover, the evident variability if the claw length was noted in the original description. It was not possible to carry out new measurements on the type material provided to us due to the unfortunate orientation of the specimens in the preparation.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe outer lunulae of the claws of pairs I\u0026ndash;III of legs are described by Kristensen (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) as \u0026ldquo;weakly serrated\u0026rdquo;. We were unable to detect serration on these elements either in the type material or in the White Sea specimens (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). The noticeable unevenness sometimes present of the lunulae edges was probably taken by the author of the description for the presence of denticles. Kristensen also described the cuticular bar at the base of the inner claws of pairs I\u0026ndash;III of legs as part of a large lunula. A repeated examination of the type material revealed the presence of a small typical lunula on the inner claws (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), often folded under the base of the claw and therefore unnoticeable. The White Sea specimens have the same claw structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The cuticular bar is not part of the claw lunula, but is homologous to other similar structures that are widespread within eutardigrades.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eFor all populations with significant number of specimens examined (two populations from the White Sea and one from the Barents Sea), the presence of a cyclomorphic pseudosimplex stage characteristic of \u003cem\u003eH. crispae\u003c/em\u003e was shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eHalobiotus arcturulius\u003c/h2\u003e\u003cp\u003eAnimals from the Laptev Sea populations match the original description of \u003cem\u003eHalobiotus arcturulius\u003c/em\u003e (Crisp \u0026amp; Kristensen \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and reinvestigated type material (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eA\u0026ndash;D). Authors of the description gave no formal differential diagnosis for the species, but all discriminative traits mentioned by them (i.e., larger body length, smooth cuticle, less pronounced AISM\u0026rsquo;s) are present in our material and confirm the presence of this species in the Laptev Sea. As in the case of the type population of this species no cyclomorphic stages were observed. Investigated specimen of \u003cem\u003eH. arcturulius\u003c/em\u003e collected by Biserov from a small lake on Yuzhny Island of the Novaya Zemlya archipelago (slide number: ZMUC TAR-773) also conforms the original description and material from the Laptev Sea in all observed characters (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eE\u0026ndash;H).\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eGenetic data on the White Sea material\u003c/h2\u003e\u003cp\u003eMolecular genetic analysis also revealed a high similarity between our material and \u003cem\u003eH. crispae.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAccording to the mitochondrial marker COI, sampled populations from the White Sea (Fettah Island and Matrenin Island) were identical to each other and completely identical to the Danish specimens (Vellerup Vig); in addition, they were extremely close to samples from the White Sea published by Biserova et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) (Olenevsky Island, \u003cem\u003ep\u003c/em\u003e-distance 0,17%) and from the type locality (Greenland, Nipisat) and another Danish location (\u0026AElig;r\u0026oslash;) (\u003cem\u003ep\u003c/em\u003e-distance 0.86% and 0.85%, respectively). This result is slightly in contrary with the statement of Biserova et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) that \u0026ldquo;cox1 gene sequences of the White Sea \u003cem\u003eH. crispae\u003c/em\u003e are 100% identical with \u003cem\u003eH. crispae\u003c/em\u003e isolates from GenBank database\u0026rdquo;, but the differences are still minor enough to consider all sequences conspecific. Median Joining haplotype network for the COI gene is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBased on the nuclear ITS-2 marker, White Sea samples from both populations were identical to each other and completely matched those from Greenland and two Danish localities. Surprisingly, sample from the other White Sea population (Olenevsky Island; Biserova et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) was significantly different from all other \u003cem\u003eH. crispae\u003c/em\u003e populations. \u003cem\u003eP\u003c/em\u003e-distance for the ITS-2 marker of this population is 8.49%, which exceed even the distance for the same marker of \u003cem\u003eH. arcturulius.