Rediscovery and neotype designation of Dichaetura surreyi Martin, 1990 (Gastrotricha: Paucitubulatina) from Northern Germany with a threefold morphological examination with a specimen-saving method

Rediscovery and neotype designation of Dichaetura surreyi Martin, 1990 (Gastrotricha: Paucitubulatina) from Northern Germany with a threefold morphological examination with a specimen-saving method | 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 Rediscovery and neotype designation of Dichaetura surreyi Martin, 1990 (Gastrotricha: Paucitubulatina) from Northern Germany with a threefold morphological examination with a specimen-saving method Axell Kou Minowa, Thiago Quintão Araújo, André Rinaldo Senna Garraffoni, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7189628/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Oct, 2025 Read the published version in Zoomorphology → Version 1 posted 18 You are reading this latest preprint version Abstract The rediscovery of Dichaetura surreyi Martin, 1990 from farm ditches in Northwest Germany (East Frisia) provides new data on freshwater meiofauna. To gain the maximum amount of detailed morphological and anatomical data from the limited number of individuals, a specimen-saving methodology is employed, combining light microscopy, confocal laser scanning microscopy and scanning electron microscopy on the same specimens. This approach refined the taxonomic understanding of Dichaetura surreyi , confirming originally described morphological features, while complementing undescribed muscular architecture and cuticular characters for the Dichaeturidae family. Genetic data on mitochondrial COI locus is provided along a gene tree with members of Paucitubulatina, hinting at a close phylogenetic position to Polymerurus under Maximum Likelihood approach. The designation of a neotype and deposit of mitochondrial barcoding sequences establishes reference for future research, addressing gaps in knowledge surrounding this poorly known family and advancing understanding of freshwater meiofaunal diversity. Freshwater Biodiversity Invertebrates Synanthropic Fauna Meifauna 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 INTRODUCTION The Northwestern German lowlands feature a diverse range of geographical physiognomies, including marshlands along the coastline of the North Sea intertwined with slightly higher leveling areas of so-called ‘Geest’, former pleistocene deposits with predominantly sandy and barren soils, and with vast moor areas (Pott 1999 ; Becker and Siegmüller 2021 ). Historically, to manage rainwater and groundwater seepage, medieval settlers established a network of channels and ditches over centuries to drain the terrain and enhance agricultural land use, such as meadow culture (Herzon and Helenius 2008 ; Verdonschot et al. 2011 ), but also for mining peat as a fuel (Hadler et al. 2025 ). These channels serve as water buffers, removing excess water during the wet season and retaining it during dry periods (Armitage et al. 2003 ). However, in the present research area, their primary function was to regulate the drainage of inland water in conjunction with the closed line of the coastal dikes, tides and storm floods (Behre 2012 , 2014 ). Contrary to the long-standing assumption that a high aquatic biodiversity is primarily found in larger water bodies, such as rivers and lakes (see, e.g., Boix et al. 2012 ), these drainage systems and small streams are not “deserts” for aquatic biota (Davies et al. 2008 ), and recent studies have shown to be important reservoirs for biodiversity, providing habitats for a surprising variety of aquatic organisms and even harboring rare species (Painter 1999 ; Malmqvist and Hoffsten 2000 ; Oertli et al. 2002 ; Armitage et al. 2003 ; Williams et al. 2004 ; Nicolet et al. 2004 ; Biggs et al. 2005 ; Heino et al. 2005 ; Davies et al. 2008 ; De Bie et al. 2008 ; Chester and Robson 2013 ; Clifford and Heffernan 2018 ), despite, or perhaps because of , the heavy human usage interference, as exemplified in the current study area by the smell of manure treatment of the surrounding pastureland. The various invertebrate taxa commonly found in man-made ditches include macroscopic animals, such as hoverfly larvae, water beetles, and gastropods (e.g., Watson and Ormerod 2004 ; Herzon and Helenius 2008 ). However, relatively few studies have assessed these communities involving multiple taxa or different trophic levels (see Twisk, Noordervliet and Keurs 2000, Armitage et al. 2003 , Davies et al. 2008 ), and even fewer studies gave attention to the community of microscopic invertebrates found in ditches (Clare and Edwards 1983 ; Richards et al. 1993 ). However, growing evidence suggests that microinvertebrates play a crucial ecological role in diverse interactions within matter and energy networks, acting as intermediaries between macrofauna and microbial loops (see Schratzberger and Ingels 2018 ), but still there is a considerable gap in knowledge of the benthic microinvertebrate community, known as meiobenthos or meiofauna, in these man-made environments (e.g., De Bie et al. 2008 ; Mioduchowska and Wojtasik 2009 ). The freshwater meiofauna assemblages typically comprise several interstitial taxa, where nematodes, rotifers, copepods and annelids are relatively inventoried (Peralta-Maraver et al. 2023 ), while some taxa, such as Gastrotricha, are still insufficiently studied in artificial environments (Franz 1950 ; Franz and Donner 1954 ; Kolicka et al. 2013 , 2016 ; Suzuki et al. 2013 ; Kolicka 2016 ; Minowa and Garraffoni 2025 ). Regarding our current study area and the particular biotope, i.e. artificial agricultural, urban and rural drainage ditches in Northwest Germany, a single study yielded the second record of a rare freshwater dwelling gastrotrich after its original description from a central Italian lake (Kieneke and Riemann 2008 ). Gastrotricha is traditionally divided into two main taxa: Macrodasyida Remane, 1925, composed of mostly vermiform or tongue-shaped animals bearing several adhesive tubes along the body axis and occurring mostly in the interstice of marine sand; and a possibly paraphyletic order Chaetonotida Remane, 1925, further divided into monogeneric suborder Multitubulatina d’Hondt, 1971 (represented by three marine species of Neodasys Remane, 1927 within family Neodasyidae Remane, 1929) and monophyletic suborder Paucitubulatina d’Hondt, 1971, represented by 512 either marine or freshwater species of 33 genera within seven families (Balsamo et al. 2019 ). Recent phylogenetic studies on Paucitubulatina support its monophyly, comprising the families Xenotrichulidae plus Muselliferidae as the sister group of a clade Oiorpatra formed by the remaining families Chaetonotidae, Dasydytidae and Neogosseidae and a few less species-rich families (Bekkouche and Worsaae 2016 ; Kolicka et al. 2020 ; Gammuto et al. 2024 ). However, several phylogenetic studies endorse the non-monophyly (polyphyly) of the hyperdiverse family Chaetonotidae, encompassing both Dasydytidae and Neogosseidae families nested within (Hochberg and Litvaitis 2000 ; Kieneke et al. 2008b ; Todaro et al. 2012 ; Kånneby 2013 ; Bekkouche and Worsaae 2016 ; Kolicka et al. 2020 ; Minowa and Garraffoni 2020 , 2022 ), rendering “Chaetonotidae” phylogenetically synonymous (i.e. same last common ancestor) to the whole Oiorpata clade (Gammuto et al. 2024 ). Nonetheless, the families Proichthydidae and Dichaeturidae are still omitted in molecular studies because of lack of molecular data. Morphological evidence suggest that Dichaeturidae is deeply nested within Paucitubulatina, with either a close affinity to the genus Ichthydium (Kieneke et al. 2008), or to a clade comprising the benthic Proichthydidae and the planktonic Neogosseidae and Dasydytidae families (Hochberg and Litvaitis 2000 ). Members of the freshwater family Dichaeturidae rank among the rarest gastrotrichs known (Balsamo et al., 2014 ). Metschnikoff ( 1865 ) originally established the genus Chaetura to allocate its first species Chaetura capricornia Metschnikoff, 1865 . Later, Murrey (1913) added a second species Chaetura piscator Murray, 1913 , a species never to be seen again. Lauterborn ( 1913 ) subsequently renamed the genus to Dichaetura to resolve a nomenclatural conflict with a genus of the bird clade of swifts (Apodidae), resulting in Dichaetura capricornia (Metschnikoff, 1865 ) and Dichaetura piscator (Murray, 1913 ). Martin ( 1981 ) examined specimens that he initially believed as belonging to the former species, however, upon further study (Martin 1990 ), he recognized distinct morphological differences and described these specimens as a new species, and formally established Dichaetura surreyi Martin, 1990 . Two decades passed before the discovery of a fourth species, Dichaetura filispina Suzuki, Maeda et Furuya, 2013. Notably, all known records of Dichaetura species ( D. filispina , D. capricornia , and D. surrey ) come from artificial or anthropogenically influenced habitats, such as rice paddies, open-air cisterns, rural ditches, and the surroundings of metropolitan regions (e.g., Kharkiv). Each record of all four species documents very low abundance, with some descriptions based on a single specimen (e.g. Metschnikoff 1865 ; Suzuki et al. 2013 ). Consequently, our understanding of their biology, life cycle, and distribution remains poorly understood. Research on these tiny invertebrates also faces inherent challenges due to their fragile nature and diminutive size, often causing specimens to be lost or destroyed during preparation procedures and examination. Additionally, preserved specimens inevitably deteriorate over time, with numerous diagnostic traits dissolving or becoming irretrievable after fixation (Giere 2009 ; Fonseca et al. 2018 ; Garraffoni et al. 2019b ). As a result, it becomes essential to extract as much information as possible, ideally following an integrative taxonomy framework (see Dayrat 2005 ), from the limited number of specimens to make it digitally accessible (Garraffoni et al. 2019b ). To address the notably low abundance of the Dichaetura surreyi in our samples, we employed a triple morphological investigation by retrieving the specimen from each method. First, we conducted detailed differential interference contrast (DIC) light microscopy on living specimens. We then performed fluorescence staining of the musculature, nuclei and cilia for observation under confocal laser scanning microscope (CLSM). Finally, we examined the same individual with a scanning electron microscope (SEM). We propose that this ‘line of production’ can be adapted for other soft-bodied micrometazoans to increase the yield of morphological and anatomical data from single specimens, particularly in cases where specimens are rare and delicate or difficult to obtain. Our integrative investigation of specimens of D. surreyi revealed further details of the cuticular armament which justify a redescription of the species. Furthermore, newly observed aspects of the species’ musculature are shedding light on the evolution of Paucitubulatina lineages and calling into question a longstanding understanding of a “second pair of adhesive tubes” on the furca of Dichaeturidae. METHODS Sampling and morphological methods Freshwater samples containing floating plants ( Lemna spp.) and submerged roots of vascular plants growing along the trench edge were collected from ditches in Logabirumer Hammrich, Leer, Northern Germany (53°13'59''N–53°14'11''N; 7°31'19''E–7°31'20''E) (Fig. 1 ) in May 15 and July 22, 2023, using a 65-µm mesh plankton net mounted on an extendable handle made of fiberglass-reinforced plastic. The filtrate was transferred into a sampling jar with ambient water using a squirt bottle for rinsing the plankton net and its collecting jar. Subsamples were transferred to Petri dishes and screened under stereomicroscopes with zoom optics (Leica M80 and M125), employing various illumination modes (substage bright field, oblique illumination and dark field) and magnifications. For microscopic examination and documentation, single specimens were isolated from the Petri dish using a capillary mouth pipette and placed on glass slides with a drop of ambient water. They were covered with a coverslip, and gently clamped by carefully removing the excess water from the edge of the coverslip using a snippet of filter paper. Observations and digital recordings were made using an Olympus BX53 microscope equipped with high-resolution objectives and DIC, and a Euromex HD-Ultra digital camera VC.3036-HDS. After documentation, each specimen was anesthetized with a few microliters of 0.25% Buccain (PUREN Pharma GmbH & Co.KG, München, Germany) before being fixed in freshly prepared 4% formaldehyde buffered in 0.1M Phosphate-buffered saline (PBS, pH 7.4) at 4°C for one hour, for further fluorescence studies according to Kieneke et al. ( 2008a ). For the examination with the CLSM, each specimen was thoroughly rinsed in 0.1M PBS at pH 7.4 to remove any remnants of the fixative. Each animal was incubated overnight in a 0.1% (v/v) Triton-X-100 solution in 0.1 M PBS for permeabilization. They were stained in a TRITC-phalloidin (#P1951, Sigma-Aldrich, Saint Louis, USA) solution (2 µl of 38 µmol/L TRITC-phalloidin in methanol, diluted in 100 µl of permeabilization buffer after evaporation of the methanol) for 3 hours at 4°C. Some specimens were counterstained with SYBR-Green I (#S9430, Sigma-Aldrich, Saint Louis, USA) nucleic acid stain (one microlitre of 10,000x stock solution added to the phalloidin solution) for another hour. A single specimen was counterstained immunohistochemically against acetylated ∝-tubuline. The indirect immunohistochemical staining was carried out with the primary antibody (monoclonal anti-∝-tubuline IgG2b, produced in mouse clone 6-11B-1, Sigma-Aldrich, Saint Louis USA, cat. nr. T7451) incubated at a dilution of 1:500 (in 0.1 M PBS with Triton-X-100; see above) and for 24 hours at 8°C, followed by the secondary, fluorescently labelled antibody (anti-mouse IgG-FITC-conjugate, produced in goat, Sigma-Aldrich, Saint Louis USA, cat. nr. F0257) incubated at a dilution of 1:100 and for 2 hours at 20°C. In the case of the immunohistochemical staining, a modified PBS buffer containing bovine serum albumin (0.2% w/v), goat serum (6% v/v), Triton-X-100 (0.5% v/v) and NaN 3 (0.05% w/v) as preservative was used to pre-incubate the specimen. All stainings were halted by thoroughly rinsing the specimens several times in 0.1M PBS. Individual specimens were mounted on microscopic slides with a hydrosoluble CitiFluor™ CFP mounting medium plus CitiFluor™ AF100 antifadent solution (#E17978-35, Science Services, Munich, Germany) with binder ring reinforcement stickers as spacers for the coverslip (See Fig. 1 of Michels and Büntzow 2010 ). Observation was carried out with a Leica TCS SP 5 confocal laser scanning microscope on a DM5000B upright microscope base. We operated the CLSM with the argon laser (488 nm) for excitation of SybrGreen I and Alexa 488 and the DPSS laser (561 nm) for excitation of TRITC using the sequential scanning modus for both detection channels, detection bandwidths of the two photomultipliers were set to 507–534 nm and 570–670 nm, respectively. Each specimen was subsequently recovered from the slide for further investigation by SEM by dissolving the mounting medium again with 0.1M PBS and lifting the coverslip, gently pushing it aside or carefully removing it with Dumont No. 7 precision tweezers, and then retrieving the animal with a copper wire loop attached to a skewer with nail polish. The image stacks of the fluorescently stained specimens were either processed as maximum intensity projections and virtual orthogonal sections with the proprietary LAS AF software of Leica (Leica Microsystems GmbH, Wetzlar, Germany), or volume-rendered as well as surface-rendered using the IsoSurface and Voltex algorithms of the Amira 5.0.0 software (Visage Imaging, Inc. San Diego, USA) and Imaris x64 v. 9.9.0 software (Oxford Instruments, Oxfordshire, UK). For the multiple staining datasets, different combinations of visualisation tools have been applied. These processed CLSM datasets have been the basis for a graphical reconstruction of the myoanatomy of D. surreyi , carried out as vector drawings. Prior to SEM investigation we applied a well-established preparation method (Kieneke and Zekely 2008 ; Kieneke et al. 2015 ; Trokhymchuk and Kieneke 2024a , b ). The animals recovered from the slides for CLSM were placed inside small tube-shaped metal containers (invented by Dr. Wilko Ahlrichs, Oldenburg, Germany) of stainless steel with both ends closed by two copper transmission electron microscopy grids (1500 mesh, hole size 9 µm, catalog number #T1500-CU, Koch Electron Microscopy, São Paulo, Brazil, and #G2670C, Plano GmbH, Wetzlar, Germany), and immersed in 5% ethanol solution. Dehydration was carried out by increasing ethanol solutions in distilled water (5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100% [v/v]; 10 minutes in each step). Critical point desiccation was carried out with a Leica CPD300 automatic critical point dryer using ethanol as intermedium. The desiccated specimens were mounted on a round glass coverslip (⌀ 12 mm) coated with a thin layer of TempFix mounting resin (catalog number #G3305, Plano GmbH, Wetzlar, Germany) using an eyelash glued to a wooden rod. The coverslip with mounted specimens was gently and shortly heated up to 50°C on a heat plate to adhere the specimen to the surface. The thin layer of TempFix was obtained by dissolving a 125 mm³ block of TempFix in 5 ml of chloroform, vigorously mixing it on a single tube vortex machine until completely dissolved, then adding 45 ml of chloroform to sum 50 ml of final solution. A volume of the solution to completely cover the round coverslip was applied with a Pasteur pipette and left to evaporate at room temperature, subsequently heated to 120°C for 60 seconds to smoothen the surface and let it cool to room temperature again before placing the specimens on it. After placing each specimen, the coverslip was adhered to a SEM stub using double-sided adhesive pads and specimens were coated with palladium-gold in a Bal-Tec SCD-050 sputter coater, operated within 150 seconds at 40 mA. SEM examination was performed with a Tescan VEGA3 Scanning Electron Microscope at the Institute Senckenberg am Meer (Wilhelmshaven, Germany), using acceleration voltages of 10 and 30kV and both the secondary and the backscattered electron detectors. Molecular data For tissue lysis and DNA extraction, two fresh specimens were added to 35µL of InstaGene™ Matrix (Catalog # 732–6030, Bio-Rad Laboratories, Hercules, USA) and incubated at 56°C for 20 min and subsequently at 100°C for 10 min. 1 µl of the DNA-containing supernatant was used as a template for the amplification. PCR reactions were prepared with illustra™ PuReTaq™ Ready-To-Go™ PCR beads (GE Healthcare UK, Little Chalfont, UK) using a final volume of 25 µl (1 µl of each primer at 10 µM, 1 µl of template DNA and 22 µl of cleaned water for molecular biology purpose). We used universal primers LCO1490 and HCO2198 for amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene (Folmer et al., 1994). The parameters for PCR were as follows: an initial denaturation of 3 min at 94°C was followed by 40 cycles of 0.5 min at 94°C, 1 min at 48°C and 1 min at 72°C, with an additional final elongation step of 7 min at 72°C. Each PCR reaction product was checked for length and approximated DNA yield by running 1 µL of the PCR product on 1% agarose gels with 1x nucleic acid stain GelRedTM (Biotium Inc., Hayward, CA, USA). Phylogenetic analyses All available Chaetonotida sequences of the COI gene were retrieved from GenBank ( https://www.ncbi.nlm.nih.gov/genbank/ ), with accession number provided in Supporting Information (Supplementary Table S1 ). The mitochondrial protein-coding COI sequences were aligned in MEGA X (Kumar et al. 2018 ) using the Invertebrate Mitochondrial genetic code and the MUSCLE algorithm for codon-based alignment. The aligned sequences were subsequently trimmed on their ends in BioEdit v.7.2.5 (Hall 1999 ) to achieve a uniform length. The final alignment comprised 662 nucleotide sites for 211 terminal sequences covering 79 species of Paucitubulatina. Phylogenetic reconstruction was conducted using the Maximum Likelihood (ML) approach in IQ-TREE v.1.6.10 (Nguyen et al. 2015 ). The best-fit substitution model was estimated with ModelFinder (Kalyaanamoorthy et al. 2017 ) under Bayesian information criterion and Akaike information criterion, selecting GTR + F + I(= 0.3191) + Γ 4 (= 0.6608). Branch support was assessed using ultrafast bootstrap approximation with 1,000 pseudo-replicates (Hoang et al. 2018 ) and Shimodaira-Hasegawa approximate likelihood ratio test with 1,000 replicates (Guindon et al. 2010 ). The resulting trees were rooted using the midpoint method in FigTree v1.4.3 ( http://tree.bio.ed.ac.uk/software/figtree/ ). Data accessibility Microphotographs of the neotype and four additional specimens were deposited in the Museu de Diversidade Biológica (MDBio) at University of Campinas (Campinas, Brazil) under accession number ZUEC-PIC 1207 and ZUEC-VID 1345 (neotype) and ZUEC-PIC ZUEC-PIC 1206, 1208–1210 and ZUEC-VID 1344, 1346–1348 (additional material). Following the suggestion of Kieneke & Nikoukar ( 2017 ), the four additional specimens shall serve as novel paratypes (“neo-paratypes”), a category that is not yet included and defined by the ICZN (international code of zoological nomenclature) but considered a quite helpful category of type specimens. The neotype specimen and the second specimen used for DNA extraction are only present as a series of digital micrographs (ZUEC-PIC 1206, 1208). The admissibility of specimens only available as a digitalized version as type specimens is comprehensively discussed in Garraffoni et al. ( 2019b ). The four gold-palladium-coated animals on an aluminum-SEM-stub are deposited in the Gastrotricha collection of the Senckenberg Research Institute and Natural History Museum (Frankfurt am Main, Germany) under accession numbers SMF XXXX-YYYY. Results Taxonomic accounts Phylum Gastrotricha Metschnikoff, 1865 Order Chaetonotida Remane, 1925 [Rao & Clausen, 1970] Suboreder Paucitubulatina d'Hondt, 1971 Family Dichaeturidae Remane 1927 Genus Dichaetura Lauterborn, 1913 Dichaetura surreyi Martin, 1990 (Figs. 2 – 11 ; Suppl. Fig. S1 –3; Table 1) Emended diagnosis Elongated, cylindrical body measuring 168–210 µm in total length, including the furca. Head region wide, with a weakly narrowed neck, and slightly broader trunk tapering to a narrow caudal region. Caudal furca 23–29 µm in length, with a cylindrical adhesive tube with almost constant width, only tapering weakly to blunt distal tip, and with a central groove from base to distal tip. Pharynx with one indistinct pharyngeal thickening on the posterior portion, connected to a cylindrical intestine by a subtle pharyngo-intestinal junction. Pharynx flanked by a pair of pharyngeal glands probably opening at the lateral edges of the buccal cavity and widening to a droplet-shaped bulb at the posterior portion of the pharynx. Head rounded, with a large cephalion and small dorsolateral, triangular pleurae and a ventral transverse rod-shaped