\u003c/em\u003e In our opinion such difference is likely to be a result of amplification or sequencing artifact. Surely, such phenomenon needs careful investigation of the additional material from the same locality. Median Joining haplotype network for the ITS-2 marker is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eB.\u003c/p\u003e\u003cp\u003eConservative marker 18S rRNA shows very low variability within all studied populations (do not exceed 0.10%). There are no data on the homological regions of the 28S rRNA marker of \u003cem\u003eH. crispae\u003c/em\u003e from outside the White Sea in GenBank. The 28S rRNA marker shows relatively high variability (\u003cem\u003ep\u003c/em\u003e-distance is 3.20%) between our results and sequences obtained by Biserova et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). We consider these results as requiring additional investigation. Complete results of the \u003cem\u003ep\u003c/em\u003e-distances calculations are presented in Supplementary file SM.01.\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparative morphometry of\u003c/b\u003e \u003cb\u003eH. crispae\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eH. arcturulius\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDirect comparison of the morphometric traits of \u003cem\u003eHalobiotus\u003c/em\u003e populations from the White Sea and the Laptev Sea revealed their similarity (see Tables\u0026nbsp;2 and 3). Meanings of the \u003cem\u003ept\u003c/em\u003e index interleave or close for most of the measurements. The most constant differences were observed in the length of the first macroplacoids (\u003cem\u003ept\u003c/em\u003e value is 8.3\u0026ndash;15.0 in \u003cem\u003eH. crispae\u003c/em\u003e and 15.3\u0026ndash;21.5 in \u003cem\u003eH. arcturulius\u003c/em\u003e), macroplacoid row length (\u003cem\u003ept\u003c/em\u003e value is 38.9\u0026ndash;52.8 in \u003cem\u003eH. crispae\u003c/em\u003e and 56.0\u0026ndash;72.2 in \u003cem\u003eH. arcturulius\u003c/em\u003e), and internal (anterior) claws secondary branch length of all legs (e.g., for the anterior claws of the hind legs secondary branch \u003cem\u003ept\u003c/em\u003e value is 34.6\u0026ndash;44.5 in \u003cem\u003eH. crispae\u003c/em\u003e and 48.3\u0026ndash;58.0 in \u003cem\u003eH. arcturulius\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eThe results of the performed statistical analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e) additionally support our conclusion of presence of two distinct species of the genus \u003cem\u003eHalobiotus\u003c/em\u003e in the fauna of Russia.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTaxonomic composition of the genus\u003c/b\u003e \u003cb\u003eHalobiotus\u003c/b\u003e \u003cb\u003ein North European seas.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIt can be confidently concluded that in the fauna of the White Sea only one species of the genus \u003cem\u003eHalobiotus\u003c/em\u003e is reliably found, namely \u003cem\u003eH. crispae\u003c/em\u003e. Considering that trustworthy morphological identification of the species of this complex is nearly impossible, only molecular taxonomy methods can answer the question of whether the Barents Sea population belongs to the same species or represents a separate taxon. But taking into account its morphological identity with the studied White Sea populations and presence of the typical cyclomorphic stages we can assume that in the Barents Sea the same species \u003cem\u003eH. crispae\u003c/em\u003e is present.\u003c/p\u003e\u003cp\u003eOur investigation of several specimens identified as \u003cem\u003eH. appelloefi\u003c/em\u003e from Sweden (Tj\u0026auml;rn\u0026ouml;; slides NHMD-633704\u0026ndash;633707) and Denmark (Helsing\u0026oslash;r; slide ZMUC TAR-181) from collection of Zoological Museum, University of Copenhagen revealed presence of the cuticular sculpture at least in some specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eA, B), which indicates that these populations should be also considered as belonging to species \u003cem\u003eH. crispae.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTaking into account that specimens of \u003cem\u003eH. crispae\u003c/em\u003e can possess nearly completely smooth cuticle with sculpture invisible both in LM and SEM (J\u0026oslash;rgensen \u0026amp; Kristensen \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; M\u0026oslash;bjerg et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Biserova et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; our data), which is the main character discriminating \u003cem\u003eH. crispae\u003c/em\u003e from the species of \u003cem\u003eH. appelloefi/stenostomus\u003c/em\u003e complex, and considering the incomplete and outdated descriptions of \u003cem\u003eH. appelloefi\u003c/em\u003e and \u003cem\u003eH. stenostomus\u003c/em\u003e both these species should be considered as \u003cem\u003enomina inquirendae\u003c/em\u003e. All records of these species should be considered doubtful, waiting for the confirmation by the methods of modern taxonomy.\u003c/p\u003e\u003cp\u003eIn concordance with Petelina \u0026amp; Tchesunov (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) we do not accept \u003cem\u003eH. geddesi\u003c/em\u003e as a valid species. Taking into account the presence of the cuticular sculpture well visible in the photographs of the type specimen (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eC\u0026ndash;F; kindly provided by R. Bertolani) this species is likely a junior synonym of \u003cem\u003eH. crispae\u003c/em\u003e. Until the new material is collected and genetically characterized from the type locality of \u003cem\u003eH. geddesi\u003c/em\u003e this species should be considered \u003cem\u003enomen inquirendum\u003c/em\u003e as proposed by Gąsiorek et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe therefore conclude that the genus \u003cem\u003eHalobiotus\u003c/em\u003e currently comprises only two adequately described species, which are reliably distinguished using both morphological and molecular methods.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eNotes on the oral cavity armature in Isohypsibioidea\u003c/h2\u003e\u003cp\u003eIn the latest revision of the Isohypsibioidea morphology and phylogeny Gąsiorek et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) stated that all genera of this clade have no more than two rows of teeth on the OCA. In their opinion this fundamental difference with the Macrobiotoidea clade which typically have three rows of teeth on OCA makes it difficult to homologize these structures between the clades. Our observations revealed presence of the three rows of teeth on the OCA of both investigated \u003cem\u003eHalobiotus\u003c/em\u003e species. In each case the OCA comprises: 1) the anteriormost row of minute teeth, located on the anterior ring of the oral cavity immediately below the peribuccal cuticular membrane, 2) the second row, consisted of larger conical teeth, located on and behind the ring fold of the oral cavity, and 3) the third row, consisted of large partially fused teeth, which forms strong mediodorsal and medioventral transverse ridges in the caudal part of the oral cavity. This configuration perfectly coincides with the typical OCA configuration within Macrobiotoidea, and in our opinion those three rows of teeth are evidently homologous within Isohypsibioidea, Eohypsibioidea and Macrobiotoidea.\u003c/p\u003e\u003cp\u003eIt is interesting to note that \u003cem\u003eHalobiotus\u003c/em\u003e is not the only taxon within Isohypsibioidea possessing strong mediodorsal and medioventral transverse ridges in the caudal part of the oral cavity. Such structures are also present in the OCA of species, currently attributed to the genus \u003cem\u003eGrevenius\u003c/em\u003e Gąsiorek, Stec, Morek \u0026amp; Michalczyk, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, more precisely, to the group of species similar to \u003cem\u003eGrevenius monoicus\u003c/em\u003e (Bertolani, 1981). This group incorporates species with rugose cuticular sculpture, three wide massive macroplacoids and OCA with strong dorsal and ventral toothed ridges (Bertolani \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eG). Three species can be currently attributed to this morphological group: \u003cem\u003eG. monoicus\u003c/em\u003e, \u003cem\u003eGrevenius baicalensis\u003c/em\u003e (Ramazzotti, 1966), and \u003cem\u003eGrevenius ladogensis\u003c/em\u003e (Tumanov, 2003). The fourth species with the same set of characters was described by Pilato et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) as \u003cem\u003eIsohypsibius occultus\u003c/em\u003e Pilato, D\u0026rsquo;Urso, Sabella \u0026amp; Lisi, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e simultaneously with the publication of the Isohypsibioidea revision by Gąsiorek et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), where all freshwater species of the former genus \u003cem\u003eIsohypsibius\u003c/em\u003e were transferred into the newly instituted genus \u003cem\u003eGrevenius\u003c/em\u003e. Taking into account the morphological similarity of \u003cem\u003eIs. occultus\u003c/em\u003e and \u003cem\u003eG. monoicus\u003c/em\u003e and the aquatic lifestyle of the former species we suppose to move this species formally into the genus \u003cem\u003eGrevenius\u003c/em\u003e as \u003cem\u003eGrevenius occultus\u003c/em\u003e (Pilato, D\u0026rsquo;Urso, Sabella \u0026amp; Lisi, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) \u003cb\u003ecomb. nov.