hypostomion. Mouth opening round, supported by strong, thorn-like braces protruding slightly from the buccal cavity. Two pairs of dorsal sensory bristles insert directly into the cuticle at the anterior head. Sensory bristles absent from neck and caudal regions. Dorsal body surface is covered with 12–19 longitudinal columns of 50–70 small round and short-spined scales with almost constant size along the body. A patch of rounded scales with elongated spines arranged in 7 columns of 6–7 scales on the posterior dorsal trunk. Ventral interciliary field covered by 8 longitudinal columns of 50–60 small, roundish, smooth scales, each with a posterior free margin extending into a sharp tip. A triangular patch of 5 transverse rows with 4 large keeled scales with a long posterior spiny process at the posterior ventral trunk. Terminal plates consist of a pair of keeled elongated scales with prominent posterior spines. Each furcal branch is medially armed with a large, curved, hook-like scale. A single, unpaired scale with a thick blunt-end spine is present medially at the furcal indentation, posterior to the terminal plates. Morphological redescription This redescription and the ranges of measurements are based on 5 adult specimens. The species has an elongated oval to cylindrical body with 168–212 µm in total length, with wide head (24–29 µm wide at U04), a weakly narrowed neck (22–27 µm wide at U17), and a slightly wider trunk (19–36 µm wide at U50) with narrower caudal region (Figs. 2 – 5 ). The caudal furca is 23–29 µm in length, with cylindrical, 11–17 µm long adhesive tubes, of almost constant width of 1–2 µm, only tapering gradually towards their distal blunt tips (Figs. 3 E, 4 C, 5 C, 6 , 7 B). The adhesive tubes are grooved from the base to the distal tip (Figs. 6 B, 9 E). The pharynx is 36–50 µm in length from the posterior edge of terminal mouth to pharyngo-intestinal junction at U26 (7–13 µm wide) with posterior pharynx thickening of 14–18 µm wide (U17). Posteriorly, the pharynx is followed by a cylindrical intestine of 89–119 µm length, which is slightly narrowing posteriorly (Figs. 3 , 4 ). The pharynx is laterally flanked by paired pharyngeal glands from U03 to U15, with unknown function (Figs. 2 , 3 A, B). The glands insert anteriorly to the lateral edges of the mouth ring, probably discharging into the buccal cavity, and widen to droplet-shaped bulbs at the posterior portion of the pharynx (Figs. 2 , 3 A, B). The animals have a rounded head with a clearly visible large cephalion (12–19 µm long x 13–20 µm wide) with rounded anterior edge, and two small lateral protrusions, followed by convex posterior edge (Figs. 2 , 3 A, 8 , 9 B). Furthermore, one pair of rather small triangular dorsolateral pleurae with fingerprint-like surface texture posterior to the cephalion and partly covered by its posterior-lateral edges (Fig. 8 A, B), and a transversal, rod-shaped hypostomion on the ventral side of the head (3–4 µm long x 8–10 µm wide) (Figs. 5 A, B, 7 A, 8 C, 9 C). The round mouth opening (6–10 µm diameter) is supported by strong and thorn-like braces that slightly emerge from the buccal cavity. Two paired dorsal sensory bristles (10–17 µm long) insert directly to the cuticle at U03 (Figs, 3B, 4B, D). There are no tactile bristles on the neck region, but one pair of tactile bristles on the rear end (Fig. 9 E). The dorsal head and body are covered by 12–19 longitudinal columns of 50–70 small (3.0 µm long x 1.0 µm wide at U17) round scales, each with a thin and short spine (2.7 µm from posterior scale edge to tip), with constant shape along the body axis, only slightly varying in size, smallest on the dorsal head, and increasing in size toward the pharyngeal region and mid trunk (3.6 µm long x 0.7 µm wide at U77) (Fig. 3 , 4 ). On the dorsal posterior trunk, approximately between U84 and U89, there is a patch of small rounded scales each with a thin elongate spine (12–15 µm), arranged in 7 columns of 6–7 scales each (Figs. 6 A, 7 C, D, 9 A–D). The ventral interciliary area is covered by 8 longitudinal columns of 50–60 small (3.6 µm long x 0.7 µm wide) roundish smooth scales with posterior portion free and extending toward a sharp tip (Figs. 6 A, 7 C, D). In the posterior ventral surface around U95, there is a triangular patch of 5 transversal rows with 4 long keeled scales with a long spiny process (Fig. 6 B, C), all distinctly larger (7.4 µm long x 1.3 µm wide) than usual ventral interciliary scales. A pair of terminal, keeled, long, and elliptical scales at U98 with a long, spiny posterior projection (i.e. terminal plates) (Fig. 6 C). Each branch of the furca’s base is medially armed with one strong and large scale with a thick curved and hook-like spine, laterally on the furcal branches there are tiny scales similar to those of the trunk (Fig. 6 B, C). Medially at the furcal indentation, posterior to the terminal plates, there is a single, unpaired scale with a thick spine with a blunt end (Fig. 5 C, 6 C). Ventral ciliation consists of a dense field of cilia posterior to the mouth (U03), continuing into two parallel bands on each side of the trunk (Figs. 6 A, 7 A, C, D, 9 A). The distance between both ciliary bands (= width of the interciliary field) is narrowing toward the posterior trunk end and measures 4.98 µm, 10.5 µm, and 4 µm at U11, U49, and U83, respectively (Figs. 6 A, 9 A). Fluorescent staining in combination with CLSM furthermore shows that cilia in the pharyngeal region also insert on the lateral sides and almost form an anterior ring of cilia that is dorsally interrupted, though (Fig. S3A–C). Phenotypic variance The shape and general characteristics among the five specimens studied are congruent, with only little total size or specific feature length variance (Table 1). However, we were able to distinguish two variants regarding differences in dorsal/dorso-lateral and ventral cuticular ornamentation that were only detectable with the resolution power of the SEM. One variant (Fig. 3 A–D) has scales with shorter, delicate spines of 3.0–4.1 µm lengths covering the dorsal, lateral and ventral body (Figs. 6 , 7 ). The other variant (Fig. 4 ) has scales bearing slightly longer and thicker spines of 5.6–7.3 µm lengths (Figs. 8 , 9 ). Myoanatomy The description of the muscular anatomy of Dichaetura surreyi is based on three adult specimens examined with the CLSM. The muscular architecture in somatic and splanchnic regions consisted of longitudinal, circular, and helicoidal muscles (Figs. 10 – 11 ). In the splanchnic region, up to 35 complete circular muscles occur along the pharynx (Fig. 11 A–C). Complete circular muscles are absent in the intestinal region. Helicoidal muscles are very thin and extend from the mouth ring up to the mid-body, wrapping all splanchnic longitudinal muscles plus one splanchnic branch of the otherwise somatic ventrolateral longitudinal muscle on the pharynx region (Fig. 11 A–C, G). Longitudinal muscles are present in dorsal, ventral, and lateral positions and either splanchnic or somatic, and mostly span the entire body length, insert anteriorly in the head region close to the mouth opening and extend posteriorly into the rearmost region of the trunk, roughly at the position of the anus, where they fuse in a single complex (Fig. 11 A–C). Only the ventrolateral longitudinal muscles extend into the furcal branches (see below). Two or three short muscle fibres occur close to the ventral anterior part of the pharynx at U02, probably inserting into the mouth opening at the ventral body. Dorsally, there are two pairs of dorsal longitudinal muscles. The first pair of these muscles is inserted anteriorly on the mouth ring lining up the entire dorsal body and possibly connecting each other at U04. This dorsal longitudinal muscle branches posteriorly in a pair of thin muscles ( Rückenhautmuskel ) at U72, which, in turn, is inserted dorsally to the integument at U41 (Fig. 11 A, F–I). These muscles, apart from the Rückenhautmuskel -branch, are splanchnic and warped by helicoidal muscles in the pharynx region but are not in the intestine region. A second pair of dorsal longitudinal muscles is anteriorly inserted to the head integument close to the insertion point of the first muscle pair, and extends in a splanchnic position along the entire gut tube (Fig. 11 A, F–I). Both dorsal longitudinal muscles seem to fuse a short distance posterior to the anus. Ventrally, there are three pairs of longitudinal muscles. A pair of ventrolateral longitudinal muscles inserts slightly ventrolateral in the furcal base and continues a short distance into the proximal portion of the adhesive tubes. Each muscle stretches along the trunk in a somatic position up to the posterior swelling of the pharynx close to the pharyngeal-intestinal junction (Fig. 11 A–C, E–I). At about U29 each ventrolateral longitudinal muscle splits into two branches. The more lateral branches insert laterally to the head integument at U07, while the more median branches follow closely the contours of the pharynx, insert in the integument close to the anterior end of the pharynx and are enwrapped by splanchnic circular and helicoidal muscles (Figs. 10 A, B, 11 A–B, E). Hence, parts of the ventrolateral longitudinal muscle pair occupy a somatic position, while others are splanchnic. There is a pair of ventral longitudinal muscles that is anteriorly inserted into the integument close to the mouth opening, splanchnic in the pharyngeal region, where it is warped by helicoidal muscles, and somatic in the intestine region. A second pair of ventral longitudinal muscles, slightly more median than the latter pair, is inserted anteriorly to the head integument and stretches along the entire gut tube, always remaining in a splanchnic position (Fig. 11 B, F, H–I). Both pairs of ventral longitudinal muscles fuse with the pair of ventrolateral longitudinal muscles in the region of the anus at U97 (Figs. 10 B, 11 I). Two pairs of smaller dorsal muscular branches emerge from the ventrolateral longitudinal muscles at U97 and insert dorsally and slightly more posterior at U98, respectively. Both branches are inserted in the integument at the level of the posteriormost patch of long and thin spines. Nuclei distribution The cellular nuclei distribution of Dichaetura surreyi is described from SYBR-Green I nucleic acid staining and reflects a general architecture within Paucitubulatina, with high density of nuclei in the anterior third of the body (pharyngeal region) and especially dorsally and dorsolaterally of the mid-pharynx region (Fig. S1 C). This dense arrangement of cell nuclei corresponds with the two hemispheres of the bilateral cerebral ganglion of Gastrotricha. Areas without any DNA-signals within this dorsal to dorso-lateral aggregation of nuclei much likely reflect the dorsal commissure(s) inside the cerebral ganglion of D. surreyi (Fig. S2A, B). Also at the posterior end there is a higher density of nuclei, probably associated with adhesive glands Fig. S2A–C). The egg was diffusely stained and fills the whole dorsal trunk (Figs. S1C, D, S2A, C), this fluorescence pattern possibly indicates a high level of gene expression since Sybr Green I also stains single-stranded DNA and RNA. Along the trunk there are only scattered nucleic patterns that probably belong to muscle, epidermis and intestinal cells (Fig. S2A–C). Taxonomic Remarks The original description and the newly recorded animals both have an elongated, cylindrical body shape, tapering towards the caudal region and only with a weak depression of the pharyngeal region. However, present specimens tend to be larger (168–212 µm in total length; current study) compared to the original description (102–150 µm overall length; Martin 1981 ). The pharynx dimensions of the original description (38 µm) fall within the newly recorded range (36–50 µm), the same is true for the head width (27 µm vs 24–29 µm). Furthermore, both sets of specimens exhibit similar patterns of the dorsal scales, with the posteriormost patch of about 70 small round scales with long, thin, slightly curved simple spines. Also the peculiar hook-shaped spined scales at the medial faces of the furca show a highly similar pattern between the original description and the specimens investigated by us. Dichaetura surreyi is well delineated from the most similar species Dichaetura filispina by the presence of numerous finely spined scales on the dorsal and lateral surfaces of the whole trunk, which are absent from the latter (Suzuki et al. 2013 ; Balsamo et al. 2014 ). Despite slight variance in cuticular ornamentation among the five specimens from the identical sampling site, all measurements and myoanatomical features are congruent, strongly indicating a single entity. The original description of Dichaetura surreyi of Martin ( 1981 ) is missing several data on structures that are recently important for a proper morphological species delineation. We were able to provide detailed counts of scale columns and the number of scales per column and to resolve the shape of the trunk scales. Furthermore, our measurements of the cephalion and hypostomion are also detailed in the new specimens, not mentioned at all in the original description, and we were able to confirm the presence of one pair of pleurae on the head section. We were also able to clarify the morphology of the basal scales bearing the hook-shaped spines at the medial margins of the furca. Therefore, providing a redescription of D. surreyi is well-justified. Designation of a neotype. The present study allows us to deposit a neotype specimen of Dichaetura surreyi according to article 75 of the International Code of Zoological Nomenclature/ICZN (International Commission on Zoological Nomenclature, 1999). Upon publication of this study, the digital neotype specimen (Fig. 3 A–D, 6 , 10 ) will be added to the scientific collection of the Museu de Diversidade Biológica (MDBio) at University of Campinas under the collection number ZUEC-PIC 1206. A digitized fresh specimen facilitates the best long-term preservation over every other method linked to a physical specimen (see Garraffoni et al. 2019). According to the ICZN, a number of qualifying conditions need to be fulfilled in order to designate a name-bearing neotype validly (Article 75.3 ICZN). We will now present our qualifying conditions to support the designation of a neotype specimen of D. surreyi . Because of the incompleteness of the original description (see ‘taxonomic remarks’ of the current study), a future delineation from further similar but distinct species cannot be guaranteed and could lead to taxonomic confusion. We designate the neotype specimen in order to corroborate the taxonomic status of D. surreyi (Article 75.3.1 ICZN). As can be read in Balsamo et al. ( 2014 ), the body cuticle made of numerous fine spined scales distinguishes D. surreyi from other species of Dichaetura (Article 75.3.2 ICZN). The designated neotype specimen is a digitized individual that was imaged alive (see above; Fig. 3 A–D, 6 , 10 ). Its collection number is ZUEC-PIC 1206 (Article 75.3.3 ICZN). Furthermore, DNA sequences of the ‘neoparatype’ specimens with the accession numbers 1206 and 1208 are available. Martin ( 1981 , 1990 ) nowhere mentioned the deposition of name-bearing type specimens, neither a holotype, nor a syntype series. As far as we know, the author had no institutional affiliation and no access to a museal scientific collection and it is plausible that type specimens were never preserved and deposited (Article 75.3.4 ICZN). Therefore, we regard the drawing of D. surreyi in Martin’s ( 1981 ) Fig. 4 as the holotype specimen that no longer exists (Article 73.1.4 ICZN). A comparison of our Fig. 2 and Fig. 4 of Martin ( 1990 ) demonstrates consistency between the original holotype specimen and the designated neotype specimen (Article 75.3.5 ICZN). The designated neotype specimen was sampled from quite a distance to the original type locality (northwest Germany versus southeast England), however, both locations are in the same climatological and vegetational zone and only a few millennia ago the British Island and Europe were connected by a wide land bridge (Gupta et al. 2017 ). Therefore the origin of the neotype specimen is suitably close enough to the original type locality (Article 75.3.6 ICZN). Both the no longer existing holotype specimen and the designated neotype specimen were furthermore extracted from similar habitats, i.e. the surface mud of stagnant water bodies and among floating and submerged vegetation (Martin 1981 , present study). The designated digital neotype specimen is the property of the scientific collection of the Museu de Diversidade Biológica (MDBio) at University of Campinas under the collection number ZUEC-PIC 1207 and is available for further scientific studies (Article 75.3.7 ICZN). Phylogenetic analyses The phylogenetic analysis of 211 COI sequences from the suborder Paucitubulatina resulted in a best-scoring ML topology with a log-likelihood of -33200.0016 (Fig. 11 ). The two Dichaetura surreyi terminals formed a fully supported clade and is placed as a sister group to Polymerurus species with weak support. In turn, this clade is the sister lineage to Dasydytidae and Neogosseidae. After midpoint rooting, the inferred phylogeny showed Xenotrichula sp. (TK2012) as sister group of the clade comprising Draculiciteria tesselata (MT63 and TK142) and the remaining members of Paucitubulatina included in our analysis. Some regions of the tree have quite long branches and some species that were included with multiple sequences even show comparatively long intraspecific branches (e.g., Polymerurus nodicaudus , Chaetonotus daphnes , Dendroichthydium ibryapora ), while others have almost no intraspecific divergence (e.g., Chaetonotus slavicus , Chaetonotus mirabilis , Heterolepidoderma acidophilum ). DISCUSSION Morphology and systematics of Dichaeturidae Updating knowledge as new evidence emerges is an integral part of the scientific process, and SEM imagery of Dichaetura surreyi furcal branches can help reinterpret earlier descriptions. Regarding the taxonomic history of the group, the most distinctive trait in Dichaeturidae has long been regarded as the presence of a “second pair of adhesive tubes on the furca” (Metschnikoff 1865 ), a condition otherwise known only in Diuronotus Todaro, Balsamo et Kristensen, 2005 (Muselliferidae) and, with even two additional pairs of ‘accessory posterior adhesive tubes’, in Aspidiophorus multitubulatus Hummon, 1974 (Chaetonotidae) among Paucitubulatina (Hummon 1974 ; Todaro et al. 2005 ). However, Martin ( 1981 ) describes these as “a hook [that] arises from the inner side of each toe and projects upwards at an angle”, rather than functional adhesive tubes. Also Todaro et al. ( 2005 ) question the existence of a second pair of posterior adhesive tubes in Dichaetura . This condition was later supported by the description of D. filispina , which bears one pair of adhesive tubes and a solid spine on each furcal branch (Suzuki et al. 2013 ), reaffirming the doubts about the existence of a second pair of adhesive tubes in Dichaeturidae (Todaro et al. 2005 ; Kieneke and Schmidt-Rhaesa 2015 ). Our SEM examinations of the furca of D. surreyi finally confirm the existence of a pair of modified scales partially “wrapping” the medial edge of each branch and bearing a pair of thick and curved, horn-shaped spines, and even with a peek under the scale. Nonetheless, future attempts for transmission electron microscope imagery of the furcal branches are highly desirable to further detail the cuticular structure of the horn-shaped spines and its basal furcal scales. Dichaetura piscator was studied in the best knowledge of its time (Murray 1913 ), but the description lacks several details and a reinvestigation using every state-of-the-art technology is urgently needed for clarifying its systematic status. However, it is hard to ignore the significant distinctions compared to other Dichaetura species: indeed, it shows closer morphological resemblance to the dubious freshwater macrodasyidan Marinellina flagellata Ruttner-Kolisko, 1955, or to a juvenile Redudasys Kisielewski, 1987, e.g., a juvenile Redudasys neotemperatus Kånneby & Kirk, 2017 (see Fig. 3 b of Kånneby and Kirk 2017 ), than to other Dichaetura . Dichaetura piscator is described as a small animal (150 µm) with a spindle-shaped body and a separated head, with irregular undulating folds, bearing numerous long sensory hairs and c-shaped curved bristles (cilia) along the body. Most importantly, D. piscator has two adhesive tubes of equal-length on each furcal branch, an undeniable pattern that could fit to a juvenile Redudasys , maybe with fixation artifacts on sensory cilia curling into c-shape (see Kieneke et al. 2013 , p. 45 Fig. 2 B, D for a comparable artifact). Conversely, Dichaetura capricornia , the first described species and type species of the genus (Remane 1927 , p. 283), originally placed to the genus Chaetura by Metschnikoff ( 1865 ), shares diagnostic features with the other members of Dichaetura (Suzuki 2018 ), such as the arrangement of ventral locomotory cilia in organized perpendicular rows along the longitudinal columns, wider on the pharyngeal region and narrowing toward pharyngo-intestinal region and the posterior end (our Fig. 5 A and Supplementary Material S3 vs Martin 1990 , p. 477, Fig. 7 and Suzuki 2018 , p. 21, Fig. 8 ), and a dorsal posterior patch of small scales with thin long spines (our Figs. 4 B, 6 C, 7 C vs Martin 1990 , p. 477, Fig. 7 ). Although there is some resemblance of the shape of the posterior furca between C. capricornia and the two species D. surreyi and D. filispina , it is definitely different. Both posterior adhesive tubes of D. capricornia appear elongated, rather thin and pointed at their distal tip (see Fig. 7 of Martin 1990 ). On the medial margin, each tube has a sharp denticle at a position where the horn-shaped spine occurs in D. surreyi and D. filispina (compare, e.g., Figs. 8.44, 8.45 and 8.47 of Balsamo et al. 2014 ). Also the median unpaired spine protruding into the indentation of the furcal bases seems to be absent in D. capricornia (see Fig. 2 of Metschnikoff 1865 and Fig. 7 of Martin 1990 ). Concludingly, it seems likely to us that both Dichaetura surreyi and D. filispina represent a pair of sister species and the genus type species D. capricornia is phylogenetically related to the latter two species. The sharp denticles of the furca of D. capricornia could either represent predecessors of the horn-shaped spines of the other two species, or it might be a vestigial form. It seems furthermore plausible that the fourth nominal species Dichaetura piscator does not belong to the genus and rather is a member of a different order of Gastrotricha, viz. the Macrodasyida and could be related to the only freshwater-dwelling genera of that clade, Redudasys and Marinellina . For testing these hypotheses of course new specimens of at least D. piscator and D. capricornia are urgently needed for comprehensive morphological and molecular investigations. We think in this context it is worth mentioning that the type locality of the latter species ( D. capricornia ) close to Kharkiv (Ukraine) is currently under heavy impact due to an unlawful war of aggression against an independent democratic state imposed