\u003c/b\u003e But in our opinion taxonomic position of the \u003cem\u003emonoicus\u003c/em\u003e morphogroup within the genus \u003cem\u003eGrevenius\u003c/em\u003e should be considered as tentative and questionable, because of the divergent configuration of the buccal apparatus, which strongly resembles the genus \u003cem\u003eHalobiotus\u003c/em\u003e, and the evidently polyphyletic nature of the genus \u003cem\u003eGrevenius\u003c/em\u003e itself (see Tumanov \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tumanov et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This problem should be solved in future using the methods of molecular phylogenetic analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eAmended diagnosis of the family Halobiotidae\u003c/h2\u003e\u003cp\u003eThe current diagnosis of the family Halobiotidae was given by Gąsiorek et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It was formulated as follows: \u0026ldquo;\u003cem\u003eMarine eutardigrades with six peribuccal lobes equipped with chemosensory organs. Two large, dome-shaped cephalic papillae present. Mouth opening surrounded by the peribuccal lamina. No ventral lamina on the buccal tube. AISM symmetrical, divided into the anterior semilunar hook and the posterior slight thickening. Claws with pseudolunulae\u003c/em\u003e\u0026rdquo;. Some of these statements need to be corrected or excluded. First, the representatives of the family are not exclusively marine \u0026ndash; \u003cem\u003eH. arcturulius\u003c/em\u003e was registered in the freshwater lake (Biserov 1999). Second, cephalic sense organs are not dome-shaped, but rather flat plates. They are definitely marked in \u003cem\u003eH. crispae\u003c/em\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;C and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC in Gąsiorek et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), being nearly completely invisible in \u003cem\u003eH. arcturulius.\u003c/em\u003e Third, AISM are symmetrical and hook-like only in \u003cem\u003eH. crispae\u003c/em\u003e, while in \u003cem\u003eH. arcturulius\u003c/em\u003e they are evidently asymmetrical and ridge-like. Fourth, presence of lunulae (pseudolunulae according to Gąsiorek et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) in our opinion should be excluded from the diagnosis, because this character can hardly be accepted as family-level, being rather variable in nearly all Eutardigrada families. On the other hand, some characters seem to be unique for the family Halobiotidae. These are the presence of the \u0026ldquo;double wall\u0026rdquo; in the anterior part of the buccal tube, presence of three rows of teeth in the OCA, and presence of three massive macroplacoids with detached caudal part of the third macroplacoid.\u003c/p\u003e\u003cp\u003eHere we propose an amended diagnosis of the family Halobiotidae: Isohypsibioidea, six peribuccal lobes equipped with chemosensory organs, mouth opening surrounded by the peribuccal lamina, no ventral lamina on the buccal tube, anterior part of the buccal tube with \u0026ldquo;double wall\u0026rdquo;, three rows of teeth in the OCA, third row in the shape of massive transverse ridges dorsally and ventrally, three massive macroplacoids with detached caudal part of the third macroplacoid, long claws with flexible main branches.