by an autocratic state. At worst, this man-made impact could result in the extinction of this species. Our primary aim of the molecular analysis is neither to reconstruct the phylogeny of Paucitubulatina, nor to definitely clarify the systematic position of Dichaetura surreyi , but the ML tree effectively demonstrates the utility of COI as a suitable barcode marker. Comparably long branches probably indicate a high level of mutation saturation of positions in the COI gene, which makes this mitochondrial gene particularly unsuitable for a reliable reconstruction of phylogeny (DeSalle et al. 2005 ). However, including multiple specimens per species, where feasible, shows a pattern of low intraspecific variation versus high interspecific divergence and therefore the existence of a ‘barcode gap’, a prerequisite for a suitable barcode sequence (Hebert et al. 2003 ). Comparative myoanatomy of Paucitubulatina The muscular architecture of Paucitubulatina has so far been documented in 23 species using epifluorescence or confocal microscopy, consistently revealing a conserved ancestral pattern of muscle architecture, consisting of circular and helicoidal muscles around the pharynx and intestine, four pairs of longitudinal muscles (dorsal, lateral, ventrolateral, and ventral), and a paired Rückenhautmuskel (Hochberg and Litvaitis 2001 , 2003 ; Hochberg 2005 ; Leasi et al. 2006 ; Kieneke et al. 2008a ; Leasi and Todaro 2008 , 2009 ; Kieneke and Ostmann 2012 ; Bekkouche and Worsaae 2016 ; Münter and Kieneke 2017 ; Minowa et al. 2025 ). The musculature of Dichaetura surreyi aligns to the aforementioned Paucitubulatina pattern, confirming the ancestral set of muscle components of the last common ancestor of Paucitubulatina. But it also displays previously undescribed muscular branchings, including two additional pairs of longitudinal muscles in the splanchnic region, one dorsal and one ventral pair, as well as two or three muscular fibers at the anterior part of the pharynx. Another notable difference to the ancestral muscular pattern is the bifurcation of the ventrolateral longitudinal muscle in the pharyngeal region. Furthermore,thin muscle branches insert into the dorsal integument at the furcal base in the area of the elongate posterior spines (dmb in Fig. 10 ), indicating a hypothesized functional role related to a protective behavior (see below). To date, only four dasydytid species have been investigated with CLSM (Kieneke et al. 2008a ; Kieneke and Ostmann 2012 ) to determine spine-associated musculature, making Dichaetura surreyi probably the second recorded Gastrotricha lineage with such a set of muscles responsible for a movement of cuticular structures. Defensive behavior Several lineages within Paucitubulatina have a protective cuticle featuring spines and scales that shield against mechanical stress from the surrounding rough environment and defense from predation; Dichaeturidae is no exception. Many Chaetonotus species, for instance, display a defensive strategy of curling into a ball—similar to a hedgehog (See Schwank 1990 ), to expose their armored dorsal cuticle, while protecting the smoother belly, offering defense against heliozoans and amoebozoans (Brunson 1949 ; Bovee and Cordell 1971 ), or, as documented most recently, against tentaculiferous ciliates of the genus Legendrea —although evade can still fail (Pomahač et al. 2023 ). Certain members of the Chaetonotus subgenus Zonochaeta further exhibit a specialized dorsal girdle of scales with longer, thicker spines that can abduct, likely aided by dorsal musculature (Kisielewski 1997 ). Likewise, some freshwater semiplanktonic and pelagic lineages in the Dasydytidae possess elongated saltatory spines associated with a specific set of muscles derived from segmentation of lateral and ventral longitudinal muscles, enabling evasive quick “jumps” in the water column when disturbed, and other lineages spreading these spines radially for protection (Kieneke et al. 2008a ; Kieneke and Ostmann 2012 ; Minowa and Garraffoni 2021 ; see also Fig. 5 b of Schwank 1990 ). In a similar fashion, living specimens of Dichaetura surreyi observed in the course of this study displayed a defensive behavior when threatened, curving the posterior portion of the body and raising the spines of the posterior patch on the dorsal furcal base (Fig. 5 B–C). Specimen-saving preparation for multiple purposes Our methodology successfully yielded behavioral and morphological information from light microscopy of a living individual, anatomical data from confocal laser scanning microscopy (CLSM), and finally high-resolution morphological data from electron scanning microscopy (SEM) of the identical specimen of tiny gastrotrichs. This approach supported the redescription of the species Dichaetura surreyi and provided further insights into its muscular architecture and morphological features, while still preserving a depositable specimen for future study. The limited availability of specimens poses a well-known challenge for microinvertebrate zoologists, especially when applying integrative taxonomy approach, and often needs creative strategies to overcome the distressing chances of losing material during or between each morphological method (George and Plum 2009 ; Garraffoni et al. 2019a ; Bosco et al. 2020 ). Declarations This work has been registered in ZooBank with registration number urn:lsid:zoobank.org:pub:049836B1-5B99-4D27-83A1-6400A3CB2955 Conflict of interest. The authors declare that they have no conflicts of interest. Funding This study was partially financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - CAPES/PrInt (nº 88887.716041/2022-00) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (nº 141482/2021-4) grant to AKM. It was also partially funded by the National Science Foundation (NSF) - DEB 2051684 (R. Hochberg). The third author (ARSG) is funded by São Paulo Research Foundation (FAPESP) (Proc: 2014/23856-0; 2018/10313-0; 2023/05724-9) and the Brazilian fostering agency ‘Conselho Nacional de Desenvolvimento Científico e Tecnológico’ through a productivity grant (CNPq proc. 04/2021). Author contributions A.K.M. conceptualized the study, collected samples, conducted laboratory examinations of the material, data collection and analysis, and drafted the manuscript. T.Q.A. contributed to data collection and analysis and co-authored the manuscript draft. A.R.S.G. participated in data collection and analysis and co-authored the manuscript draft. A.K. conceptualized and supervised the study, collected samples, and co-authored the manuscript draft. All authors reviewed and approved the final manuscript. References Armitage PD, Szoszkiewicz K, Blackburn JH, Nesbitt I (2003) Ditch communities: a major contributor to floodplain biodiversity. Aquat Conserv Mar Freshw Ecosyst 13:165–185. https://doi.org/10.1002/aqc.549 Balsamo M, d’Hondt J-L, Grilli P (2019) Phylum Gastrotricha. In: Rogers DC, Thorp J (eds) Thorp and Covich’s Freshwater Invertebrates. Elsevier, Amsterdam, Netherlands, pp 149–218 Balsamo M, Grilli P, Guidi L, d’Hondt J-L (2014) Gastrotricha: Biology, ecology and systematics. Families Dasydytidae, Dichaeturidae, Neogosseidae, Proichthydiidae. In: Identification Guides to the Plankton and Benthos of Inland Waters. Backhuys Publishers, Weikersheim, Germany Becker T, Siegmüller A (2021) The coastal lowland of northwestern Germany as an archive of Holocene landscape evolution: basis for a spatial evaluation of Stone Age settlement patterns in the Dornumer tidal basin. Neth J Geosci. https://doi.org/10.1017/njg.2020.17 Behre K-E (2012) Die Geschichte der Landschaft um den Jadebusen: Freisland-Wilhelmshaven-Wesemarsch. Brune-Mettcker Druck-und Verlagsgesellschaft mbH, Wilhelmshaven Behre K-E (2014) Ostfriesland: die Geschichte seiner Landschaft und ihrer Besiedlung. Brune-Mettcker Druck-und Verlagsgesellschaft mbH, Wilhelmshaven Bekkouche N, Worsaae K (2016) Neuromuscular study of early branching Diuronotus aspetos (Paucitubulatina) yields insights into the evolution of organs systems in Gastrotricha. Zool Lett 2:21. https://doi.org/10.1186/s40851-016-0054-3 Biggs J, Williams P, Whitfield M, et al (2005) 15 years of pond assessment in Britain: results and lessons learned from the work of Pond Conservation. Aquat Conserv Mar Freshw Ecosyst 15:693–714. https://doi.org/10.1002/aqc.745 Boix D, Biggs J, Céréghino R, et al (2012) Pond research and management in Europe: “Small is Beautiful.” Hydrobiologia 689:1–9. https://doi.org/10.1007/s10750-012-1015-2 Bosco I, Lourenco AP, Guidi L, et al (2020) Integrative description of a new species of Acanthodasys Remane, 1927 (Gastrotricha, Macrodasyida, Thaumastodermatidae) based on four distinct morphological techniques and molecular data. Zool Anz 286:31–42. https://doi.org/10.1016/j.jcz.2020.03.003 Bovee EC, Cordell DL (1971) Feeding on Gastrotrichs by the Heliozoon Actinophrys sol . Trans Am Microsc Soc 90:365–369. https://doi.org/10.2307/3225197 Brunson RB (1949) The life history and ecology of two North American gastrotrichs. Trans Am Microsc Soc 68:1–20 Chester ET, Robson BJ (2013) Anthropogenic refuges for freshwater biodiversity: Their ecological characteristics and management. Biol Conserv 166:64–75. https://doi.org/10.1016/j.biocon.2013.06.016 Clare P, Edwards RW (1983) The macroinvertebrate fauna of the drainage channels of the Gwent Levels, South Wales. Freshw Biol 13:205–225. https://doi.org/10.1111/j.1365-2427.1983.tb00672.x Clifford CC, Heffernan JB (2018) Artificial aquatic ecosystems. Water 10:1096. https://doi.org/10.3390/w10081096 Davies B, Biggs J, Williams P, et al (2008) Comparative biodiversity of aquatic habitats in the European agricultural landscape. Agric Ecosyst Environ 125:1–8. https://doi.org/10.1016/j.agee.2007.10.006 Dayrat B (2005) Towards integrative taxonomy. Biol J Linn Soc 85:407–417. https://doi.org/10.1111/j.1095-8312.2005.00503.x De Bie T, Declerck S, Martens K, et al (2008) A comparative analysis of cladoceran communities from different water body types: patterns in community composition and diversity. Hydrobiologia 597:19–27. https://doi.org/10.1007/s10750-007-9222-y DeSalle R, Egan MG, Siddall M (2005) The unholy trinity: taxonomy, species delimitation and DNA barcoding. Phylosophical Trans R Soc B 1905–1916. https://doi.org/10.1098/rstb.2005.1722 Fonseca G, Fontaneto D, Di Domenico M (2018) Addressing biodiversity shortfalls in meiofauna. J Exp Mar Biol Ecol 502:26–38. https://doi.org/10.1016/j.jembe.2017.05.007 Franz H (1950) Bodenzoologie als Grundlage der Bodenpflege mit besonderer Beruecksichtigung der Bodenfauna in den Ostalpen und im Donaubecken. Akademie, Berlin Franz H, Donner J (1954) Gastrotricha. In: Franz H (ed) Die Nordostalpen im Spiegel ihrer Landtierwelt. Wager University Press, Innsbrucks, pp 1–158 Gammuto L, Serra V, Petroni G, Todaro MA (2024) Molecular phylogenetic position and description of a new genus and species of freshwater Chaetonotidae (Gastrotricha: Chaetonotida: Paucitubulatina), and the annotation of its mitochondrial genome. Invertebr Syst 38:IS23059. https://doi.org/10.1071/IS23059 Garraffoni ARS, Araújo TQ, Lourenço AP, et al (2019a) Integrative taxonomy of a new Redudasys species (Gastrotricha: Macrodasyida) sheds light on the invasion of freshwater habitats by macrodasyids. Sci Rep 9:2067. https://doi.org/10.1038/s41598-018-38033-0 Garraffoni ARS, Kieneke A, Kolicka M, et al (2019b) ICZN Declaration 45: a remedy for the nomenclatural and typification dilemma regarding soft-bodied meiofaunal organisms? Mar Biodivers 49:2199–2207. https://doi.org/10.1007/s12526-019-00983-7 George KH, Plum C (2009) Description of two new species of Dorsiceratus Drzycimski, 1967 (Copepoda: Harpacticoida: Ancorabolidae) from Sedlo and Seine Seamounts (Northeastern Atlantic) and remarks on the phylogenetic status of the genus. Zootaxa 2096:257–286 Giere O (2009) Meiobenthology. Springer, Berlin, Heidelberg Guindon S, Dufayard J-F, Lefort V, et al (2010) New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Syst Biol 59:307–321. https://doi.org/10.1093/sysbio/syq010 Gupta S, Collier JS, Garcia-Moreno D, et al (2017) Two-stage opening of the Dover Strait and the origin of island Britain. Nat Commun 8:15101. https://doi.org/10.1038/ncomms15101 Hadler H, Reiß A, Willershäuser T, et al (2025) Medieval Overexploitation of Peat Triggered Large-Scale Drowning and Permanent Land Loss in Coastal North Frisia (Wadden Sea Region, Germany). Geosciences 15:1. https://doi.org/10.3390/geosciences15010001 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98 Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc Biol Sci 270:313–321. https://doi.org/10.1098/rspb.2002.2218 Heino J, Parviainen J, Paavola R, et al (2005) Characterizing macroinvertebrate assemblage structure in relation to stream size and tributary position. Hydrobiologia 539:121–130. https://doi.org/10.1007/s10750-004-3914-3 Herzon I, Helenius J (2008) Agricultural drainage ditches, their biological importance and functioning. Biol Conserv 141:1171–1183. https://doi.org/doi:10.1016/j.biocon.2008.03.005 Hoang DT, Chernomor O, Von Haeseler A, et al (2018) UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol 35:518–522 Hochberg R (2005) Musculature of the primitive gastrotrich Neodasys (Chaetonotida): functional adaptations to the interstitial environment and phylogenetic significance. Mar Biol 146:315–323. https://doi.org/10.1007/s00227-004-1437-0 Hochberg R, Litvaitis M (2000) Phylogeny of Gastrotricha: a morphology-based framework of gastrotrich relationships. 198:299–305. https://doi.org/10.2307/1542532 Hochberg R, Litvaitis MK (2003) Organization of muscles in Chaetonotida paucitubulatina (Gastrotricha). Meiofauna Mar 12:47–58 Hochberg R, Litvaitis MK (2001) A Muscular Double Helix in Gastrotricha. Zool Anz - J Comp Zool 240:61–68. https://doi.org/10.1078/0044-5231-00006 Hummon WD (1974) Gastrotricha from Beaufort, North Carolina, USA. 15:85–92 Kalyaanamoorthy S, Minh BQ, Wong TK, et al (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14:587–589 Kånneby T (2013) New species and records of freshwater Chaetonotus (Gastrotricha: Chaetonotidae) from Sweden. Zootaxa 3701:551–588. https://doi.org/10.11646/zootaxa.3701.5.3 Kånneby T, Kirk JJ (2017) A new species of Redudasys (Gastrotricha: Macrodasyida: Redudasyidae) from the United States. Proc Biol Soc Wash 130:128–139. https://doi.org/10.2988/17-00006 Kieneke A, Arbizu PM, Riemann O (2008a) Body musculature of Stylochaeta scirtetica Brunson, 1950 and Dasydytes ( Setodytes ) tongiorgii (Balsamo, 1982) (Gastrotricha: Dasydytidae): A functional approach. Zool Anz 247:147–158. https://doi.org/10.1016/j.jcz.2007.11.001 Kieneke A, Nikoukar H (2017) Integrative morphological and molecular investigation of Turbanella hyalina Schultze, 1853 (Gastrotricha: Macrodasyida), including a redescription of the species. Zool Anz 267:168–186. https://doi.org/10.1016/j.jcz.2017.03.005 Kieneke A, Ostmann A (2012) Structure, function and evolution of somatic musculature in Dasydytidae (Paucitubulatina, Gastrotricha). Zoomorphology 131:95–114. https://doi.org/10.1007/s00435-012-0152-5 Kieneke A, Riemann O (2008) First record of rare dasydytid gastrotrich Setopus tongiorgii (Balsamo, 1982) from central Europe with some biological data. Zootaxa 1680:59–61. https://doi.org/10.11646/zootaxa.1680.1.4 Kieneke A, Riemann O, Ahlrichs WH (2008b) Novel implications for the basal internal relationships of Gastrotricha revealed by an analysis of morphological characters. Zool Scr 37:429–460. https://doi.org/10.1111/j.1463-6409.2008.00334.x Kieneke A, Rothe BH, Schmidt-Rhaesa A (2013) Record and description of Anandrodasys agadasys (Gastrotricha: Redudasyidae) from Lee Stocking Island (Bahamas), with remarks on populations from different geographic areas. Meiofauna Mar 20:39–48 Kieneke A, Schmidt-Rhaesa A (2015) Gastrotricha. In: Handbook of Zoology. Gastrotricha, Cycloneuralia and Gnathifera. de Gruyer, Berlin, pp 1–134 Kieneke A, Schmidt-Rhaesa A, Hochberg R (2015) A new species of Cephalodasys (Gastrotricha, Macrodasyida) from the Caribbean Sea with a determination key to species of the genus. Zootaxa 3947:367–385. https://doi.org/10.11646/zootaxa.3947.3.4 Kieneke A, Zekely J (2008) Desmodasys abyssalis sp. nov.—first record of a deep-sea gastrotrich from hydrothermal vents. Mar Biodivers Rec 1:. https://doi.org/10.1017/S1755267207008950 Kisielewski J (1997) On the subgeneric division of genus Chaetonotus Ehrenberg (Gastrotricha). Ann Zool 46:145–151 Kolicka M (2016) Gastrotrichs in bromeliads—newly recorded Chaetonotus ( Hystricochaetonotus ) furcatus Kisielewski, 1991 (Chaetonotida) from the Łódź Palm House. Zoosystema 38:141–155. https://doi.org/10.5252/z2016n1a5 Kolicka M, Dabert M, Olszanowski Z, Dabert J (2020) Sweet or salty? The origin of freshwater gastrotrichs (Gastrotricha, Chaetonotida) revealed by molecular phylogenetic analysis. Cladistics 36:458–480. https://doi.org/10.1111/cla.12424 Kolicka M, Gwiazdowicz DJ, Hupało K, et al (2016) Hidden invertebrate diversity - phytotelmata in Bromeliaceae from palm houses and florist wholesalers (Poland). Biologia (Bratisl) 71:194–203. https://doi.org/10.1515/biolog-2016-0026 Kolicka M, Kisielewski J, Nesteruk T, Zawierucha K (2013) Gastrotricha from the Poznań Palm House--one new subgenus and three new species of freshwater Chaetonotida (Gastrotricha). Zootaxa 3717:231–279. https://doi.org/10.11646/zootaxa.3717.2.7 Kumar S, Stecher G, Li M, et al (2018) MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096 Lauterborn R (1913) Gastrotricha. In: Handwoerterbuch der Naturwissenschaft. Gustav Fischer Verlag, Jena Leasi F, Rothe BH, Schmidt-Rhaesa A, Todaro MA (2006) The musculature of three species of gastrotrichs surveyed with confocal laser scanning microscopy (CLSM). Acta Zool 87:171–180. https://doi.org/10.1111/j.1463-6395.2006.00230.x Leasi F, Todaro MA (2009) Meiofaunal cryptic species revealed by confocal microscopy: the case of Xenotrichula intermedia (Gastrotricha). Mar Biol 156:1335–1346 Leasi F, Todaro MA (2008) The muscular system of Musellifer delamarei (Renaud-Mornant, 1968) and other chaetonotidans with implications for the phylogeny and systematization of the Paucitubulatina (Gastrotricha). Biol J Linn Soc 94:379–398. https://doi.org/10.1111/j.1095-8312.2008.00974.x Malmqvist B, Hoffsten P-O (2000) Macroinvertebrate taxonomic richness, community structure and nestedness in Swedish streams. Arch Für Hydrobiol 29–54. https://doi.org/10.1127/archiv-hydrobiol/150/2000/29 Martin LV (1990) Further observations on Gastrotrichs in Surrey and a provisional British list. Microscopy 36:415–425 Martin LV (1981) Gastrotrichs found in Surrey. Microscopy 34:286–301 Metschnikoff E (1865) Über einige wenig bekannte niedere Thierformen. Z Für Wiss Zool 15:450–463 Michels J, Büntzow M (2010) Assessment of Congo red as a fluorescence marker for the exoskeleton of small crustaceans and the cuticle of polychaetes. J Microsc 238:95–101. https://doi.org/10.1111/j.1365-2818.2009.03360.x Minowa AK, Garraffoni ARS (2025) Expanding the knowledge of Brazilian Gastrotrich biodiversity: Freshwater Paucitubulatina (Gastrotricha: Chaetonotida) from Northeastern Brazil. Zool Curitiba 42:e24025. https://doi.org/10.1590/S1984-4689.v42.e24025 Minowa AK, Garraffoni ARS (2020) Assessing biodiversity shortfalls of freshwater meiofauna from the Atlantic Forest: New species, distribution patterns and the first total-evidence phylogeny of semiplanktonic Gastrotricha. Mol Phylogenet Evol 152:106926. https://doi.org/10.1016/j.ympev.2020.106926 Minowa AK, Garraffoni ARS (2022) New data on Brazilian semiplanktonic gastrotrichs (Gastrotricha: Chaetonotida). Zootaxa 5209:45–68. https://doi.org/10.11646/zootaxa.5209.1.3 Minowa AK, Garraffoni ARS (2021) Seek and you shall find: New species of the rare genus Ornamentula (Gastrotricha: Chaetonotida) and first record outside of type-locality. Zoologia 38:1–9. https://doi.org/10.3897/ZOOLOGIA.38.E56781 Minowa AK, Kieneke A, Campos A, et al (2025) New branch on the Tree of Life of Gastrotricha: Establishment of a new genus for limno-terrestrial species. Zool J Linn Soc 2:zlae166. https://doi.org/10.1093/zoolinnean/zlae166 Mioduchowska M, Wojtasik B (2009) Copepoda-Cyclopoida of water bodies of the coast of Gdańsk. Teka Kom Ochr Kszt Środ Przyr 6:189–199 Münter L, Kieneke A (2017) Novel myo-anatomical insights to the Xenotrichula intermedia species complex (Gastrotricha: Paucitubulatina): Implications for a pan-European species and reconsideration of muscle homology among Paucitubulatina. Proc Biol Soc Wash 130:166–186. https://doi.org/10.2988/17-00013 Murray J (1913) Gastrotricha. J Quekett Microsc Club 12:211–238 Nguyen LT, Schmidt HA, Von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268–274. https://doi.org/10.1093/molbev/msu300 Nicolet P, Biggs J, Fox G, et al (2004) The wetland plant and macroinvertebrate assemblages of temporary ponds in England and Wales. Biol Conserv 120:261–278. https://doi.org/10.1016/j.biocon.2004.03.010 Oertli B, Joye DA, Castella E, et al (2002) Does size matter? The relationship between pond area and biodiversity. Biol Conserv 104:59–70. https://doi.org/10.1016/S0006-3207(01)00154-9 Painter D (1999) Macroinvertebrate distributions and the conservation value of aquatic Coleoptera, Mollusca and Odonata in the ditches of traditionally managed and grazing fen at Wicken Fen, UK. J Appl Ecol 36:33–48. https://doi.org/10.1046/j.1365-2664.1999.00376.x Peralta-Maraver I, Traunspurger W, Robertson AL, et al (2023) Freshwater Meiofauna—A Biota with Different Rules? In: Giere O, Schratzberger M (eds) New Horizons in Meiobenthos Research: Profiles, Patterns and Potentials. Springer International Publishing, Cham, pp 153–173 Pomahač O, Méndez-Sánchez D, Poláková K, et al (2023) Rediscovery of remarkably rare anaerobic tentaculiferous ciliate genera Legendrea and Dactylochlamys (Ciliophora: Litostomatea). Biology 12:707 Pott R (1999) Nordwestdeutsches Tiefland zwischen Ems und Weser. Ulmer, Stuttgart, Jena, New York Remane A (1927) Beitrage zur Systematik der Susswassergastrotrichen. Zool Jahrb Abt Für Syst Oekologie Geogr Tiere 53:268–320 Richards C, Host GE, Arthur JW (1993) Identification of predominant environmental factors structuring stream macroinvertebrate communities within a large agricultural catchment. Freshw Biol 29:285–294. https://doi.org/10.1111/j.1365-2427.1993.tb00764.x Schratzberger M, Ingels J (2018) Meiofauna matters: the roles of meiofauna in benthic ecosystems. J Exp Mar Biol Ecol 502:12–25. https://doi.org/10.1016/j.jembe.2017.01.007 Schwank P (1990) Gastrotricha. In: Schwoerbel J, Zwick P (eds) Süßwasserfauna von Mitteleuropa. Gastrotricha und Nemertini. Gustav Fischer Verlag, Stuttgart, Jena, New York, pp 1–252 Suzuki T (2018) 水田に棲みつく多様なイタチムシたち. 日本の科学者 53:16–21. https://doi.org/10.60233/jjsci.53.4_16 Suzuki TG, Maeda M, Furuya H (2013) Two new Japanse species of Gastrotricha (Chaetonotida, Chaetonotidae, Lepidodermella and Dichaeturidae, Dichaetura ), with Comments on the Diversity of Gastrotrichs in Rice Paddies. Zootaxa 3691:229–239. https://doi.org/10.11646/zootaxa.3691.2.3 Todaro MA, Balsamo M, Kristensen RM (2005) A new genus of marine chaetonotids (Gastrotricha), with a description of two new species from Greenland and Denmark. J Mar Biol Assoc U K 85:1391–1400. https://doi.org/10.1017/S0025315405012579 Todaro MA, Zotto MD, Jondelius U, et al (2012) Gastrotricha: A Marine Sister for a Freshwater Puzzle. 