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEcology of\u003c/b\u003e \u003cb\u003eHalobiotus crispae\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eH. arcturulius\u003c/b\u003e \u003cb\u003eon Arctic tidal flats\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTardigrade assemblages sampled quantitatively along two high-latitude intertidal transects were both monospecific with exclusively \u003cem\u003eH. crispae\u003c/em\u003e for the Barents Sea and \u003cem\u003eH. arcturulius\u003c/em\u003e for the Laptev Sea coast. Our findings are consistent with previous records of these species in high latitudes. Indeed, monospecific assemblages of \u003cem\u003eH. crispae\u003c/em\u003e have been documented in intertidal and shallow subtidal environments across the Holarctic. These include locations in Greenland, Spitsbergen, Arctic Canada, Norway, Alaska, Sweden, Denmark, and the White Sea (Kristensen \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; M\u0026oslash;bjerg et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Smykla et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Halberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Biserova et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Similarly, monospecific assemblages of \u003cem\u003eH. arcturulius\u003c/em\u003e have been reported from intertidal zone of Greenland, Spitsbergen, the Kara and Laptev Seas, and tidal flats of Alaska (Feder \u0026amp; Paul \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1980\u003c/span\u003e as \u003cem\u003eHypsibius appelloefi\u003c/em\u003e; Crisp \u0026amp; Kristensen \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Mokievsky \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Biserov \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). All findings of \u003cem\u003eH. arcturulius\u003c/em\u003e, including our own, were located in estuarine areas or near freshwater streams, suggesting a specific habitat preference. The prevalence of monospecific assemblages is often associated with harsh environments, such as the intertidal zone, where stress is caused by the regular fluctuation of environmental factors\u0026rsquo; impact (e.g., Kemp et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). At high latitudes, this stress is amplified by extreme climates, which can favor the dominance of highly adapted stress-tolerant species.\u003c/p\u003e\u003cp\u003eOur data revealed a stark contrast in the population densities of the two species. Densities of \u003cem\u003eH. arcturulius\u003c/em\u003e in the Laptev Sea intertidal were more than an order of magnitude higher than those of \u003cem\u003eH. crispae\u003c/em\u003e in the Barents Sea tidal flat. The Laptev Sea population of \u003cem\u003eH. arcturulius\u003c/em\u003e surpassed the maximal densities previously described for this species in other regions, such as Greenland (5.8\u0026ndash;11.6 ind./10 cm\u003csup\u003e3\u003c/sup\u003e; Crisp \u0026amp; Kristensen \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and Spitsbergen (maximum: 40 ind./10 cm\u003csup\u003e3\u003c/sup\u003e, mean density per transect 9 ind./10 cm\u003csup\u003e3\u003c/sup\u003e; Mokievsky \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), suggesting the local conditions in the Laptev Sea are more favorable for \u003cem\u003eH. arcturulius\u003c/em\u003e. In contrast, the Barents Sea site was sparsely populated by \u003cem\u003eH. crispae\u003c/em\u003e, although comparisons with other regions are hindered by the scarcity of quantitative data and inconsistent methodologies. A study from Vellerup Vig, Denmark, recorded high population densities of up to 1,300 individuals per sample but omitted sample volumes, which prevents direct comparisons (Halberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Another study conducted in Spitsbergen reported 268 specimens of \u003cem\u003eH. crispae\u003c/em\u003e found in a transect of ten 10 cm\u003csup\u003e3\u003c/sup\u003e samples, with no reported densities per sample (Smykla et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The resulting average density was 26.8 ind./10 cm\u003csup\u003e3\u003c/sup\u003e, which is more than 10 times higher than the average density of this species found in the Barents Sea. The lower abundance in our study may be related to a less preferred biotope and/or may reflect the uneven distribution of this species, which has been previously reported for \u003cem\u003eH. arcturulius\u003c/em\u003e (Crisp \u0026amp; Kristensen \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Mokievsky \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) and which is typical for Arctic meiobenthos (Szymelfenig et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Weslawski et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Urban-Malinga et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Urban-Malinga \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe highest mean tardigrade densities were observed at the lowest tidal stations on the transect in both seas. Specimens of \u003cem\u003eH. crispae\u003c/em\u003e were restricted to lower elevations and absent from the upper, vegetated zones, whereas \u003cem\u003eH. arcturulius\u003c/em\u003e displayed a broader range, extending to the upper salt