7:e31740. https://doi.org/10.1371/journal.pone.0031740 Trokhymchuk R, Kieneke A (2024a) New records of deep-sea Gastrotricha and Tardigrada from Iberian and Canary Basins (Northeast Atlantic) with comments on abyssal meiofauna composition and the meiofauna paradox. J VNKarazin Kharkiv Natl Univ Ser «Biology» 43:66–84. https://doi.org/10.26565/2075-5457-2024-43-6 Trokhymchuk R, Kieneke A (2024b) Novel distribution records of marine Tardigrada from abyssal sediments of the Northwest Atlantic Ocean. Org Divers Evol. https://doi.org/10.1007/s13127-024-00641-2 Twisk W, Noordervliet MAW, ter Keurs WJ (2000) Effects of ditch management on caddisfly, dragonfly and amphibian larvae in intensively farmed peat areas. Aquat Ecol 34:397–411. https://doi.org/10.1023/A:1011430831180 Verdonschot RCM, Keizer-vlek HE, Verdonschot PFM (2011) Biodiversity value of agricultural drainage ditches: a comparative analysis of the aquatic invertebrate fauna of ditches and small lakes. Aquat Conserv Mar Freshw Ecosyst 21:715–727. https://doi.org/10.1002/aqc.1220 Watson AM, Ormerod SJ (2004) The distribution of three uncommon freshwater gastropods in the drainage ditches of British grazing marshes. Biol Conserv 118:455–466. https://doi.org/10.1016/j.biocon.2003.09.021 Williams P, Whitfield M, Biggs J, et al (2004) Comparative biodiversity of rivers, streams, ditches and ponds in an agricultural landscape in Southern England. Biol Conserv 115:329–341. https://doi.org/10.1016/S0006-3207(03)00153-8 Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1Dichaeturamorphometric.xlsx Table 1. Morphometric and morphological features of Dichaetura surreyi specimens analyzed in this study. Measurements (in μm) are based on five individuals: The columns present values for each specimen, followed by observed ranges and averages. Dash (–) indicates unmeasured data. SupplementarytableS1.GenBank.xlsx Supplementary Table S1. Accession numbers in Genbank of of cytochrome c oxidase subunit I (COI) sequences used in the present study. The table includes the family assignment, species or operational taxonomic unit. Sequences belong to members of the order Chaetonotida, primarily from the family Chaetonotidae, with additional representatives from Dasydytidae, Neogosseidae, and Xenotrichulidae. FigS1Supplementarymaterial.jpg Supplementary Material S1. Yield of the threefold morphological method, with A. optical microscopy with differential interference contrast lenses of fresh living animal, B–D. Maximum intensity projection of myoanatomy and nuclei staying from confocal laser scanning microscopy revealing the musculature architecture and distribution of nuclei with high density on anterior portion (ganglia) and posterior portion (adhesive gland), and E. scanning electron microscopy displaying cuticular ornamentation. Scale: 30 µm. FigS2Supplementarymaterial.jpg Supplementary Material S2. CLSM of phalloidin stained muscle and SYBR Green counter-stained nuclei of Dichaetura surreyi . A. Volume rendered 3D image of dorsal view of the whole specimen with actin filaments in orange-red and DNA in blueish tones. B. Same specimen, again in dorsal view with muscular architecture (yellow) and nuclei distribution (bright blue), both surface-rendered and C. ventral view with muscular (actin) patterns surface-rendered (yellow) and nuclei (DNA) volume-rendered (blueish). Abbreviations: ag: nuclei associated with adhesive glands, cg: cerebral ganglion, dlm: dorsal longitudinal muscles, R: Rückenhautmuskel , vlm: ventrolateral muscles. Scale: 30 µm. FigS3Supplementarymaterial.jpg Supplementary Material S3. Volume-rendered 3D image from CLSM image stack of Dichaetura surreyi with phalloidin stained muscles (orange-red) and counter-stained cilia with acetylated α-tubulin immunoreactivity (blueish). A. Dorsolateral view of the whole specimen, B. lateral view, and C. ventral view. Scale: 30 µm. 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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-7189628","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":495785539,"identity":"9d386fcc-4992-4458-bbb4-59680c8fcea0","order_by":0,"name":"Axell Kou Minowa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIie2PsQrCMBCGFcGp6iqI+gpKIVOoD+KSEqiLvkGHuthFnH0J184ngbgEuwZcLL5AJzfFqwqK0KqbYL4l5Pg/7v5SyWD4TcqwB3wq1w/NBlN45wB7KF6mBB8qN8RtUhRvLDkAU9RdhbX1wfJjZxUK3OLTYZ7SVJIB054biTq3LbnjkXJRkd4kyHM28x6wVKBikda4uuMEUCkHIlfpikb6pJy3nMRJsdLbzLG+viuTGThEv9nSVxIPU56Nim2fFpwRjVtYQZeO5sk+lbQdxaqfLI/OgMQjnPg0v/4r7jXJPo1nDL4JGwwGw39wAWNzb3M2w3ssAAAAAElFTkSuQmCC","orcid":"","institution":"State University of Campinas","correspondingAuthor":true,"prefix":"","firstName":"Axell","middleName":"Kou","lastName":"Minowa","suffix":""},{"id":495785540,"identity":"8d6f19d1-f232-46ea-8c88-037ae6aefee8","order_by":1,"name":"Thiago Quintão Araújo","email":"","orcid":"","institution":"University of Massachusetts Lowell","correspondingAuthor":false,"prefix":"","firstName":"Thiago","middleName":"Quintão","lastName":"Araújo","suffix":""},{"id":495785541,"identity":"e2633b40-426c-4609-8a4e-5c3419b83759","order_by":2,"name":"André Rinaldo Senna Garraffoni","email":"","orcid":"","institution":"State University of Campinas","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"Rinaldo Senna","lastName":"Garraffoni","suffix":""},{"id":495785542,"identity":"e9f013bf-4de3-43ad-9cfa-93cd38d2f256","order_by":3,"name":"Alexander Kieneke","email":"","orcid":"","institution":"German Center for Marine Biodiversity Research (DZMB)","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Kieneke","suffix":""}],"badges":[],"createdAt":"2025-07-22 17:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7189628/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7189628/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00435-025-00748-w","type":"published","date":"2025-10-17T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88624906,"identity":"82a144b1-da95-4238-aecc-6b4a0886ce13","added_by":"auto","created_at":"2025-08-08 12:41:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4414301,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of the study area in Logabirumer Hammrich, Leer, Northern Germany (red dot on the inset map), with the sampling site indicated by a dashed box (A). Satellite view of the sampling location along a farming ditch, showing six sampling points (LO-1 to LO-6) (B). Due to a drop in the water level, the sampling had shifted northward between May 15 (LO-1) and May 22, 2023 (LO-2 to LO-6). Field view of the ditch running through the agricultural landscape (C), with close-up of the water body at LO-1 (D). The map of Leer (Ostfriesland, Germany) (A–B), has been reproduced thanks to OpenStreetMap and is licensed under The Open Database License (ODbL).\u003c/p\u003e","description":"","filename":"Fig1Dichaeturarecords.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/917b3e690f6e61d6df763704.jpg"},{"id":88626448,"identity":"752682e2-d711-41b8-8741-4fd3504c16b1","added_by":"auto","created_at":"2025-08-08 12:57:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2484570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDichaetura surreyi\u003c/em\u003e schematic illustration. A. Dorsal body view, B. Ventral body view. Dotted lines indicate the area of the insertion of ciliary rows. Scale: 50 µm\u003c/p\u003e","description":"","filename":"Fig2IllustrationSchemmeDichaetura.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/a68f2135aaf4f493d2fb9706.jpg"},{"id":88625493,"identity":"8ccc6613-851f-4b8d-80bc-f7037375dfb8","added_by":"auto","created_at":"2025-08-08 12:49:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2945892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDichaetura surreyi\u003c/em\u003ephotomicrographs (differential interference contrast). A. Mid-plane optical section of internal organs, B. Dorsal view. C Mid-plane view, D. Ventral view, E. Ventral view. Abbreviation: bfs, basal furcal spines; ce, cephalion; cb, cephalic bristle; dp, dorsal scales patch; fs, furca spine; hy, hypostomion; mo, mouth ring; vs, ventral scales; pl, pleurae. Scale: 50 μm.\u003c/p\u003e","description":"","filename":"Fig3LMDichaeturaoverall.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/b1495b832c1a955ae417d00c.jpg"},{"id":88624878,"identity":"453a571b-50ad-479b-be12-7c0595fa1e0a","added_by":"auto","created_at":"2025-08-08 12:41:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1768612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDichaetura surreyi\u003c/em\u003ephotomicrographs (differential interference contrast). A\u003cstrong\u003e–\u003c/strong\u003eC Lateral optical section view. Abbreviation: at, adhesive tubes; bfs, basal furcal spines; cb, cephalic bristle; ce, cephalion; cr, ciliary rows; dp, dorsal scales patch; fs, furca spine; vs, ventral scales; pl, pleurae; ts, terminal scales. Scale: 50 μm.\u003c/p\u003e","description":"","filename":"Fig4LMDichaeturalateral.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/60413b265e302e7511e67d5f.jpg"},{"id":88624929,"identity":"7f574105-8c8c-4f22-a0b7-698131696c71","added_by":"auto","created_at":"2025-08-08 12:41:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":811042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDichaetura surreyi\u003c/em\u003ephotomicrographs (differential interference contrast). A–B Anterior head ventral optical section (U00 to U33). C. Ventral optical section of posterior furca (from U78 to end). Abbreviation: bfs, basal furcal spine; cb, cephalic bristle; fs, furca spine; hy, hypostomion; mo, mouth ring; pg, pharyngeal glands; PhIj, pharyngo-intestinal junction. Scale: 10 μm.\u003c/p\u003e","description":"","filename":"Fig5LMheadfurca.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/dae942e5ea5e88658b53b24b.jpg"},{"id":88624890,"identity":"9cf5c434-1bf3-41a9-983b-40004b972378","added_by":"auto","created_at":"2025-08-08 12:41:10","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3780149,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDichaetura surreyi \u003c/em\u003eScanning electron microscopic images. A. ventral view of the complete specimen. B. Close-up of ventrolateral posterior end, showing ventral ciliation, interciliary cuticle coverage, posteriormost keeled scales and terminal plates (from U71 to end). C. Close-up of ventral posterior end (by mechanically tilting the stub within the SEM machine), ventral ciliation and keeled terminal scales, hook-shaped basal furcal medial scale (from U91 to end). Abbreviation: bfs; basal furcal spine; ce, cephalion; dp, dorsal spines patch; fs, furcal hook-shape spine; hy, hypostomion; ks, keeled ventral posterior scales; mo, mouth ring; ts, terminal scales; vs, ventral scales. Scale: 10 μm.\u003c/p\u003e","description":"","filename":"Fig6SEMDichaeturaventral.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/ebdcbf57d5e89a11e84ec1df.jpg"},{"id":88625505,"identity":"0bca4a82-a8bb-4c9a-abff-db74cd6f4377","added_by":"auto","created_at":"2025-08-08 12:49:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4000492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDichaetura surreyi \u003c/em\u003escanning electron microscope images. A. Ventral view of the anterior head, close-up of ventral head, mouth entrance and cephalion (U00–U33). B. Lateral view of the posterior end, with insertion as scale morphology of the posterior spines patch (from U71 to end). C. Close-up of ventral interciliary cuticle (U08–U16), D. Close-up of ventral interciliary cuticle (U49–U61). Abbreviation: at, anterior ciliary tufts. bfs; basal furcal spine; ce, cephalion; dp, dorsal spines patch; fs, furcal hook-shape spine; hy, hypostomion; mo, mouth ring. Scales A, B: 10 μm; C, D: 5 µm.\u003c/p\u003e","description":"","filename":"Fig7SEMDichaeturadorsaltwisted.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/c72509418f41d2cfccd3fed8.jpg"},{"id":88624922,"identity":"b4a86dad-1018-4795-87d3-523937c7b65f","added_by":"auto","created_at":"2025-08-08 12:41:11","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2517799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDichaetura surreyi\u003c/em\u003e scanning electron microscope image. A. Close-up of the dorsolateral head (U00–U06). B. Lateral view of the head. C. Close-up of the ventrolateral head (anterior end to U29). Abbreviation: cb, cephalic sensory bristle; ce, cephalion; hp, hypopleurae; hy, hypostomion; mo, mouth ring. Scales: 2 µm.\u003c/p\u003e","description":"","filename":"Fig8SEMDichaeturadorsallateralhead.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/f304884d09b75cc978c335bf.jpg"},{"id":88624904,"identity":"cf685ea0-80a8-42f2-a2d5-6a960cb1f8ec","added_by":"auto","created_at":"2025-08-08 12:41:10","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3886083,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDichaetura surreyi\u003c/em\u003e scanning electron microscope image. A. Ventral overview. B. Ventrolateral close-up of head, mouth entrance and cephalion (U00–U32). C. Ventral close-up of head, mouth entrance and hypostomion (U00–U35). D. Close-up of ventral interciliary cuticle (U45–U69). E. Dorsolateral close-up of posterior end (U89 to end), featuring the insertion of posterior sensory bristle. Abbreviation: ce, cephalion; hy: hypostomion; pb: posterior sensory bristle. Scales: 5 µm.\u003c/p\u003e","description":"","filename":"Fig9SEMDichaeturaventralside.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/30e0d62cf1b0682e13e4b88c.jpg"},{"id":88624896,"identity":"47fab01d-abd0-41a9-ae79-9b0697c5add8","added_by":"auto","created_at":"2025-08-08 12:41:10","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2583203,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDichaetura surreyi\u003c/em\u003e schematic illustration of muscular organization. A. Dorsal body view, B. Ventral body view. Abbreviations: dlm: dorsal longitudinal muscles, dmb: dorsal muscular branch, hm: helicoidal muscles, pmp: pharyngeal muscular fibers, R: \u003cem\u003eRückenhautmuskel\u003c/em\u003e, sdlm: second dorsal longitudinal muscles, vlm: ventrolateral muscles, vm-I: ventral longitudinal muscles, vm-II: second ventral longitudinal muscles. Scale: 50 µm\u003c/p\u003e","description":"","filename":"Fig10IllustrationSchemmeDichaetura3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/03f3612518ac5f383bd99ff8.jpg"},{"id":88626450,"identity":"fc75f9a6-4850-4375-b86a-8352bc2dc809","added_by":"auto","created_at":"2025-08-08 12:57:11","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":2155644,"visible":true,"origin":"","legend":"\u003cp\u003eMuscular architecture of \u003cem\u003eDichaetura surreyi\u003c/em\u003e. \u003cstrong\u003eA–C. \u003c/strong\u003eVoltex rendered reconstruction of dorsal and ventral muscular architecture (f-actin stained with fluorescence-labeled phalloidin), artificially coloured. \u003cstrong\u003eD–H. \u003c/strong\u003eTransversal cross-section in different regions along the body. \u003cstrong\u003eI\u003c/strong\u003e. Lateral view of longitudinal section of posterior furcal muscular architecture (dashed inset in C). Abbreviations: dlm: dorsal longitudinal muscles, dmb: dorsal muscular branch, hm: helicoidal muscles, pmp: pharyngeal muscular fibers, R: \u003cem\u003eRückenhautmuskel\u003c/em\u003e, sdlm: second dorsal longitudinal muscles, vlm: ventrolateral muscles, vm-I: ventral longitudinal muscles, vm-II: second ventral longitudinal muscles, horizontal arrows: helicoidal muscle. Scale: A–H 30 µm; I: 5 µm.\u003c/p\u003e","description":"","filename":"Fig11CLSMDichaeturaTQA20250306.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/424fcec3ddd6d6a655d8dcc3.jpg"},{"id":88624920,"identity":"9bab52d9-7c58-42fe-80f6-4c4d5ad0cdb5","added_by":"auto","created_at":"2025-08-08 12:41:11","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1972965,"visible":true,"origin":"","legend":"\u003cp\u003eBest-scoring maximum likelihood phylogenetic tree of Paucitubulatins inferred from COI sequences using IQ-TREE. Notes with full statistical support are indicated by black solid circles. Sequences of \u003cem\u003eDichaetura surreyi \u003c/em\u003egenerated in this study are highlighted in red. The scale bar represents ten substitutions per one hundred nucleotide positions. GenBank accession numbers are provided in Supporting Information, Table S1.\u003c/p\u003e","description":"","filename":"Fig12COIphylogeny.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/d8e98c6a006fa4a37a7e734c.jpg"},{"id":93955988,"identity":"aa10f025-1d6f-4bc7-85bc-fe6be33058d0","added_by":"auto","created_at":"2025-10-20 16:08:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":34234318,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/cd606be3-036c-4018-8429-08c97d8e268a.pdf"},{"id":88624912,"identity":"13f42988-09b1-4ef0-8467-a0e3621b3490","added_by":"auto","created_at":"2025-08-08 12:41:11","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":46097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Morphometric and morphological features of \u003cem\u003eDichaetura surreyi\u003c/em\u003e specimens analyzed in this study. Measurements (in μm) are based on five individuals: The columns present values for each specimen, followed by observed ranges and averages. Dash (–) indicates unmeasured data.\u003c/p\u003e","description":"","filename":"Table1Dichaeturamorphometric.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/fdd43cc133917adc5dded2b1.xlsx"},{"id":88624923,"identity":"18d946de-d5d5-4316-8f46-b305e9222fd1","added_by":"auto","created_at":"2025-08-08 12:41:11","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table S1. \u003c/strong\u003eAccession numbers in Genbank of of cytochrome c oxidase subunit I (COI) sequences used in the present study. The table includes the family assignment, species or operational taxonomic unit. Sequences belong to members of the order Chaetonotida, primarily from the family Chaetonotidae, with additional representatives from Dasydytidae, Neogosseidae, and Xenotrichulidae.\u003c/p\u003e","description":"","filename":"SupplementarytableS1.GenBank.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/4cb7244c6b1b162e843a473b.xlsx"},{"id":88624914,"identity":"24d614ba-736d-4e58-8606-5be94863bddb","added_by":"auto","created_at":"2025-08-08 12:41:11","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4865766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Material S1. \u003c/strong\u003eYield of the threefold morphological method, with \u003cstrong\u003eA. \u003c/strong\u003eoptical microscopy with differential interference contrast lenses of fresh living animal, \u003cstrong\u003eB–D. \u003c/strong\u003eMaximum intensity projection of myoanatomy and nuclei staying from confocal laser scanning microscopy revealing the musculature architecture and distribution of nuclei with high density on anterior portion (ganglia) and posterior portion (adhesive gland), and \u003cstrong\u003eE. \u003c/strong\u003escanning electron microscopy displaying cuticular ornamentation. Scale: 30 µm.\u003c/p\u003e","description":"","filename":"FigS1Supplementarymaterial.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/bf657f977340cd060931e3db.jpg"},{"id":88624883,"identity":"ba05f21e-67e6-40b2-9b3c-1a89c859a20a","added_by":"auto","created_at":"2025-08-08 12:41:09","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1823189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Material S2. \u003c/strong\u003eCLSM of phalloidin stained muscle and SYBR Green counter-stained nuclei of \u003cem\u003eDichaetura surreyi\u003c/em\u003e. \u003cstrong\u003eA. \u003c/strong\u003eVolume rendered 3D image of dorsal view of the whole specimen with actin filaments in orange-red and DNA in blueish tones. \u003cstrong\u003eB. \u003c/strong\u003eSame specimen, again in dorsal view with muscular architecture (yellow) and nuclei distribution (bright blue), both surface-rendered and \u003cstrong\u003eC. \u003c/strong\u003eventral view with muscular (actin) patterns surface-rendered (yellow) and nuclei (DNA) volume-rendered (blueish). Abbreviations: ag: nuclei associated with adhesive glands, cg: cerebral ganglion, dlm: dorsal longitudinal muscles, R: \u003cem\u003eRückenhautmuskel\u003c/em\u003e, vlm: ventrolateral muscles. Scale: 30 µm.\u003c/p\u003e","description":"","filename":"FigS2Supplementarymaterial.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/20ff95f9391eebb467e6db39.jpg"},{"id":88624888,"identity":"71bc8502-b19c-4c85-ba38-1868eb69e2fb","added_by":"auto","created_at":"2025-08-08 12:41:10","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":884547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Material S3. \u003c/strong\u003eVolume-rendered 3D image from CLSM image stack of \u003cem\u003eDichaetura surreyi \u003c/em\u003ewith phalloidin stained muscles (orange-red) and counter-stained cilia with acetylated α-tubulin immunoreactivity (blueish). \u003cstrong\u003eA. \u003c/strong\u003eDorsolateral view of the whole specimen, \u003cstrong\u003eB. \u003c/strong\u003elateral view, and \u003cstrong\u003eC\u003c/strong\u003e. ventral view. Scale: 30 µm.\u003c/p\u003e","description":"","filename":"FigS3Supplementarymaterial.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7189628/v1/7141295ab0200c6e89e8563e.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rediscovery and neotype designation of Dichaetura surreyi Martin, 1990 (Gastrotricha: Paucitubulatina) from Northern Germany with a threefold morphological examination with a specimen-saving method","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe Northwestern German lowlands feature a diverse range of geographical physiognomies, including marshlands along the coastline of the North Sea intertwined with slightly higher leveling areas of so-called ‘Geest’, former pleistocene deposits with predominantly sandy and barren soils, and with vast moor areas (Pott \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Becker and Siegmüller \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Historically, to manage rainwater and groundwater seepage, medieval settlers established a network of channels and ditches over centuries to drain the terrain and enhance agricultural land use, such as meadow culture (Herzon and Helenius \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Verdonschot et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), but also for mining peat as a fuel (Hadler et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These channels serve as water buffers, removing excess water during the wet season and retaining it during dry periods (Armitage et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). However, in the present research area, their primary function was to regulate the drainage of inland water in conjunction with the closed line of the coastal dikes, tides and storm floods (Behre \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eContrary to the long-standing assumption that a high aquatic biodiversity is primarily found in larger water bodies, such as rivers and lakes (see, e.g., Boix et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), these drainage systems and small streams are not “deserts” for aquatic biota (Davies et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and recent studies have shown to be important reservoirs for biodiversity, providing habitats for a surprising variety of aquatic organisms and even harboring rare species (Painter \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Malmqvist and Hoffsten \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Oertli et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Armitage et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Williams et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Nicolet et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Biggs et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Heino et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Davies et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; De Bie et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Chester and Robson \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Clifford and Heffernan \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), despite, or perhaps \u003cem\u003ebecause of\u003c/em\u003e, the heavy human usage interference, as exemplified in the current study area by the smell of manure treatment of the surrounding pastureland.