marsh. Mokievsky (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) also reported peak densities of \u003cem\u003eH. arcturulius\u003c/em\u003e at the low intertidal of Spitsbergen with its sparse abundance on the other stations. The preference of both species for lower tidal elevations is likely due to more frequent and prolonged submersion during tidal cycles, which provides more favorable and stable environmental conditions. Such pattern of vertical zonation of benthic organisms is typical for Arctic intertidal communities (Thyrring et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe presence of exuviae with eggs confirms that the sampling periods captured the reproductive phase of \u003cem\u003eH. crispae\u003c/em\u003e in the Barents Sea (mid-July) and \u003cem\u003eH. arcturulius\u003c/em\u003e in the Laptev Sea (mid-September). These findings align with reports of abundant exuviae with eggs of \u003cem\u003eH. arcturulius\u003c/em\u003e in eastern Greenland from mid-July to mid-August (Crisp \u0026amp; Kristensen \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and in western Greenland from June to August (M\u0026oslash;bjerg et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These records suggest that reproduction in Arctic populations occurs from mid-summer to early autumn. In contrast, temperate-latitude populations of \u003cem\u003eH. crispae\u003c/em\u003e reproduce from mid-January to mid-April (M\u0026oslash;bjerg et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Halberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This latitudinal pattern of reproductive timing likely reflects an adaptive response to regional climatic conditions, particularly the short Arctic summer preceding the seasonal freezing of intertidal sediments, which suppresses tardigrade activity.\u003c/p\u003e\u003cp\u003eOur study revealed contrasting densities of two tardigrade species in the Arctic intertidal zone: low densities of \u003cem\u003eH. crispae\u003c/em\u003e in the Barents Sea and high densities of \u003cem\u003eH. arcturulius\u003c/em\u003e in the Laptev Sea. The high abundance of \u003cem\u003eH. arcturulius\u003c/em\u003e suggests that, despite the highly stressed high-latitude intertidal environment of the Laptev Sea, there are likely to be favorable local conditions that support high population densities. However, the factors determining the distribution and abundance of both studied species, as well as marine tardigrades in general, remain unclear and require further study.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eSupplementary materials\u003c/h2\u003e\u003cp\u003eSM.01. Uncorrected genetic \u003cem\u003ep\u003c/em\u003e-distances between the genes\u0026rsquo; sequences of the studied specimens and sequences of the genus \u003cem\u003eHalobiotus\u003c/em\u003e available in GenBank.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eSamples from the Barents Sea intertidal were obtained in 2019 during the coastal expedition under the RFBR project 18-54-20001 (SPbU, PI Andrey Granovitch). Laptev Sea samples were collected during a cruise aboard the R/V \u003cem\u003eAkademik Mstislav Keldysh\u003c/em\u003e in 2017 operated by the P.P. Shirshov Institute of Oceanology RAS. We gratefully acknowledge these research opportunities. We are also grateful to Dmitrii Mikhailov for collecting samples from the Bolshoy Begichev Island (Laptev Sea), Valeria Khabibulina (St Petersburg State University) for collecting samples from the Matrenin Island (White Sea), and to Luidmila A. Segrienko (Petrozavodsk University) and Vladislav V. Petrovsky (Komarov Botanical Institute RAS) for salt marsh plants identification. We would like to thank Reinhardt M\u0026oslash;bjerg Kristensen and Martin Vinther S\u0026oslash;rensen (Zoological Museum, University of Copenhagen) for the loan of the patatypes of \u003cem\u003eHalobiotus crispae\u003c/em\u003e and \u003cem\u003eH. arcturulius\u003c/em\u003e and additional specimens of the genus \u003cem\u003eHalobiotus\u003c/em\u003e. We also want to thank Roberto Bertolani (University of Modena and Reggio Emilia, Italy) for providing the photographs of the type specimen of \u003cem\u003eH. geddesi\u003c/em\u003e. Specimens of \u003cem\u003eGrevenius\u003c/em\u003e cf. \u003cem\u003emonoicus\u003c/em\u003e were provided by Elena Chertoprud (A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences). This study was carried out with the use of the equipment of the Core Facilities Centre \u0026ldquo;Centre for Molecular and Cell Technologies\u0026rdquo; of St Petersburg State University (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://researchpark.spbu.ru/index.php/en/biomed-eng\u003c/span\u003e\u003cspan address=\"https://researchpark.spbu.ru/index.php/en/biomed-eng\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and \u0026ldquo;Taxon\u0026rdquo; Research Resource Center of the Zoological Institute of the Russian Academy of Sciences (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ckp-rf.ru/ckp/3038/\u003c/span\u003e\u003cspan address=\"http://www.ckp-rf.ru/ckp/3038/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The study was supported by the Russian Science Foundation, grant No. 25-74-20033 \u0026ldquo;Evolutionary transformations of nanostructural elements of the Ecdysozoa cuticle using the example of the integumentary structures of tardigrades (Tardigrada)\u0026rdquo;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAstrin JJ, St\u0026uuml;ben PE (2008) Phylogeny in cryptic weevils: molecules, morphology and new genera of western Palaearctic Cryptorhynchinae (Coleoptera: Curculionidae). 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Springer-, New York\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"e3a43ed5-eec0-4c74-adc2-eb7ba9207bf3","identifier":"10.13039/501100006769","name":"Russian Science Foundation","awardNumber":"25-74-20033","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Saint Petersburg State University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"morphology, morphometry, ecology, tardigrades, Halobiotidae, White Sea, Laptev Sea, Arctic fauna","lastPublishedDoi":"10.21203/rs.3.rs-8224833/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8224833/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this paper we present the results of taxonomic and ecological investigation of several populations of marine tardigrades of the genus \u003cem\u003eHalobiotus\u003c/em\u003e inhabiting the seas of Russian Arctic. We studied representatives of the genus \u003cem\u003eHalobiotus\u003c/em\u003e collected from three localities of the White Sea, from the Barents Sea and from the Laptev Sea. Morphological analysis was performed using the methods of light and scanning electron microscopy, and supplemented with statistical analysis of the morphometric data. For the comparative genetic analysis, we obtained data on mitochondrial COI gene and on 18S rRNA, 28rRNA and ITS-2 sequences. Our study revealed the presence of two species, clearly differentiated both morphologically and genetically in the fauna of Arctic seas of Russia. Comparison of our specimens with the available type material of the previously described \u003cem\u003eHalobiotus\u003c/em\u003e species and the obtained gene sequences with those deposited in GenBank made it possible to attribute the White Sea and the Barents Sea populations to the species \u003cem\u003eHalobiotus crispae\u003c/em\u003e, while the Laptev Sea population was recognized as belonging to \u003cem\u003eHalobiotus arcturulius\u003c/em\u003e. We provide here emended morphological descriptions for both species based on the in-depth study of the obtained material together with the type specimens of both species. In our opinion, the genus \u003cem\u003eHalobiotus\u003c/em\u003e currently includes only two adequately described species, other species of this genus should be considered \u003cem\u003enomina inquirendae.\u003c/em\u003e We provide a new diagnosis for the family Halobiotidae based on the new data, and specify taxonomic position of the species \u003cem\u003eIsohypsibius occultus\u003c/em\u003e as \u003cem\u003eGrevenius occultus\u003c/em\u003e \u003cb\u003ecomb. nov.\u003c/b\u003e We also provide new biological data for the \u003cem\u003eHalobiotus\u003c/em\u003e spp. populations from the Barents Sea and the Laptev Sea.\u003c/p\u003e","manuscriptTitle":"Tardigrades of the genus Halobiotus (Eutardigrada, Isohypsibioidea) inhabiting Arctic seas – new taxonomic and biological data","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-12 13:21:46","doi":"10.21203/rs.3.rs-8224833/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5965d028-2cf4-40e2-b54c-32035f814a8d","owner":[],"postedDate":"December 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58735391,"name":"Animal Science"},{"id":58735392,"name":"Marine and Freshwater Biology"},{"id":58735393,"name":"Taxonomy"}],"tags":[],"updatedAt":"2025-12-12T13:21:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-12 13:21:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8224833","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8224833","identity":"rs-8224833","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}