\u003c/p\u003e\u003cp\u003eThe various invertebrate taxa commonly found in man-made ditches include macroscopic animals, such as hoverfly larvae, water beetles, and gastropods (e.g., Watson and Ormerod \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Herzon and Helenius \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, relatively few studies have assessed these communities involving multiple taxa or different trophic levels (see Twisk, Noordervliet and Keurs 2000, Armitage et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Davies et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and even fewer studies gave attention to the community of microscopic invertebrates found in ditches (Clare and Edwards \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Richards et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). However, growing evidence suggests that microinvertebrates play a crucial ecological role in diverse interactions within matter and energy networks, acting as intermediaries between macrofauna and microbial loops (see Schratzberger and Ingels \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), but still there is a considerable gap in knowledge of the benthic microinvertebrate community, known as meiobenthos or meiofauna, in these man-made environments (e.g., De Bie et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Mioduchowska and Wojtasik \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe freshwater meiofauna assemblages typically comprise several interstitial taxa, where nematodes, rotifers, copepods and annelids are relatively inventoried (Peralta-Maraver et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), while some taxa, such as Gastrotricha, are still insufficiently studied in artificial environments (Franz \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1950\u003c/span\u003e; Franz and Donner \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1954\u003c/span\u003e; Kolicka et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Suzuki et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kolicka \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Minowa and Garraffoni \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Regarding our current study area and the particular biotope, i.e. artificial agricultural, urban and rural drainage ditches in Northwest Germany, a single study yielded the second record of a rare freshwater dwelling gastrotrich after its original description from a central Italian lake (Kieneke and Riemann \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGastrotricha is traditionally divided into two main taxa: Macrodasyida Remane, 1925, composed of mostly vermiform or tongue-shaped animals bearing several adhesive tubes along the body axis and occurring mostly in the interstice of marine sand; and a possibly paraphyletic order Chaetonotida Remane, 1925, further divided into monogeneric suborder Multitubulatina d’Hondt, 1971 (represented by three marine species of \u003cem\u003eNeodasys\u003c/em\u003e Remane, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1927\u003c/span\u003e within family Neodasyidae Remane, 1929) and monophyletic suborder Paucitubulatina d’Hondt, 1971, represented by 512 either marine or freshwater species of 33 genera within seven families (Balsamo et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Recent phylogenetic studies on Paucitubulatina support its monophyly, comprising the families Xenotrichulidae plus Muselliferidae as the sister group of a clade Oiorpatra formed by the remaining families Chaetonotidae, Dasydytidae and Neogosseidae and a few less species-rich families (Bekkouche and Worsaae \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kolicka et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gammuto et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, several phylogenetic studies endorse the non-monophyly (polyphyly) of the hyperdiverse family Chaetonotidae, encompassing both Dasydytidae and Neogosseidae families nested within (Hochberg and Litvaitis \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Kieneke et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2008b\u003c/span\u003e; Todaro et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kånneby \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Bekkouche and Worsaae \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kolicka et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Minowa and Garraffoni \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), rendering “Chaetonotidae” phylogenetically synonymous (i.e. same last common ancestor) to the whole Oiorpata clade (Gammuto et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nonetheless, the families Proichthydidae and Dichaeturidae are still omitted in molecular studies because of lack of molecular data. Morphological evidence suggest that Dichaeturidae is deeply nested within Paucitubulatina, with either a close affinity to the genus \u003cem\u003eIchthydium\u003c/em\u003e (Kieneke et al. 2008), or to a clade comprising the benthic Proichthydidae and the planktonic Neogosseidae and Dasydytidae families (Hochberg and Litvaitis \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMembers of the freshwater family Dichaeturidae rank among the rarest gastrotrichs known (Balsamo et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Metschnikoff (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1865\u003c/span\u003e) originally established the genus \u003cem\u003eChaetura\u003c/em\u003e to allocate its first species \u003cem\u003eChaetura capricornia\u003c/em\u003e Metschnikoff, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1865\u003c/span\u003e. Later, Murrey (1913) added a second species \u003cem\u003eChaetura piscator\u003c/em\u003e Murray, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1913\u003c/span\u003e, a species never to be seen again. Lauterborn (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1913\u003c/span\u003e) subsequently renamed the genus to \u003cem\u003eDichaetura\u003c/em\u003e to resolve a nomenclatural conflict with a genus of the bird clade of swifts (Apodidae), resulting in \u003cem\u003eDichaetura capricornia\u003c/em\u003e (Metschnikoff, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1865\u003c/span\u003e) and \u003cem\u003eDichaetura piscator\u003c/em\u003e (Murray, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1913\u003c/span\u003e). Martin (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) examined specimens that he initially believed as belonging to the former species, however, upon further study (Martin \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), he recognized distinct morphological differences and described these specimens as a new species, and formally established \u003cem\u003eDichaetura surreyi\u003c/em\u003e Martin, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e. Two decades passed before the discovery of a fourth species, \u003cem\u003eDichaetura filispina\u003c/em\u003e Suzuki, Maeda et Furuya, 2013. Notably, all known records of \u003cem\u003eDichaetura\u003c/em\u003e species (\u003cem\u003eD. filispina\u003c/em\u003e, \u003cem\u003eD. capricornia\u003c/em\u003e, and \u003cem\u003eD. surrey\u003c/em\u003e) come from artificial or anthropogenically influenced habitats, such as rice paddies, open-air cisterns, rural ditches, and the surroundings of metropolitan regions (e.g., Kharkiv). Each record of all four species documents very low abundance, with some descriptions based on a single specimen (e.g. Metschnikoff \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1865\u003c/span\u003e; Suzuki et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Consequently, our understanding of their biology, life cycle, and distribution remains poorly understood.\u003c/p\u003e\u003cp\u003eResearch on these tiny invertebrates also faces inherent challenges due to their fragile nature and diminutive size, often causing specimens to be lost or destroyed during preparation procedures and examination. Additionally, preserved specimens inevitably deteriorate over time, with numerous diagnostic traits dissolving or becoming irretrievable after fixation (Giere \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Fonseca et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Garraffoni et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). As a result, it becomes essential to extract as much information as possible, ideally following an integrative taxonomy framework (see Dayrat \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), from the limited number of specimens to make it digitally accessible (Garraffoni et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo address the notably low abundance of the \u003cem\u003eDichaetura surreyi\u003c/em\u003e in our samples, we employed a triple morphological investigation by retrieving the specimen from each method. First, we conducted detailed differential interference contrast (DIC) light microscopy on living specimens. We then performed fluorescence staining of the musculature, nuclei and cilia for observation under confocal laser scanning microscope (CLSM). Finally, we examined the same individual with a scanning electron microscope (SEM). We propose that this ‘line of production’ can be adapted for other soft-bodied micrometazoans to increase the yield of morphological and anatomical data from single specimens, particularly in cases where specimens are rare and delicate or difficult to obtain. Our integrative investigation of specimens of \u003cem\u003eD. surreyi\u003c/em\u003e revealed further details of the cuticular armament which justify a redescription of the species. Furthermore, newly observed aspects of the species’ musculature are shedding light on the evolution of Paucitubulatina lineages and calling into question a longstanding understanding of a “second pair of adhesive tubes” on the furca of Dichaeturidae.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cb\u003eSampling and morphological methods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFreshwater samples containing floating plants (\u003cem\u003eLemna\u003c/em\u003e spp.) and submerged roots of vascular plants growing along the trench edge were collected from ditches in Logabirumer Hammrich, Leer, Northern Germany (53°13'59''N–53°14'11''N; 7°31'19''E–7°31'20''E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in May 15 and July 22, 2023, using a 65-µm mesh plankton net mounted on an extendable handle made of fiberglass-reinforced plastic. The filtrate was transferred into a sampling jar with ambient water using a squirt bottle for rinsing the plankton net and its collecting jar. Subsamples were transferred to Petri dishes and screened under stereomicroscopes with zoom optics (Leica M80 and M125), employing various illumination modes (substage bright field, oblique illumination and dark field) and magnifications. For microscopic examination and documentation, single specimens were isolated from the Petri dish using a capillary mouth pipette and placed on glass slides with a drop of ambient water. They were covered with a coverslip, and gently clamped by carefully removing the excess water from the edge of the coverslip using a snippet of filter paper. Observations and digital recordings were made using an Olympus BX53 microscope equipped with high-resolution objectives and DIC, and a Euromex HD-Ultra digital camera VC.3036-HDS. After documentation, each specimen was anesthetized with a few microliters of 0.25% Buccain (PUREN Pharma GmbH \u0026amp; Co.KG, München, Germany) before being fixed in freshly prepared 4% formaldehyde buffered in 0.1M Phosphate-buffered saline (PBS, pH 7.4) at 4°C for one hour, for further fluorescence studies according to Kieneke et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008a\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor the examination with the CLSM, each specimen was thoroughly rinsed in 0.1M PBS at pH 7.4 to remove any remnants of the fixative. Each animal was incubated overnight in a 0.1% (v/v) Triton-X-100 solution in 0.1 M PBS for permeabilization. They were stained in a TRITC-phalloidin (#P1951, Sigma-Aldrich, Saint Louis, USA) solution (2 µl of 38 µmol/L TRITC-phalloidin in methanol, diluted in 100 µl of permeabilization buffer after evaporation of the methanol) for 3 hours at 4°C. Some specimens were counterstained with SYBR-Green I (#S9430, Sigma-Aldrich, Saint Louis, USA) nucleic acid stain (one microlitre of 10,000x stock solution added to the phalloidin solution) for another hour. A single specimen was counterstained immunohistochemically against acetylated ∝-tubuline. The indirect immunohistochemical staining was carried out with the primary antibody (monoclonal anti-∝-tubuline IgG2b, produced in mouse clone 6-11B-1, Sigma-Aldrich, Saint Louis USA, cat. nr. T7451) incubated at a dilution of 1:500 (in 0.1 M PBS with Triton-X-100; see above) and for 24 hours at 8°C, followed by the secondary, fluorescently labelled antibody (anti-mouse IgG-FITC-conjugate, produced in goat, Sigma-Aldrich, Saint Louis USA, cat. nr. F0257) incubated at a dilution of 1:100 and for 2 hours at 20°C. In the case of the immunohistochemical staining, a modified PBS buffer containing bovine serum albumin (0.2% w/v), goat serum (6% v/v), Triton-X-100 (0.5% v/v) and NaN\u003csub\u003e3\u003c/sub\u003e (0.05% w/v) as preservative was used to pre-incubate the specimen. All stainings were halted by thoroughly rinsing the specimens several times in 0.1M PBS. Individual specimens were mounted on microscopic slides with a hydrosoluble CitiFluor™ CFP mounting medium plus CitiFluor™ AF100 antifadent solution (#E17978-35, Science Services, Munich, Germany) with binder ring reinforcement stickers as spacers for the coverslip (See Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e1\u003c/span\u003e of Michels and Büntzow \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eObservation was carried out with a Leica TCS SP 5 confocal laser scanning microscope on a DM5000B upright microscope base. We operated the CLSM with the argon laser (488 nm) for excitation of SybrGreen I and Alexa 488 and the DPSS laser (561 nm) for excitation of TRITC using the sequential scanning modus for both detection channels, detection bandwidths of the two photomultipliers were set to 507–534 nm and 570–670 nm, respectively. Each specimen was subsequently recovered from the slide for further investigation by SEM by dissolving the mounting medium again with 0.1M PBS and lifting the coverslip, gently pushing it aside or carefully removing it with Dumont No. 7 precision tweezers, and then retrieving the animal with a copper wire loop attached to a skewer with nail polish.\u003c/p\u003e\u003cp\u003eThe image stacks of the fluorescently stained specimens were either processed as maximum intensity projections and virtual orthogonal sections with the proprietary LAS AF software of Leica (Leica Microsystems GmbH, Wetzlar, Germany), or volume-rendered as well as surface-rendered using the IsoSurface and Voltex algorithms of the Amira 5.0.0 software (Visage Imaging, Inc. San Diego, USA) and Imaris x64 v. 9.9.0 software (Oxford Instruments, Oxfordshire, UK). For the multiple staining datasets, different combinations of visualisation tools have been applied. These processed CLSM datasets have been the basis for a graphical reconstruction of the myoanatomy of \u003cem\u003eD. surreyi\u003c/em\u003e, carried out as vector drawings.\u003c/p\u003e\u003cp\u003ePrior to SEM investigation we applied a well-established preparation method (Kieneke and Zekely \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kieneke et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Trokhymchuk and Kieneke \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003eb\u003c/span\u003e). The animals recovered from the slides for CLSM were placed inside small tube-shaped metal containers (invented by Dr. Wilko Ahlrichs, Oldenburg, Germany) of stainless steel with both ends closed by two copper transmission electron microscopy grids (1500 mesh, hole size 9 µm, catalog number #T1500-CU, Koch Electron Microscopy, São Paulo, Brazil, and #G2670C, Plano GmbH, Wetzlar, Germany), and immersed in 5% ethanol solution. Dehydration was carried out by increasing ethanol solutions in distilled water (5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100% [v/v]; 10 minutes in each step). Critical point desiccation was carried out with a Leica CPD300 automatic critical point dryer using ethanol as intermedium. The desiccated specimens were mounted on a round glass coverslip (⌀ 12 mm) coated with a thin layer of TempFix mounting resin (catalog number #G3305, Plano GmbH, Wetzlar, Germany) using an eyelash glued to a wooden rod. The coverslip with mounted specimens was gently and shortly heated up to 50°C on a heat plate to adhere the specimen to the surface. The thin layer of TempFix was obtained by dissolving a 125 mm³ block of TempFix in 5 ml of chloroform, vigorously mixing it on a single tube vortex machine until completely dissolved, then adding 45 ml of chloroform to sum 50 ml of final solution. A volume of the solution to completely cover the round coverslip was applied with a Pasteur pipette and left to evaporate at room temperature, subsequently heated to 120°C for 60 seconds to smoothen the surface and let it cool to room temperature again before placing the specimens on it. After placing each specimen, the coverslip was adhered to a SEM stub using double-sided adhesive pads and specimens were coated with palladium-gold in a Bal-Tec SCD-050 sputter coater, operated within 150 seconds at 40 mA. SEM examination was performed with a Tescan VEGA3 Scanning Electron Microscope at the Institute Senckenberg am Meer (Wilhelmshaven, Germany), using acceleration voltages of 10 and 30kV and both the secondary and the backscattered electron detectors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular data\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor tissue lysis and DNA extraction, two fresh specimens were added to 35µL of InstaGene™ Matrix (Catalog # 732–6030, Bio-Rad Laboratories, Hercules, USA) and incubated at 56°C for 20 min and subsequently at 100°C for 10 min. 1 µl of the DNA-containing supernatant was used as a template for the amplification. PCR reactions were prepared with illustra™ PuReTaq™ Ready-To-Go™ PCR beads (GE Healthcare UK, Little Chalfont, UK) using a final volume of 25 µl (1 µl of each primer at 10 µM, 1 µl of template DNA and 22 µl of cleaned water for molecular biology purpose).\u003c/p\u003e\u003cp\u003eWe used universal primers LCO1490 and HCO2198 for amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene (Folmer et al., 1994). The parameters for PCR were as follows: an initial denaturation of 3 min at 94°C was followed by 40 cycles of 0.5 min at 94°C, 1 min at 48°C and 1 min at 72°C, with an additional final elongation step of 7 min at 72°C. Each PCR reaction product was checked for length and approximated DNA yield by running 1 µL of the PCR product on 1% agarose gels with 1x nucleic acid stain GelRedTM (Biotium Inc., Hayward, CA, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll available Chaetonotida sequences of the COI gene were retrieved from GenBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genbank/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with accession number provided in Supporting Information (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The mitochondrial protein-coding COI sequences were aligned in MEGA X (Kumar et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) using the Invertebrate Mitochondrial genetic code and the MUSCLE algorithm for codon-based alignment. The aligned sequences were subsequently trimmed on their ends in BioEdit v.7.2.5 (Hall \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) to achieve a uniform length. The final alignment comprised 662 nucleotide sites for 211 terminal sequences covering 79 species of Paucitubulatina.\u003c/p\u003e\u003cp\u003ePhylogenetic reconstruction was conducted using the Maximum Likelihood (ML) approach in IQ-TREE v.1.6.10 (Nguyen et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The best-fit substitution model was estimated with ModelFinder (Kalyaanamoorthy et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) under Bayesian information criterion and Akaike information criterion, selecting GTR + F + I(= 0.3191) + Γ\u003csub\u003e4\u003c/sub\u003e(= 0.6608). Branch support was assessed using ultrafast bootstrap approximation with 1,000 pseudo-replicates (Hoang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and Shimodaira-Hasegawa approximate likelihood ratio test with 1,000 replicates (Guindon et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The resulting trees were rooted using the midpoint method in FigTree v1.4.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eData accessibility\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMicrophotographs of the neotype and four additional specimens were deposited in the Museu de Diversidade Biológica (MDBio) at University of Campinas (Campinas, Brazil) under accession number ZUEC-PIC 1207 and ZUEC-VID 1345 (neotype) and ZUEC-PIC ZUEC-PIC 1206, 1208–1210 and ZUEC-VID 1344, 1346–1348 (additional material). Following the suggestion of Kieneke \u0026amp; Nikoukar (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the four additional specimens shall serve as novel paratypes (“neo-paratypes”), a category that is not yet included and defined by the ICZN (international code of zoological nomenclature) but considered a quite helpful category of type specimens. The neotype specimen and the second specimen used for DNA extraction are only present as a series of digital micrographs (ZUEC-PIC 1206, 1208). The admissibility of specimens only available as a digitalized version as type specimens is comprehensively discussed in Garraffoni et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). The four gold-palladium-coated animals on an aluminum-SEM-stub are deposited in the Gastrotricha collection of the Senckenberg Research Institute and Natural History Museum (Frankfurt am Main, Germany) under accession numbers SMF XXXX-YYYY.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eTaxonomic accounts\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePhylum Gastrotricha Metschnikoff, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1865\u003c/span\u003e\u003c/p\u003e\u003cp\u003eOrder Chaetonotida Remane, 1925 [Rao \u0026amp; Clausen, 1970]\u003c/p\u003e\u003cp\u003eSuboreder Paucitubulatina d'Hondt, 1971\u003c/p\u003e\u003cp\u003eFamily Dichaeturidae Remane \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1927\u003c/span\u003e\u003c/p\u003e\u003cp\u003eGenus \u003cem\u003eDichaetura\u003c/em\u003e Lauterborn, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1913\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eDichaetura surreyi\u003c/em\u003e Martin, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003e; Suppl. Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u0026ndash;3; Table\u0026nbsp;1)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEmended diagnosis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eElongated, cylindrical body measuring 168\u0026ndash;210 \u0026micro;m in total length, including the furca. Head region wide, with a weakly narrowed neck, and slightly broader trunk tapering to a narrow caudal region. Caudal furca 23\u0026ndash;29 \u0026micro;m in length, with a cylindrical adhesive tube with almost constant width, only tapering weakly to blunt distal tip, and with a central groove from base to distal tip. Pharynx with one indistinct pharyngeal thickening on the posterior portion, connected to a cylindrical intestine by a subtle pharyngo-intestinal junction. Pharynx flanked by a pair of pharyngeal glands probably opening at the lateral edges of the buccal cavity and widening to a droplet-shaped bulb at the posterior portion of the pharynx. Head rounded, with a large cephalion and small dorsolateral, triangular pleurae and a ventral transverse rod-shaped hypostomion. Mouth opening round, supported by strong, thorn-like braces protruding slightly from the buccal cavity. Two pairs of dorsal sensory bristles insert directly into the cuticle at the anterior head. Sensory bristles absent from neck and caudal regions. Dorsal body surface is covered with 12\u0026ndash;19 longitudinal columns of 50\u0026ndash;70 small round and short-spined scales with almost constant size along the body. A patch of rounded scales with elongated spines arranged in 7 columns of 6\u0026ndash;7 scales on the posterior dorsal trunk. Ventral interciliary field covered by 8 longitudinal columns of 50\u0026ndash;60 small, roundish, smooth scales, each with a posterior free margin extending into a sharp tip. A triangular patch of 5 transverse rows with 4 large keeled scales with a long posterior spiny process at the posterior ventral trunk. Terminal plates consist of a pair of keeled elongated scales with prominent posterior spines. Each furcal branch is medially armed with a large, curved, hook-like scale. A single, unpaired scale with a thick blunt-end spine is present medially at the furcal indentation, posterior to the terminal plates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMorphological redescription\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis redescription and the ranges of measurements are based on 5 adult specimens. The species has an elongated oval to cylindrical body with 168\u0026ndash;212 \u0026micro;m in total length, with wide head (24\u0026ndash;29 \u0026micro;m wide at U04), a weakly narrowed neck (22\u0026ndash;27 \u0026micro;m wide at U17), and a slightly wider trunk (19\u0026ndash;36 \u0026micro;m wide at U50) with narrower caudal region (Figs.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The caudal furca is 23\u0026ndash;29 \u0026micro;m in length, with cylindrical, 11\u0026ndash;17 \u0026micro;m long adhesive tubes, of almost constant width of 1\u0026ndash;2 \u0026micro;m, only tapering gradually towards their distal blunt tips (Figs.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The adhesive tubes are grooved from the base to the distal tip (Figs.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e9\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eThe pharynx is 36\u0026ndash;50 \u0026micro;m in length from the posterior edge of terminal mouth to pharyngo-intestinal junction at U26 (7\u0026ndash;13 \u0026micro;m wide) with posterior pharynx thickening of 14\u0026ndash;18 \u0026micro;m wide (U17). Posteriorly, the pharynx is followed by a cylindrical intestine of 89\u0026ndash;119 \u0026micro;m length, which is slightly narrowing posteriorly (Figs.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The pharynx is laterally flanked by paired pharyngeal glands from U03 to U15, with unknown function (Figs.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). The glands insert anteriorly to the lateral edges of the mouth ring, probably discharging into the buccal cavity, and widen to droplet-shaped bulbs at the posterior portion of the pharynx (Figs.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003eThe animals have a rounded head with a clearly visible large cephalion (12\u0026ndash;19 \u0026micro;m long x 13\u0026ndash;20 \u0026micro;m wide) with rounded anterior edge, and two small lateral protrusions, followed by convex posterior edge (Figs.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Furthermore, one pair of rather small triangular dorsolateral pleurae with fingerprint-like surface texture posterior to the cephalion and partly covered by its posterior-lateral edges (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B), and a transversal, rod-shaped hypostomion on the ventral side of the head (3\u0026ndash;4 \u0026micro;m long x 8\u0026ndash;10 \u0026micro;m wide) (Figs.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, \u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). The round mouth opening (6\u0026ndash;10 \u0026micro;m diameter) is supported by strong and thorn-like braces that slightly emerge from the buccal cavity. Two paired dorsal sensory bristles (10\u0026ndash;17 \u0026micro;m long) insert directly to the cuticle at U03 (Figs, 3B, 4B, D). There are no tactile bristles on the neck region, but one pair of tactile bristles on the rear end (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e9\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe dorsal head and body are covered by 12\u0026ndash;19 longitudinal columns of 50\u0026ndash;70 small (3.0 \u0026micro;m long x 1.0 \u0026micro;m wide at U17) round scales, each with a thin and short spine (2.7 \u0026micro;m from posterior scale edge to tip), with constant shape along the body axis, only slightly varying in size, smallest on the dorsal head, and increasing in size toward the pharyngeal region and mid trunk (3.6 \u0026micro;m long x 0.7 \u0026micro;m wide at U77) (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e). On the dorsal posterior trunk, approximately between U84 and U89, there is a patch of small rounded scales each with a thin elongate spine (12\u0026ndash;15 \u0026micro;m), arranged in 7 columns of 6\u0026ndash;7 scales each (Figs.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D, \u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026ndash;D).\u003c/p\u003e\u003cp\u003eThe ventral interciliary area is covered by 8 longitudinal columns of 50\u0026ndash;60 small (3.6 \u0026micro;m long x 0.7 \u0026micro;m wide) roundish smooth scales with posterior portion free and extending toward a sharp tip (Figs.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). In the posterior ventral surface around U95, there is a triangular patch of 5 transversal rows with 4 long keeled scales with a long spiny process (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C), all distinctly larger (7.4 \u0026micro;m long x 1.3 \u0026micro;m wide) than usual ventral interciliary scales. A pair of terminal, keeled, long, and elliptical scales at U98 with a long, spiny posterior projection (i.e. terminal plates) (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Each branch of the furca\u0026rsquo;s base is medially armed with one strong and large scale with a thick curved and hook-like spine, laterally on the furcal branches there are tiny scales similar to those of the trunk (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). Medially at the furcal indentation, posterior to the terminal plates, there is a single, unpaired scale with a thick spine with a blunt end (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eVentral ciliation consists of a dense field of cilia posterior to the mouth (U03), continuing into two parallel bands on each side of the trunk (Figs.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, C, D, \u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). The distance between both ciliary bands (=\u0026thinsp;width of the interciliary field) is narrowing toward the posterior trunk end and measures 4.98 \u0026micro;m, 10.5 \u0026micro;m, and 4 \u0026micro;m at U11, U49, and U83, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Fluorescent staining in combination with CLSM furthermore shows that cilia in the pharyngeal region also insert on the lateral sides and almost form an anterior ring of cilia that is dorsally interrupted, though (Fig. S3A\u0026ndash;C).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhenotypic variance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe shape and general characteristics among the five specimens studied are congruent, with only little total size or specific feature length variance (Table\u0026nbsp;1). However, we were able to distinguish two variants regarding differences in dorsal/dorso-lateral and ventral cuticular ornamentation that were only detectable with the resolution power of the SEM. One variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;D) has scales with shorter, delicate spines of 3.0\u0026ndash;4.1 \u0026micro;m lengths covering the dorsal, lateral and ventral body (Figs.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The other variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e) has scales bearing slightly longer and thicker spines of 5.6\u0026ndash;7.3 \u0026micro;m lengths (Figs.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMyoanatomy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe description of the muscular anatomy of \u003cem\u003eDichaetura surreyi\u003c/em\u003e is based on three adult specimens examined with the CLSM. The muscular architecture in somatic and splanchnic regions consisted of longitudinal, circular, and helicoidal muscles (Figs.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003e). In the splanchnic region, up to 35 complete circular muscles occur along the pharynx (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003eA\u0026ndash;C). Complete circular muscles are absent in the intestinal region. Helicoidal muscles are very thin and extend from the mouth ring up to the mid-body, wrapping all splanchnic longitudinal muscles plus one splanchnic branch of the otherwise somatic ventrolateral longitudinal muscle on the pharynx region (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003eA\u0026ndash;C, G). Longitudinal muscles are present in dorsal, ventral, and lateral positions and either splanchnic or somatic, and mostly span the entire body length, insert anteriorly in the head region close to the mouth opening and extend posteriorly into the rearmost region of the trunk, roughly at the position of the anus, where they fuse in a single complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003eA\u0026ndash;C). Only the ventrolateral longitudinal muscles extend into the furcal branches (see below). Two or three short muscle fibres occur close to the ventral anterior part of the pharynx at U02, probably inserting into the mouth opening at the ventral body.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDorsally, there are two pairs of dorsal longitudinal muscles. The first pair of these muscles is inserted anteriorly on the mouth ring lining up the entire dorsal body and possibly connecting each other at U04. This dorsal longitudinal muscle branches posteriorly in a pair of thin muscles (\u003cem\u003eR\u0026uuml;ckenhautmuskel\u003c/em\u003e) at U72, which, in turn, is inserted dorsally to the integument at U41 (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003eA, F\u0026ndash;I). These muscles, apart from the \u003cem\u003eR\u0026uuml;ckenhautmuskel\u003c/em\u003e-branch, are splanchnic and warped by helicoidal muscles in the pharynx region but are not in the intestine region. A second pair of dorsal longitudinal muscles is anteriorly inserted to the head integument close to the insertion point of the first muscle pair, and extends in a splanchnic position along the entire gut tube (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003eA, F\u0026ndash;I). Both dorsal longitudinal muscles seem to fuse a short distance posterior to the anus.\u003c/p\u003e\u003cp\u003eVentrally, there are three pairs of longitudinal muscles. A pair of ventrolateral longitudinal muscles inserts slightly ventrolateral in the furcal base and continues a short distance into the proximal portion of the adhesive tubes. Each muscle stretches along the trunk in a somatic position up to the posterior swelling of the pharynx close to the pharyngeal-intestinal junction (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003eA\u0026ndash;C, E\u0026ndash;I). At about U29 each ventrolateral longitudinal muscle splits into two branches. The more lateral branches insert laterally to the head integument at U07, while the more median branches follow closely the contours of the pharynx, insert in the integument close to the anterior end of the pharynx and are enwrapped by splanchnic circular and helicoidal muscles (Figs.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e10\u003c/span\u003eA, B, \u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003eA\u0026ndash;B, E). Hence, parts of the ventrolateral longitudinal muscle pair occupy a somatic position, while others are splanchnic. There is a pair of ventral longitudinal muscles that is anteriorly inserted into the integument close to the mouth opening, splanchnic in the pharyngeal region, where it is warped by helicoidal muscles, and somatic in the intestine region. A second pair of ventral longitudinal muscles, slightly more median than the latter pair, is inserted anteriorly to the head integument and stretches along the entire gut tube, always remaining in a splanchnic position (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003eB, F, H\u0026ndash;I). Both pairs of ventral longitudinal muscles fuse with the pair of ventrolateral longitudinal muscles in the region of the anus at U97 (Figs.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e10\u003c/span\u003eB, \u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003eI). Two pairs of smaller dorsal muscular branches emerge from the ventrolateral longitudinal muscles at U97 and insert dorsally and slightly more posterior at U98, respectively. Both branches are inserted in the integument at the level of the posteriormost patch of long and thin spines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNuclei distribution\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cellular nuclei distribution of \u003cem\u003eDichaetura surreyi\u003c/em\u003e is described from SYBR-Green I nucleic acid staining and reflects a general architecture within Paucitubulatina, with high density of nuclei in the anterior third of the body (pharyngeal region) and especially dorsally and dorsolaterally of the mid-pharynx region (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). This dense arrangement of cell nuclei corresponds with the two hemispheres of the bilateral cerebral ganglion of Gastrotricha. Areas without any DNA-signals within this dorsal to dorso-lateral aggregation of nuclei much likely reflect the dorsal commissure(s) inside the cerebral ganglion of \u003cem\u003eD. surreyi\u003c/em\u003e (Fig. S2A, B). Also at the posterior end there is a higher density of nuclei, probably associated with adhesive glands Fig. S2A\u0026ndash;C). The egg was diffusely stained and fills the whole dorsal trunk (Figs. S1C, D, S2A, C), this fluorescence pattern possibly indicates a high level of gene expression since Sybr Green I also stains single-stranded DNA and RNA. Along the trunk there are only scattered nucleic patterns that probably belong to muscle, epidermis and intestinal cells (Fig. S2A\u0026ndash;C).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTaxonomic Remarks\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe original description and the newly recorded animals both have an elongated, cylindrical body shape, tapering towards the caudal region and only with a weak depression of the pharyngeal region. However, present specimens tend to be larger (168\u0026ndash;212 \u0026micro;m in total length; current study) compared to the original description (102\u0026ndash;150 \u0026micro;m overall length; Martin \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). The pharynx dimensions of the original description (38 \u0026micro;m) fall within the newly recorded range (36\u0026ndash;50 \u0026micro;m), the same is true for the head width (27 \u0026micro;m vs 24\u0026ndash;29 \u0026micro;m). Furthermore, both sets of specimens exhibit similar patterns of the dorsal scales, with the posteriormost patch of about 70 small round scales with long, thin, slightly curved simple spines. Also the peculiar hook-shaped spined scales at the medial faces of the furca show a highly similar pattern between the original description and the specimens investigated by us. \u003cem\u003eDichaetura surreyi\u003c/em\u003e is well delineated from the most similar species \u003cem\u003eDichaetura filispina\u003c/em\u003e by the presence of numerous finely spined scales on the dorsal and lateral surfaces of the whole trunk, which are absent from the latter (Suzuki et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Balsamo et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Despite slight variance in cuticular ornamentation among the five specimens from the identical sampling site, all measurements and myoanatomical features are congruent, strongly indicating a single entity.\u003c/p\u003e\u003cp\u003eThe original description of \u003cem\u003eDichaetura surreyi\u003c/em\u003e of Martin (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) is missing several data on structures that are recently important for a proper morphological species delineation. We were able to provide detailed counts of scale columns and the number of scales per column and to resolve the shape of the trunk scales. Furthermore, our measurements of the cephalion and hypostomion are also detailed in the new specimens, not mentioned at all in the original description, and we were able to confirm the presence of one pair of pleurae on the head section. We were also able to clarify the morphology of the basal scales bearing the hook-shaped spines at the medial margins of the furca. Therefore, providing a redescription of \u003cem\u003eD. surreyi\u003c/em\u003e is well-justified.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDesignation of a neotype.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe present study allows us to deposit a neotype specimen of \u003cem\u003eDichaetura surreyi\u003c/em\u003e according to article 75 of the International Code of Zoological Nomenclature/ICZN (International Commission on Zoological Nomenclature, 1999). Upon publication of this study, the digital neotype specimen (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;D, \u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e10\u003c/span\u003e) will be added to the scientific collection of the Museu de Diversidade Biol\u0026oacute;gica (MDBio) at University of Campinas under the collection number ZUEC-PIC 1206. A digitized fresh specimen facilitates the best long-term preservation over every other method linked to a physical specimen (see Garraffoni et al. 2019). According to the ICZN, a number of qualifying conditions need to be fulfilled in order to designate a name-bearing neotype validly (Article 75.3 ICZN). We will now present our qualifying conditions to support the designation of a neotype specimen of \u003cem\u003eD. surreyi\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eBecause of the incompleteness of the original description (see \u0026lsquo;taxonomic remarks\u0026rsquo; of the current study), a future delineation from further similar but distinct species cannot be guaranteed and could lead to taxonomic confusion. We designate the neotype specimen in order to corroborate the taxonomic status of \u003cem\u003eD. surreyi\u003c/em\u003e (Article 75.3.1 ICZN). As can be read in Balsamo et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), the body cuticle made of numerous fine spined scales distinguishes \u003cem\u003eD. surreyi\u003c/em\u003e from other species of \u003cem\u003eDichaetura\u003c/em\u003e (Article 75.3.2 ICZN). The designated neotype specimen is a digitized individual that was imaged alive (see above; Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;D, \u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Its collection number is ZUEC-PIC 1206 (Article 75.3.3 ICZN). Furthermore, DNA sequences of the \u0026lsquo;neoparatype\u0026rsquo; specimens with the accession numbers 1206 and 1208 are available. Martin (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1981\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) nowhere mentioned the deposition of name-bearing type specimens, neither a holotype, nor a syntype series. As far as we know, the author had no institutional affiliation and no access to a museal scientific collection and it is plausible that type specimens were never preserved and deposited (Article 75.3.4 ICZN). Therefore, we regard the drawing of \u003cem\u003eD. surreyi\u003c/em\u003e in Martin\u0026rsquo;s (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e as the holotype specimen that no longer exists (Article 73.1.4 ICZN). A comparison of our Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e of Martin (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) demonstrates consistency between the original holotype specimen and the designated neotype specimen (Article 75.3.5 ICZN). The designated neotype specimen was sampled from quite a distance to the original type locality (northwest Germany versus southeast England), however, both locations are in the same climatological and vegetational zone and only a few millennia ago the British Island and Europe were connected by a wide land bridge (Gupta et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore the origin of the neotype specimen is suitably close enough to the original type locality (Article 75.3.6 ICZN). Both the no longer existing holotype specimen and the designated neotype specimen were furthermore extracted from similar habitats, i.e. the surface mud of stagnant water bodies and among floating and submerged vegetation (Martin \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1981\u003c/span\u003e, present study). The designated digital neotype specimen is the property of the scientific collection of the Museu de Diversidade Biol\u0026oacute;gica (MDBio) at University of Campinas under the collection number ZUEC-PIC 1207 and is available for further scientific studies (Article 75.3.7 ICZN).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe phylogenetic analysis of 211 COI sequences from the suborder Paucitubulatina resulted in a best-scoring ML topology with a log-likelihood of -33200.0016 (Fig.\u0026nbsp;\u003cspan refid=\"Fig22\" class=\"InternalRef\"\u003e11\u003c/span\u003e). The two \u003cem\u003eDichaetura surreyi\u003c/em\u003e terminals formed a fully supported clade and is placed as a sister group to \u003cem\u003ePolymerurus\u003c/em\u003e species with weak support. In turn, this clade is the sister lineage to Dasydytidae and Neogosseidae. After midpoint rooting, the inferred phylogeny showed \u003cem\u003eXenotrichula\u003c/em\u003e sp. (TK2012) as sister group of the clade comprising \u003cem\u003eDraculiciteria tesselata\u003c/em\u003e (MT63 and TK142) and the remaining members of Paucitubulatina included in our analysis. Some regions of the tree have quite long branches and some species that were included with multiple sequences even show comparatively long intraspecific branches (e.g., \u003cem\u003ePolymerurus nodicaudus\u003c/em\u003e, \u003cem\u003eChaetonotus daphnes\u003c/em\u003e, \u003cem\u003eDendroichthydium ibryapora\u003c/em\u003e), while others have almost no intraspecific divergence (e.g., \u003cem\u003eChaetonotus slavicus\u003c/em\u003e, \u003cem\u003eChaetonotus mirabilis\u003c/em\u003e, \u003cem\u003eHeterolepidoderma acidophilum\u003c/em\u003e).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cb\u003eMorphology and systematics of Dichaeturidae\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUpdating knowledge as new evidence emerges is an integral part of the scientific process, and SEM imagery of \u003cem\u003eDichaetura surreyi\u003c/em\u003e furcal branches can help reinterpret earlier descriptions. Regarding the taxonomic history of the group, the most distinctive trait in Dichaeturidae has long been regarded as the presence of a \u0026ldquo;second pair of adhesive tubes on the furca\u0026rdquo; (Metschnikoff \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1865\u003c/span\u003e), a condition otherwise known only in \u003cem\u003eDiuronotus\u003c/em\u003e Todaro, Balsamo \u003cem\u003eet\u003c/em\u003e Kristensen, 2005 (Muselliferidae) and, with even two additional pairs of \u0026lsquo;accessory posterior adhesive tubes\u0026rsquo;, in \u003cem\u003eAspidiophorus multitubulatus\u003c/em\u003e Hummon, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1974\u003c/span\u003e (Chaetonotidae) among Paucitubulatina (Hummon \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Todaro et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, Martin (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) describes these as \u0026ldquo;a hook [that] arises from the inner side of each toe and projects upwards at an angle\u0026rdquo;, rather than functional adhesive tubes. Also Todaro et al. (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) question the existence of a second pair of posterior adhesive tubes in \u003cem\u003eDichaetura\u003c/em\u003e. This condition was later supported by the description of \u003cem\u003eD. filispina\u003c/em\u003e, which bears one pair of adhesive tubes and a solid spine on each furcal branch (Suzuki et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), reaffirming the doubts about the existence of a second pair of adhesive tubes in Dichaeturidae (Todaro et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kieneke and Schmidt-Rhaesa \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Our SEM examinations of the furca of \u003cem\u003eD. surreyi\u003c/em\u003e finally confirm the existence of a pair of modified scales partially \u0026ldquo;wrapping\u0026rdquo; the medial edge of each branch and bearing a pair of thick and curved, horn-shaped spines, and even with a peek under the scale. Nonetheless, future attempts for transmission electron microscope imagery of the furcal branches are highly desirable to further detail the cuticular structure of the horn-shaped spines and its basal furcal scales.\u003c/p\u003e\u003cp\u003e\u003cem\u003eDichaetura piscator\u003c/em\u003e was studied in the best knowledge of its time (Murray \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1913\u003c/span\u003e), but the description lacks several details and a reinvestigation using every state-of-the-art technology is urgently needed for clarifying its systematic status. However, it is hard to ignore the significant distinctions compared to other \u003cem\u003eDichaetura\u003c/em\u003e species: indeed, it shows closer morphological resemblance to the dubious freshwater macrodasyidan \u003cem\u003eMarinellina flagellata\u003c/em\u003e Ruttner-Kolisko, 1955, or to a juvenile \u003cem\u003eRedudasys\u003c/em\u003e Kisielewski, 1987, e.g., a juvenile \u003cem\u003eRedudasys neotemperatus\u003c/em\u003e K\u0026aring;nneby \u0026amp; Kirk, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e3\u003c/span\u003eb of K\u0026aring;nneby and Kirk \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), than to other \u003cem\u003eDichaetura\u003c/em\u003e. \u003cem\u003eDichaetura piscator\u003c/em\u003e is described as a small animal (150 \u0026micro;m) with a spindle-shaped body and a separated head, with irregular undulating folds, bearing numerous long sensory hairs and c-shaped curved bristles (cilia) along the body. Most importantly, \u003cem\u003eD. piscator\u003c/em\u003e has two adhesive tubes of equal-length on each furcal branch, an undeniable pattern that could fit to a juvenile \u003cem\u003eRedudasys\u003c/em\u003e, maybe with fixation artifacts on sensory cilia curling into c-shape (see Kieneke et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, p. 45 Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D for a comparable artifact). Conversely, \u003cem\u003eDichaetura capricornia\u003c/em\u003e, the first described species and type species of the genus (Remane \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1927\u003c/span\u003e, p. 283), originally placed to the genus \u003cem\u003eChaetura\u003c/em\u003e by Metschnikoff (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1865\u003c/span\u003e), shares diagnostic features with the other members of \u003cem\u003eDichaetura\u003c/em\u003e (Suzuki \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), such as the arrangement of ventral locomotory cilia in organized perpendicular rows along the longitudinal columns, wider on the pharyngeal region and narrowing toward pharyngo-intestinal region and the posterior end (our Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Supplementary Material S3 vs Martin \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, p. 477, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Suzuki \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, p. 21, Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e8\u003c/span\u003e), and a dorsal posterior patch of small scales with thin long spines (our Figs.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003eC vs Martin \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, p. 477, Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Although there is some resemblance of the shape of the posterior furca between \u003cem\u003eC. capricornia\u003c/em\u003e and the two species \u003cem\u003eD. surreyi\u003c/em\u003e and \u003cem\u003eD. filispina\u003c/em\u003e, it is definitely different. Both posterior adhesive tubes of \u003cem\u003eD. capricornia\u003c/em\u003e appear elongated, rather thin and pointed at their distal tip (see Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003e of Martin \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). On the medial margin, each tube has a sharp denticle at a position where the horn-shaped spine occurs in \u003cem\u003eD. surreyi\u003c/em\u003e and \u003cem\u003eD. filispina\u003c/em\u003e (compare, e.g., Figs.\u0026nbsp;8.44, 8.45 and 8.47 of Balsamo et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Also the median unpaired spine protruding into the indentation of the furcal bases seems to be absent in \u003cem\u003eD. capricornia\u003c/em\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e2\u003c/span\u003e of Metschnikoff \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1865\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003e of Martin \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConcludingly, it seems likely to us that both \u003cem\u003eDichaetura surreyi\u003c/em\u003e and \u003cem\u003eD. filispina\u003c/em\u003e represent a pair of sister species and the genus type species \u003cem\u003eD. capricornia\u003c/em\u003e is phylogenetically related to the latter two species. The sharp denticles of the furca of \u003cem\u003eD. capricornia\u003c/em\u003e could either represent predecessors of the horn-shaped spines of the other two species, or it might be a vestigial form. It seems furthermore plausible that the fourth nominal species \u003cem\u003eDichaetura piscator\u003c/em\u003e does not belong to the genus and rather is a member of a different order of Gastrotricha, viz. the Macrodasyida and could be related to the only freshwater-dwelling genera of that clade, \u003cem\u003eRedudasys\u003c/em\u003e and \u003cem\u003eMarinellina\u003c/em\u003e. For testing these hypotheses of course new specimens of at least \u003cem\u003eD. piscator\u003c/em\u003e and \u003cem\u003eD. capricornia\u003c/em\u003e are urgently needed for comprehensive morphological and molecular investigations. We think in this context it is worth mentioning that the type locality of the latter species (\u003cem\u003eD. capricornia\u003c/em\u003e) close to Kharkiv (Ukraine) is currently under heavy impact due to an unlawful war of aggression against an independent democratic state imposed by an autocratic state. At worst, this man-made impact could result in the extinction of this species.\u003c/p\u003e\u003cp\u003eOur primary aim of the molecular analysis is neither to reconstruct the phylogeny of Paucitubulatina, nor to definitely clarify the systematic position of \u003cem\u003eDichaetura surreyi\u003c/em\u003e, but the ML tree effectively demonstrates the utility of COI as a suitable barcode marker. Comparably long branches probably indicate a high level of mutation saturation of positions in the COI gene, which makes this mitochondrial gene particularly unsuitable for a reliable reconstruction of phylogeny (DeSalle et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, including multiple specimens per species, where feasible, shows a pattern of low intraspecific variation versus high interspecific divergence and therefore the existence of a \u0026lsquo;barcode gap\u0026rsquo;, a prerequisite for a suitable barcode sequence (Hebert et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparative myoanatomy of Paucitubulatina\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe muscular architecture of Paucitubulatina has so far been documented in 23 species using epifluorescence or confocal microscopy, consistently revealing a conserved ancestral pattern of muscle architecture, consisting of circular and helicoidal muscles around the pharynx and intestine, four pairs of longitudinal muscles (dorsal, lateral, ventrolateral, and ventral), and a paired \u003cem\u003eR\u0026uuml;ckenhautmuskel\u003c/em\u003e (Hochberg and Litvaitis \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hochberg \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Leasi et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kieneke et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008a\u003c/span\u003e; Leasi and Todaro \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kieneke and Ostmann \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Bekkouche and Worsaae \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; M\u0026uuml;nter and Kieneke \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Minowa et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The musculature of \u003cem\u003eDichaetura surreyi\u003c/em\u003e aligns to the aforementioned Paucitubulatina pattern, confirming the ancestral set of muscle components of the last common ancestor of Paucitubulatina. But it also displays previously undescribed muscular branchings, including two additional pairs of longitudinal muscles in the splanchnic region, one dorsal and one ventral pair, as well as two or three muscular fibers at the anterior part of the pharynx. Another notable difference to the ancestral muscular pattern is the bifurcation of the ventrolateral longitudinal muscle in the pharyngeal region. Furthermore,thin muscle branches insert into the dorsal integument at the furcal base in the area of the elongate posterior spines (dmb in Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e10\u003c/span\u003e), indicating a hypothesized functional role related to a protective behavior (see below). To date, only four dasydytid species have been investigated with CLSM (Kieneke et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008a\u003c/span\u003e; Kieneke and Ostmann \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) to determine spine-associated musculature, making \u003cem\u003eDichaetura surreyi\u003c/em\u003e probably the second recorded Gastrotricha lineage with such a set of muscles responsible for a movement of cuticular structures.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDefensive behavior\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeveral lineages within Paucitubulatina have a protective cuticle featuring spines and scales that shield against mechanical stress from the surrounding rough environment and defense from predation; Dichaeturidae is no exception. Many \u003cem\u003eChaetonotus\u003c/em\u003e species, for instance, display a defensive strategy of curling into a ball\u0026mdash;similar to a hedgehog (See Schwank \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), to expose their armored dorsal cuticle, while protecting the smoother belly, offering defense against heliozoans and amoebozoans (Brunson \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1949\u003c/span\u003e; Bovee and Cordell \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1971\u003c/span\u003e), or, as documented most recently, against tentaculiferous ciliates of the genus \u003cem\u003eLegendrea\u003c/em\u003e\u0026mdash;although evade can still fail (Pomahač et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Certain members of the \u003cem\u003eChaetonotus\u003c/em\u003e subgenus \u003cem\u003eZonochaeta\u003c/em\u003e further exhibit a specialized dorsal girdle of scales with longer, thicker spines that can abduct, likely aided by dorsal musculature (Kisielewski \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Likewise, some freshwater semiplanktonic and pelagic lineages in the Dasydytidae possess elongated saltatory spines associated with a specific set of muscles derived from segmentation of lateral and ventral longitudinal muscles, enabling evasive quick \u0026ldquo;jumps\u0026rdquo; in the water column when disturbed, and other lineages spreading these spines radially for protection (Kieneke et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008a\u003c/span\u003e; Kieneke and Ostmann \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Minowa and Garraffoni \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; see also Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e5\u003c/span\u003eb of Schwank \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). In a similar fashion, living specimens of \u003cem\u003eDichaetura surreyi\u003c/em\u003e observed in the course of this study displayed a defensive behavior when threatened, curving the posterior portion of the body and raising the spines of the posterior patch on the dorsal furcal base (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;C).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpecimen-saving preparation for multiple purposes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur methodology successfully yielded behavioral and morphological information from light microscopy of a living individual, anatomical data from confocal laser scanning microscopy (CLSM), and finally high-resolution morphological data from electron scanning microscopy (SEM) of the identical specimen of tiny gastrotrichs. This approach supported the redescription of the species \u003cem\u003eDichaetura surreyi\u003c/em\u003e and provided further insights into its muscular architecture and morphological features, while still preserving a depositable specimen for future study. The limited availability of specimens poses a well-known challenge for microinvertebrate zoologists, especially when applying integrative taxonomy approach, and often needs creative strategies to overcome the distressing chances of losing material during or between each morphological method (George and Plum \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Garraffoni et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Bosco et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis work has been registered in ZooBank with registration number urn:lsid:zoobank.org:pub:049836B1-5B99-4D27-83A1-6400A3CB2955\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest.\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partially financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - CAPES/PrInt (nº 88887.716041/2022-00) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (nº 141482/2021-4) grant to AKM. It was also partially funded by the National Science Foundation (NSF) - DEB 2051684 (R. Hochberg). The third author (ARSG) is funded by São Paulo Research Foundation (FAPESP) (Proc: 2014/23856-0; 2018/10313-0; 2023/05724-9) and the Brazilian fostering agency ‘Conselho Nacional de Desenvolvimento Científico e Tecnológico’ through a productivity grant (CNPq proc. 04/2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eA.K.M. conceptualized the study, collected samples, conducted laboratory examinations of the material, data collection and analysis, and drafted the manuscript. T.Q.A. contributed to data collection and analysis and co-authored the manuscript draft. A.R.S.G. participated in data collection and analysis and co-authored the manuscript draft. A.K. conceptualized and supervised the study, collected samples, and co-authored the manuscript draft. All authors reviewed and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArmitage PD, Szoszkiewicz K, Blackburn JH, Nesbitt I (2003) Ditch communities: a major contributor to floodplain biodiversity. Aquat Conserv Mar Freshw Ecosyst 13:165\u0026ndash;185. https://doi.org/10.1002/aqc.549\u003c/li\u003e\n\u003cli\u003eBalsamo M, d\u0026rsquo;Hondt J-L, Grilli P (2019) Phylum Gastrotricha. In: Rogers DC, Thorp J (eds) Thorp and Covich\u0026rsquo;s Freshwater Invertebrates. Elsevier, Amsterdam, Netherlands, pp 149\u0026ndash;218\u003c/li\u003e\n\u003cli\u003eBalsamo M, Grilli P, Guidi L, d\u0026rsquo;Hondt J-L (2014) Gastrotricha: Biology, ecology and systematics. Families Dasydytidae, Dichaeturidae, Neogosseidae, Proichthydiidae. In: Identification Guides to the Plankton and Benthos of Inland Waters. Backhuys Publishers, Weikersheim, Germany\u003c/li\u003e\n\u003cli\u003eBecker T, Siegm\u0026uuml;ller A (2021) The coastal lowland of northwestern Germany as an archive of Holocene landscape evolution: basis for a spatial evaluation of Stone Age settlement patterns in the Dornumer tidal basin. Neth J Geosci. https://doi.org/10.1017/njg.2020.17\u003c/li\u003e\n\u003cli\u003eBehre K-E (2012) Die Geschichte der Landschaft um den Jadebusen: Freisland-Wilhelmshaven-Wesemarsch. Brune-Mettcker Druck-und Verlagsgesellschaft mbH, Wilhelmshaven\u003c/li\u003e\n\u003cli\u003eBehre K-E (2014) Ostfriesland: die Geschichte seiner Landschaft und ihrer Besiedlung. Brune-Mettcker Druck-und Verlagsgesellschaft mbH, Wilhelmshaven\u003c/li\u003e\n\u003cli\u003eBekkouche N, Worsaae K (2016) Neuromuscular study of early branching \u003cem\u003eDiuronotus aspetos\u003c/em\u003e (Paucitubulatina) yields insights into the evolution of organs systems in Gastrotricha. Zool Lett 2:21. https://doi.org/10.1186/s40851-016-0054-3\u003c/li\u003e\n\u003cli\u003eBiggs J, Williams P, Whitfield M, et al (2005) 15 years of pond assessment in Britain: results and lessons learned from the work of Pond Conservation. Aquat Conserv Mar Freshw Ecosyst 15:693\u0026ndash;714. https://doi.org/10.1002/aqc.745\u003c/li\u003e\n\u003cli\u003eBoix D, Biggs J, C\u0026eacute;r\u0026eacute;ghino R, et al (2012) Pond research and management in Europe: \u0026ldquo;Small is Beautiful.\u0026rdquo; Hydrobiologia 689:1\u0026ndash;9. https://doi.org/10.1007/s10750-012-1015-2\u003c/li\u003e\n\u003cli\u003eBosco I, Lourenco AP, Guidi L, et al (2020) Integrative description of a new species of \u003cem\u003eAcanthodasys\u003c/em\u003e Remane, 1927 (Gastrotricha, Macrodasyida, Thaumastodermatidae) based on four distinct morphological techniques and molecular data. Zool Anz 286:31\u0026ndash;42. https://doi.org/10.1016/j.jcz.2020.03.003\u003c/li\u003e\n\u003cli\u003eBovee EC, Cordell DL (1971) Feeding on Gastrotrichs by the Heliozoon \u003cem\u003eActinophrys sol\u003c/em\u003e. Trans Am Microsc Soc 90:365\u0026ndash;369. https://doi.org/10.2307/3225197\u003c/li\u003e\n\u003cli\u003eBrunson RB (1949) The life history and ecology of two North American gastrotrichs. Trans Am Microsc Soc 68:1\u0026ndash;20\u003c/li\u003e\n\u003cli\u003eChester ET, Robson BJ (2013) Anthropogenic refuges for freshwater biodiversity: Their ecological characteristics and management. Biol Conserv 166:64\u0026ndash;75. https://doi.org/10.1016/j.biocon.2013.06.016\u003c/li\u003e\n\u003cli\u003eClare P, Edwards RW (1983) The macroinvertebrate fauna of the drainage channels of the Gwent Levels, South Wales. Freshw Biol 13:205\u0026ndash;225. https://doi.org/10.1111/j.1365-2427.1983.tb00672.x\u003c/li\u003e\n\u003cli\u003eClifford CC, Heffernan JB (2018) Artificial aquatic ecosystems. Water 10:1096. https://doi.org/10.3390/w10081096\u003c/li\u003e\n\u003cli\u003eDavies B, Biggs J, Williams P, et al (2008) Comparative biodiversity of aquatic habitats in the European agricultural landscape. Agric Ecosyst Environ 125:1\u0026ndash;8. https://doi.org/10.1016/j.agee.2007.10.006\u003c/li\u003e\n\u003cli\u003eDayrat B (2005) Towards integrative taxonomy. Biol J Linn Soc 85:407\u0026ndash;417. https://doi.org/10.1111/j.1095-8312.2005.00503.x\u003c/li\u003e\n\u003cli\u003eDe Bie T, Declerck S, Martens K, et al (2008) A comparative analysis of cladoceran communities from different water body types: patterns in community composition and diversity. Hydrobiologia 597:19\u0026ndash;27. https://doi.org/10.1007/s10750-007-9222-y\u003c/li\u003e\n\u003cli\u003eDeSalle R, Egan MG, Siddall M (2005) The unholy trinity: taxonomy, species delimitation and DNA barcoding. Phylosophical Trans R Soc B 1905\u0026ndash;1916. https://doi.org/10.1098/rstb.2005.1722\u003c/li\u003e\n\u003cli\u003eFonseca G, Fontaneto D, Di Domenico M (2018) Addressing biodiversity shortfalls in meiofauna. J Exp Mar Biol Ecol 502:26\u0026ndash;38. https://doi.org/10.1016/j.jembe.2017.05.007\u003c/li\u003e\n\u003cli\u003eFranz H (1950) Bodenzoologie als Grundlage der Bodenpflege mit besonderer Beruecksichtigung der Bodenfauna in den Ostalpen und im Donaubecken. Akademie, Berlin\u003c/li\u003e\n\u003cli\u003eFranz H, Donner J (1954) Gastrotricha. In: Franz H (ed) Die Nordostalpen im Spiegel ihrer Landtierwelt. Wager University Press, Innsbrucks, pp 1\u0026ndash;158\u003c/li\u003e\n\u003cli\u003eGammuto L, Serra V, Petroni G, Todaro MA (2024) Molecular phylogenetic position and description of a new genus and species of freshwater Chaetonotidae (Gastrotricha: Chaetonotida: Paucitubulatina), and the annotation of its mitochondrial genome. Invertebr Syst 38:IS23059. https://doi.org/10.1071/IS23059\u003c/li\u003e\n\u003cli\u003eGarraffoni ARS, Ara\u0026uacute;jo TQ, Louren\u0026ccedil;o AP, et al (2019a) Integrative taxonomy of a new \u003cem\u003eRedudasys\u003c/em\u003e species (Gastrotricha: Macrodasyida) sheds light on the invasion of freshwater habitats by macrodasyids. Sci Rep 9:2067. https://doi.org/10.1038/s41598-018-38033-0\u003c/li\u003e\n\u003cli\u003eGarraffoni ARS, Kieneke A, Kolicka M, et al (2019b) ICZN Declaration 45: a remedy for the nomenclatural and typification dilemma regarding soft-bodied meiofaunal organisms? Mar Biodivers 49:2199\u0026ndash;2207. https://doi.org/10.1007/s12526-019-00983-7\u003c/li\u003e\n\u003cli\u003eGeorge KH, Plum C (2009) Description of two new species of \u003cem\u003eDorsiceratus\u003c/em\u003e Drzycimski, 1967 (Copepoda: Harpacticoida: Ancorabolidae) from Sedlo and Seine Seamounts (Northeastern Atlantic) and remarks on the phylogenetic status of the genus. Zootaxa 2096:257\u0026ndash;286\u003c/li\u003e\n\u003cli\u003eGiere O (2009) Meiobenthology. Springer, Berlin, Heidelberg\u003c/li\u003e\n\u003cli\u003eGuindon S, Dufayard J-F, Lefort V, et al (2010) New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Syst Biol 59:307\u0026ndash;321. https://doi.org/10.1093/sysbio/syq010\u003c/li\u003e\n\u003cli\u003eGupta S, Collier JS, Garcia-Moreno D, et al (2017) Two-stage opening of the Dover Strait and the origin of island Britain. Nat Commun 8:15101. https://doi.org/10.1038/ncomms15101\u003c/li\u003e\n\u003cli\u003eHadler H, Rei\u0026szlig; A, Willersh\u0026auml;user T, et al (2025) Medieval Overexploitation of Peat Triggered Large-Scale Drowning and Permanent Land Loss in Coastal North Frisia (Wadden Sea Region, Germany). Geosciences 15:1. https://doi.org/10.3390/geosciences15010001\u003c/li\u003e\n\u003cli\u003eHall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95\u0026ndash;98\u003c/li\u003e\n\u003cli\u003eHebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc Biol Sci 270:313\u0026ndash;321. https://doi.org/10.1098/rspb.2002.2218\u003c/li\u003e\n\u003cli\u003eHeino J, Parviainen J, Paavola R, et al (2005) Characterizing macroinvertebrate assemblage structure in relation to stream size and tributary position. Hydrobiologia 539:121\u0026ndash;130. https://doi.org/10.1007/s10750-004-3914-3\u003c/li\u003e\n\u003cli\u003eHerzon I, Helenius J (2008) Agricultural drainage ditches, their biological importance and functioning. Biol Conserv 141:1171\u0026ndash;1183. https://doi.org/doi:10.1016/j.biocon.2008.03.005\u003c/li\u003e\n\u003cli\u003eHoang DT, Chernomor O, Von Haeseler A, et al (2018) UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol 35:518\u0026ndash;522\u003c/li\u003e\n\u003cli\u003eHochberg R (2005) Musculature of the primitive gastrotrich \u003cem\u003eNeodasys\u003c/em\u003e (Chaetonotida): functional adaptations to the interstitial environment and phylogenetic significance. Mar Biol 146:315\u0026ndash;323. https://doi.org/10.1007/s00227-004-1437-0\u003c/li\u003e\n\u003cli\u003eHochberg R, Litvaitis M (2000) Phylogeny of Gastrotricha: a morphology-based framework of gastrotrich relationships. 198:299\u0026ndash;305. https://doi.org/10.2307/1542532\u003c/li\u003e\n\u003cli\u003eHochberg R, Litvaitis MK (2003) Organization of muscles in Chaetonotida paucitubulatina (Gastrotricha). Meiofauna Mar 12:47\u0026ndash;58\u003c/li\u003e\n\u003cli\u003eHochberg R, Litvaitis MK (2001) A Muscular Double Helix in Gastrotricha. Zool Anz - J Comp Zool 240:61\u0026ndash;68. https://doi.org/10.1078/0044-5231-00006\u003c/li\u003e\n\u003cli\u003eHummon WD (1974) Gastrotricha from Beaufort, North Carolina, USA. 15:85\u0026ndash;92\u003c/li\u003e\n\u003cli\u003eKalyaanamoorthy S, Minh BQ, Wong TK, et al (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14:587\u0026ndash;589\u003c/li\u003e\n\u003cli\u003eK\u0026aring;nneby T (2013) New species and records of freshwater \u003cem\u003eChaetonotus\u003c/em\u003e (Gastrotricha: Chaetonotidae) from Sweden. Zootaxa 3701:551\u0026ndash;588. https://doi.org/10.11646/zootaxa.3701.5.3\u003c/li\u003e\n\u003cli\u003eK\u0026aring;nneby T, Kirk JJ (2017) A new species of \u003cem\u003eRedudasys\u003c/em\u003e (Gastrotricha: Macrodasyida: Redudasyidae) from the United States. Proc Biol Soc Wash 130:128\u0026ndash;139. https://doi.org/10.2988/17-00006\u003c/li\u003e\n\u003cli\u003eKieneke A, Arbizu PM, Riemann O (2008a) Body musculature of \u003cem\u003eStylochaeta scirtetica\u003c/em\u003e Brunson, 1950 and \u003cem\u003eDasydytes\u003c/em\u003e (\u003cem\u003eSetodytes\u003c/em\u003e) \u003cem\u003etongiorgii\u003c/em\u003e (Balsamo, 1982) (Gastrotricha: Dasydytidae): A functional approach. Zool Anz 247:147\u0026ndash;158. https://doi.org/10.1016/j.jcz.2007.11.001\u003c/li\u003e\n\u003cli\u003eKieneke A, Nikoukar H (2017) Integrative morphological and molecular investigation of \u003cem\u003eTurbanella hyalina\u003c/em\u003e Schultze, 1853 (Gastrotricha: Macrodasyida), including a redescription of the species. Zool Anz 267:168\u0026ndash;186. https://doi.org/10.1016/j.jcz.2017.03.005\u003c/li\u003e\n\u003cli\u003eKieneke A, Ostmann A (2012) Structure, function and evolution of somatic musculature in Dasydytidae (Paucitubulatina, Gastrotricha). Zoomorphology 131:95\u0026ndash;114. https://doi.org/10.1007/s00435-012-0152-5\u003c/li\u003e\n\u003cli\u003eKieneke A, Riemann O (2008) First record of rare dasydytid gastrotrich \u003cem\u003eSetopus tongiorgii\u003c/em\u003e (Balsamo, 1982) from central Europe with some biological data. Zootaxa 1680:59\u0026ndash;61. https://doi.org/10.11646/zootaxa.1680.1.4\u003c/li\u003e\n\u003cli\u003eKieneke A, Riemann O, Ahlrichs WH (2008b) Novel implications for the basal internal relationships of Gastrotricha revealed by an analysis of morphological characters. Zool Scr 37:429\u0026ndash;460. https://doi.org/10.1111/j.1463-6409.2008.00334.x\u003c/li\u003e\n\u003cli\u003eKieneke A, Rothe BH, Schmidt-Rhaesa A (2013) Record and description of \u003cem\u003eAnandrodasys agadasys\u003c/em\u003e (Gastrotricha: Redudasyidae) from Lee Stocking Island (Bahamas), with remarks on populations from different geographic areas. Meiofauna Mar 20:39\u0026ndash;48\u003c/li\u003e\n\u003cli\u003eKieneke A, Schmidt-Rhaesa A (2015) Gastrotricha. In: Handbook of Zoology. Gastrotricha, Cycloneuralia and Gnathifera. de Gruyer, Berlin, pp 1\u0026ndash;134\u003c/li\u003e\n\u003cli\u003eKieneke A, Schmidt-Rhaesa A, Hochberg R (2015) A new species of \u003cem\u003eCephalodasys\u003c/em\u003e (Gastrotricha, Macrodasyida) from the Caribbean Sea with a determination key to species of the genus. Zootaxa 3947:367\u0026ndash;385. https://doi.org/10.11646/zootaxa.3947.3.4\u003c/li\u003e\n\u003cli\u003eKieneke A, Zekely J (2008) \u003cem\u003eDesmodasys abyssalis\u003c/em\u003e sp. nov.\u0026mdash;first record of a deep-sea gastrotrich from hydrothermal vents. Mar Biodivers Rec 1:. https://doi.org/10.1017/S1755267207008950\u003c/li\u003e\n\u003cli\u003eKisielewski J (1997) On the subgeneric division of genus \u003cem\u003eChaetonotus\u003c/em\u003e Ehrenberg (Gastrotricha). Ann Zool 46:145\u0026ndash;151\u003c/li\u003e\n\u003cli\u003eKolicka M (2016) Gastrotrichs in bromeliads\u0026mdash;newly recorded \u003cem\u003eChaetonotus\u003c/em\u003e (\u003cem\u003eHystricochaetonotus\u003c/em\u003e) \u003cem\u003efurcatus\u003c/em\u003e Kisielewski, 1991 (Chaetonotida) from the Ł\u0026oacute;dź Palm House. Zoosystema 38:141\u0026ndash;155. https://doi.org/10.5252/z2016n1a5\u003c/li\u003e\n\u003cli\u003eKolicka M, Dabert M, Olszanowski Z, Dabert J (2020) Sweet or salty? The origin of freshwater gastrotrichs (Gastrotricha, Chaetonotida) revealed by molecular phylogenetic analysis. Cladistics 36:458\u0026ndash;480. https://doi.org/10.1111/cla.12424\u003c/li\u003e\n\u003cli\u003eKolicka M, Gwiazdowicz DJ, Hupało K, et al (2016) Hidden invertebrate diversity - phytotelmata in Bromeliaceae from palm houses and florist wholesalers (Poland). Biologia (Bratisl) 71:194\u0026ndash;203. https://doi.org/10.1515/biolog-2016-0026\u003c/li\u003e\n\u003cli\u003eKolicka M, Kisielewski J, Nesteruk T, Zawierucha K (2013) Gastrotricha from the Poznań Palm House--one new subgenus and three new species of freshwater Chaetonotida (Gastrotricha). Zootaxa 3717:231\u0026ndash;279. https://doi.org/10.11646/zootaxa.3717.2.7\u003c/li\u003e\n\u003cli\u003eKumar S, Stecher G, Li M, et al (2018) MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol 35:1547\u0026ndash;1549. https://doi.org/10.1093/molbev/msy096\u003c/li\u003e\n\u003cli\u003eLauterborn R (1913) Gastrotricha. In: Handwoerterbuch der Naturwissenschaft. Gustav Fischer Verlag, Jena\u003c/li\u003e\n\u003cli\u003eLeasi F, Rothe BH, Schmidt-Rhaesa A, Todaro MA (2006) The musculature of three species of gastrotrichs surveyed with confocal laser scanning microscopy (CLSM). Acta Zool 87:171\u0026ndash;180. https://doi.org/10.1111/j.1463-6395.2006.00230.x\u003c/li\u003e\n\u003cli\u003eLeasi F, Todaro MA (2009) Meiofaunal cryptic species revealed by confocal microscopy: the case of \u003cem\u003eXenotrichula intermedia\u003c/em\u003e(Gastrotricha). Mar Biol 156:1335\u0026ndash;1346\u003c/li\u003e\n\u003cli\u003eLeasi F, Todaro MA (2008) The muscular system of \u003cem\u003eMusellifer delamarei\u003c/em\u003e (Renaud-Mornant, 1968) and other chaetonotidans with implications for the phylogeny and systematization of the Paucitubulatina (Gastrotricha). Biol J Linn Soc 94:379\u0026ndash;398. https://doi.org/10.1111/j.1095-8312.2008.00974.x\u003c/li\u003e\n\u003cli\u003eMalmqvist B, Hoffsten P-O (2000) Macroinvertebrate taxonomic richness, community structure and nestedness in Swedish streams. Arch F\u0026uuml;r Hydrobiol 29\u0026ndash;54. https://doi.org/10.1127/archiv-hydrobiol/150/2000/29\u003c/li\u003e\n\u003cli\u003eMartin LV (1990) Further observations on Gastrotrichs in Surrey and a provisional British list. Microscopy 36:415\u0026ndash;425\u003c/li\u003e\n\u003cli\u003eMartin LV (1981) Gastrotrichs found in Surrey. Microscopy 34:286\u0026ndash;301\u003c/li\u003e\n\u003cli\u003eMetschnikoff E (1865) \u0026Uuml;ber einige wenig bekannte niedere Thierformen. Z F\u0026uuml;r Wiss Zool 15:450\u0026ndash;463\u003c/li\u003e\n\u003cli\u003eMichels J, B\u0026uuml;ntzow M (2010) Assessment of Congo red as a fluorescence marker for the exoskeleton of small crustaceans and the cuticle of polychaetes. J Microsc 238:95\u0026ndash;101. https://doi.org/10.1111/j.1365-2818.2009.03360.x\u003c/li\u003e\n\u003cli\u003eMinowa AK, Garraffoni ARS (2025) Expanding the knowledge of Brazilian Gastrotrich biodiversity: Freshwater Paucitubulatina (Gastrotricha: Chaetonotida) from Northeastern Brazil. Zool Curitiba 42:e24025. https://doi.org/10.1590/S1984-4689.v42.e24025\u003c/li\u003e\n\u003cli\u003eMinowa AK, Garraffoni ARS (2020) Assessing biodiversity shortfalls of freshwater meiofauna from the Atlantic Forest: New species, distribution patterns and the first total-evidence phylogeny of semiplanktonic Gastrotricha. Mol Phylogenet Evol 152:106926. https://doi.org/10.1016/j.ympev.2020.106926\u003c/li\u003e\n\u003cli\u003eMinowa AK, Garraffoni ARS (2022) New data on Brazilian semiplanktonic gastrotrichs (Gastrotricha: Chaetonotida). Zootaxa 5209:45\u0026ndash;68. https://doi.org/10.11646/zootaxa.5209.1.3\u003c/li\u003e\n\u003cli\u003eMinowa AK, Garraffoni ARS (2021) Seek and you shall find: New species of the rare genus \u003cem\u003eOrnamentula\u003c/em\u003e (Gastrotricha: Chaetonotida) and first record outside of type-locality. Zoologia 38:1\u0026ndash;9. https://doi.org/10.3897/ZOOLOGIA.38.E56781\u003c/li\u003e\n\u003cli\u003eMinowa AK, Kieneke A, Campos A, et al (2025) New branch on the Tree of Life of Gastrotricha: Establishment of a new genus for limno-terrestrial species. Zool J Linn Soc 2:zlae166. https://doi.org/10.1093/zoolinnean/zlae166\u003c/li\u003e\n\u003cli\u003eMioduchowska M, Wojtasik B (2009) Copepoda-Cyclopoida of water bodies of the coast of Gdańsk. Teka Kom Ochr Kszt Środ Przyr 6:189\u0026ndash;199\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;nter L, Kieneke A (2017) Novel myo-anatomical insights to the \u003cem\u003eXenotrichula intermedia\u003c/em\u003e species complex (Gastrotricha: Paucitubulatina): Implications for a pan-European species and reconsideration of muscle homology among Paucitubulatina. Proc Biol Soc Wash 130:166\u0026ndash;186. https://doi.org/10.2988/17-00013\u003c/li\u003e\n\u003cli\u003eMurray J (1913) Gastrotricha. J Quekett Microsc Club 12:211\u0026ndash;238\u003c/li\u003e\n\u003cli\u003eNguyen LT, Schmidt HA, Von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268\u0026ndash;274. https://doi.org/10.1093/molbev/msu300\u003c/li\u003e\n\u003cli\u003eNicolet P, Biggs J, Fox G, et al (2004) The wetland plant and macroinvertebrate assemblages of temporary ponds in England and Wales. Biol Conserv 120:261\u0026ndash;278. https://doi.org/10.1016/j.biocon.2004.03.010\u003c/li\u003e\n\u003cli\u003eOertli B, Joye DA, Castella E, et al (2002) Does size matter? The relationship between pond area and biodiversity. Biol Conserv 104:59\u0026ndash;70. https://doi.org/10.1016/S0006-3207(01)00154-9\u003c/li\u003e\n\u003cli\u003ePainter D (1999) Macroinvertebrate distributions and the conservation value of aquatic Coleoptera, Mollusca and Odonata in the ditches of traditionally managed and grazing fen at Wicken Fen, UK. J Appl Ecol 36:33\u0026ndash;48. https://doi.org/10.1046/j.1365-2664.1999.00376.x\u003c/li\u003e\n\u003cli\u003ePeralta-Maraver I, Traunspurger W, Robertson AL, et al (2023) Freshwater Meiofauna\u0026mdash;A Biota with Different Rules? In: Giere O, Schratzberger M (eds) New Horizons in Meiobenthos Research: Profiles, Patterns and Potentials. Springer International Publishing, Cham, pp 153\u0026ndash;173\u003c/li\u003e\n\u003cli\u003ePomahač O, M\u0026eacute;ndez-S\u0026aacute;nchez D, Pol\u0026aacute;kov\u0026aacute; K, et al (2023) Rediscovery of remarkably rare anaerobic tentaculiferous ciliate genera \u003cem\u003eLegendrea\u003c/em\u003e and \u003cem\u003eDactylochlamys\u003c/em\u003e (Ciliophora: Litostomatea). Biology 12:707\u003c/li\u003e\n\u003cli\u003ePott R (1999) Nordwestdeutsches Tiefland zwischen Ems und Weser. Ulmer, Stuttgart, Jena, New York\u003c/li\u003e\n\u003cli\u003eRemane A (1927) Beitrage zur Systematik der Susswassergastrotrichen. Zool Jahrb Abt F\u0026uuml;r Syst Oekologie Geogr Tiere 53:268\u0026ndash;320\u003c/li\u003e\n\u003cli\u003eRichards C, Host GE, Arthur JW (1993) Identification of predominant environmental factors structuring stream macroinvertebrate communities within a large agricultural catchment. Freshw Biol 29:285\u0026ndash;294. https://doi.org/10.1111/j.1365-2427.1993.tb00764.x\u003c/li\u003e\n\u003cli\u003eSchratzberger M, Ingels J (2018) Meiofauna matters: the roles of meiofauna in benthic ecosystems. J Exp Mar Biol Ecol 502:12\u0026ndash;25. https://doi.org/10.1016/j.jembe.2017.01.007\u003c/li\u003e\n\u003cli\u003eSchwank P (1990) Gastrotricha. In: Schwoerbel J, Zwick P (eds) S\u0026uuml;\u0026szlig;wasserfauna von Mitteleuropa. Gastrotricha und Nemertini. Gustav Fischer Verlag, Stuttgart, Jena, New York, pp 1\u0026ndash;252\u003c/li\u003e\n\u003cli\u003eSuzuki T (2018) 水田に棲みつく多様なイタチムシたち. 日本の科学者 53:16\u0026ndash;21. https://doi.org/10.60233/jjsci.53.4_16\u003c/li\u003e\n\u003cli\u003eSuzuki TG, Maeda M, Furuya H (2013) Two new Japanse species of Gastrotricha (Chaetonotida, Chaetonotidae, \u003cem\u003eLepidodermella\u003c/em\u003e and Dichaeturidae, \u003cem\u003eDichaetura\u003c/em\u003e), with Comments on the Diversity of Gastrotrichs in Rice Paddies. Zootaxa 3691:229\u0026ndash;239. https://doi.org/10.11646/zootaxa.3691.2.3\u003c/li\u003e\n\u003cli\u003eTodaro MA, Balsamo M, Kristensen RM (2005) A new genus of marine chaetonotids (Gastrotricha), with a description of two new species from Greenland and Denmark. J Mar Biol Assoc U K 85:1391\u0026ndash;1400. https://doi.org/10.1017/S0025315405012579\u003c/li\u003e\n\u003cli\u003eTodaro MA, Zotto MD, Jondelius U, et al (2012) Gastrotricha: A Marine Sister for a Freshwater Puzzle. 7:e31740. https://doi.org/10.1371/journal.pone.0031740\u003c/li\u003e\n\u003cli\u003eTrokhymchuk R, Kieneke A (2024a) New records of deep-sea Gastrotricha and Tardigrada from Iberian and Canary Basins (Northeast Atlantic) with comments on abyssal meiofauna composition and the meiofauna paradox. J VNKarazin Kharkiv Natl Univ Ser \u0026laquo;Biology\u0026raquo; 43:66\u0026ndash;84. https://doi.org/10.26565/2075-5457-2024-43-6\u003c/li\u003e\n\u003cli\u003eTrokhymchuk R, Kieneke A (2024b) Novel distribution records of marine Tardigrada from abyssal sediments of the Northwest Atlantic Ocean. Org Divers Evol. https://doi.org/10.1007/s13127-024-00641-2\u003c/li\u003e\n\u003cli\u003eTwisk W, Noordervliet MAW, ter Keurs WJ (2000) Effects of ditch management on caddisfly, dragonfly and amphibian larvae in intensively farmed peat areas. Aquat Ecol 34:397\u0026ndash;411. https://doi.org/10.1023/A:1011430831180\u003c/li\u003e\n\u003cli\u003eVerdonschot RCM, Keizer-vlek HE, Verdonschot PFM (2011) Biodiversity value of agricultural drainage ditches: a comparative analysis of the aquatic invertebrate fauna of ditches and small lakes. Aquat Conserv Mar Freshw Ecosyst 21:715\u0026ndash;727. https://doi.org/10.1002/aqc.1220\u003c/li\u003e\n\u003cli\u003eWatson AM, Ormerod SJ (2004) The distribution of three uncommon freshwater gastropods in the drainage ditches of British grazing marshes. Biol Conserv 118:455\u0026ndash;466. https://doi.org/10.1016/j.biocon.2003.09.021\u003c/li\u003e\n\u003cli\u003eWilliams P, Whitfield M, Biggs J, et al (2004) Comparative biodiversity of rivers, streams, ditches and ponds in an agricultural landscape in Southern England. Biol Conserv 115:329\u0026ndash;341. https://doi.org/10.1016/S0006-3207(03)00153-8\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"zoomorphology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"zomo","sideBox":"Learn more about [Zoomorphology](http://link.springer.com/journal/435)","snPcode":"435","submissionUrl":"https://submission.nature.com/new-submission/435/3","title":"Zoomorphology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Freshwater, Biodiversity, Invertebrates, Synanthropic Fauna, Meifauna","lastPublishedDoi":"10.21203/rs.3.rs-7189628/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7189628/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe rediscovery of \u003cem\u003eDichaetura surreyi\u003c/em\u003e Martin, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e from farm ditches in Northwest Germany (East Frisia) provides new data on freshwater meiofauna. To gain the maximum amount of detailed morphological and anatomical data from the limited number of individuals, a specimen-saving methodology is employed, combining light microscopy, confocal laser scanning microscopy and scanning electron microscopy on the same specimens. This approach refined the taxonomic understanding of \u003cem\u003eDichaetura surreyi\u003c/em\u003e, confirming originally described morphological features, while complementing undescribed muscular architecture and cuticular characters for the Dichaeturidae family. Genetic data on mitochondrial COI locus is provided along a gene tree with members of Paucitubulatina, hinting at a close phylogenetic position to \u003cem\u003ePolymerurus\u003c/em\u003e under Maximum Likelihood approach. The designation of a neotype and deposit of mitochondrial barcoding sequences establishes reference for future research, addressing gaps in knowledge surrounding this poorly known family and advancing understanding of freshwater meiofaunal diversity.\u003c/p\u003e","manuscriptTitle":"Rediscovery and neotype designation of Dichaetura surreyi Martin, 1990 (Gastrotricha: Paucitubulatina) from Northern Germany with a threefold morphological examination with a specimen-saving method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 12:41:02","doi":"10.21203/rs.3.rs-7189628/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-27T02:09:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T10:27:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-14T08:52:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-13T16:43:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-13T08:39:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-13T06:03:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86630694698973159524738306574787729862","date":"2025-08-05T05:58:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-04T15:03:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84350077495657250608055595770463174844","date":"2025-08-04T13:23:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82860087478576835722084501131837822820","date":"2025-08-03T16:04:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100889822569638420222448826715612438671","date":"2025-08-03T12:01:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163880960003405077509517688706311018664","date":"2025-08-03T11:01:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4010295581129120735081655844745692943","date":"2025-08-03T09:19:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252417808154195143842583699812485653563","date":"2025-08-02T19:59:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-02T17:18:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-24T14:08:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-24T14:06:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Zoomorphology","date":"2025-07-22T17:43:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"zoomorphology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"zomo","sideBox":"Learn more about [Zoomorphology](http://link.springer.com/journal/435)","snPcode":"435","submissionUrl":"https://submission.nature.com/new-submission/435/3","title":"Zoomorphology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a7aa9eff-fbf2-4400-a0cf-7cf733813bda","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:01:41+00:00","versionOfRecord":{"articleIdentity":"rs-7189628","link":"https://doi.org/10.1007/s00435-025-00748-w","journal":{"identity":"zoomorphology","isVorOnly":false,"title":"Zoomorphology"},"publishedOn":"2025-10-17 15:57:38","publishedOnDateReadable":"October 17th, 2025"},"versionCreatedAt":"2025-08-08 12:41:02","video":"","vorDoi":"10.1007/s00435-025-00748-w","vorDoiUrl":"https://doi.org/10.1007/s00435-025-00748-w","workflowStages":[]},"version":"v1","identity":"rs-7189628","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7189628","identity":"rs-7189628","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}