Integrative description of a new species of Minibiotus (Tardigrada: Macrobiotidae) from Salta City (Argentina)

77 European Journal of Taxonomy 958: 77–113 ISSN 2118-9773 https://doi.org/10.5852/ejt.2024.958.2663 www.europeanjournaloftaxonomy.eu 2024 · Rocha A. et al. This work is licensed under a Creative Commons Attribution License (CC BY 4.0). Research article urn:lsid:zoobank.org:pub:94D3C714-B808-436F-B84B-1398196B1E09 Integrative description of a new species of Minibiotus (Tardigrada: Macrobiotidae) from Salta City (Argentina) Alejandra ROCHA 1, Irene DOMA 2, Daniele CAMARDA 3, Belen OSTERTAG 4, Florencia MEIER 5, Federica FRIGIERI 6, Michele CESARI 7 & Oscar LISI 8,* 1,2,4 National University of La Pampa, Faculty of Exact and Natural Sciences, Uruguay Avenue 151, CP L6300DUG, Santa Rosa, La Pampa, Argentina. 3,4,8 University of Catania, Department of Biological, Geological and Environmental Sciences, Section of Animal Biology, Via Androne 81, 95124, Catania, Italy. 4,5 National Scientific and Technical Research Council (CONICET), Godoy Cruz Street 2290, CP C1425FQB, Buenos Aires, Argentina. 6,7 University of Modena and Reggio Emilia, Department of Life Sciences, Via G. Campi 213/d, 41125, Modena, Italy. 7 National Biodiversity Future Center (NBFC), Piazza Marina 61, 90133, Palermo, Italy. * Corresponding author: [email protected] 1 Email: [email protected] 2 Email: [email protected] 3 Email: [email protected] 4 Email: [email protected] 5 Email: m.meier[email protected] 6 Email: [email protected] 7 Email: [email protected] 1 urn:lsid:zoobank.org:author:37D7AFFE-F588-478D-91AE-6E8BEA8754F9 2 urn:lsid:zoobank.org:author:C8122DC4-DE1C-4DB9-9CFA-E461D2F8F136 3 urn:lsid:zoobank.org:author:5D617255-0390-4370-A335-69590E61870C 4 urn:lsid:zoobank.org:author:7E8091DC-BFEA-42FE-AAE6-F5A2DB49958F 5 urn:lsid:zoobank.org:author:6CBE552A-4217-44B7-B444-67369F926ADB 6 urn:lsid:zoobank.org:author:92C61F57-2A95-40D2-8917-60660575F2E5 7 urn:lsid:zoobank.org:author:416958C5-064F-4E10-BA23-DA81B63D7946 8 urn:lsid:zoobank.org:author:1EE34EFB-1742-49A8-BC11-109B67E2BEA3 Abstract. The limno-terrestrial tardigrade fauna of Argentina has been investigated methodically and with modern criteria just in the last two decades, but current knowledge is still incomplete. So far, about 119 limno-terrestrial species are known for the country, of which only 6 belong to the genus Minibiotus R.O. Schuster, 1980. Until 1988, this genus was monotypic, with only Minibiotus intermedius (Plate, 1888), but today the number of species of the genus has risen to 55. In the present contribution, we describe with an integrated approach (PCM, SEM, morphometry and DNA analysis with COI, ITS2, 18S and 28S genes) a new species of Minibiotus from Salta City (Argentina). Minibiotus dispositus sp. nov. has ten transverse bands of variously shaped cuticular pores, arranged in transverse rows, with European Journal of Taxonomy 958: 77–113 (2024) 78 differences between smaller and larger specimens. Three macroplacoids and a microplacoid are present in the pharynx. The eggs have small conical processes and granulated chorion. The new species is morphologically and morphometrically well differentiated from all other species of the genus, and genetically from the up to date sequenced species. The new species description gave the occasion to broaden knowledge on taxonomy, morphology and faunistics of the genus Minibiotus, and on the tardigrade fauna of Argentina and the Neotropical region. Keywords. Tardigrades, South America, taxonomy, morphology, DNA analysis. Rocha A., Doma I., Camarda D., Ostertag B., Meier F., Frigieri F., Cesari M. & Lisi O. 2024. Integrative description of a new species of Minibiotus (Tardigrada: Macrobiotidae) from Salta City (Argentina). European Journal of Taxonomy 958: 77–113. https://doi.org/10.5852/ejt.2024.958.2663 Introduction Tardigrada Doyre, 1840 is a phylum of microscopic animals (usually 250–600 μm in length) belonging to the superclade Ecdysozoa Aguinaldo et al. 1997, inhabiting terrestrial, freshwater and marine environments throughout the world (Nelson et al. 2015; Schill et al. 2018). To date, 1488 species have been formally described (Degma & Guidetti 2009–2024) and this number is systematically growing. The tardigrade fauna of the Republic of Argentina has begun to be investigated continuously just over the last two decades; however, the current state of knowledge is very far from complete. Up to date, 119 limno-terrestrial species have been reported for the country (Rocha et al. 2023) of which only 6 belong to the genus Minibiotus R.O. Schuster, 1980, with M. claxtonae Rossi, Claps & Ardohain, 2009 and M. pseudostellarus Roszkowska, Stec, Ciobanu & Kaczmarek, 2016 currently considered endemic. This genus is characterized by the presence of an antero-ventral mouth with 10 peribuccal ‘papulae’ but without lamellae; a short, rigid, and narrow buccal tube usually with 2 bends; a relatively cephalic stylet support insertion point, and an extra thickening on the buccal tube wall immediately caudal to the stylet support insertion point (Claxton 1998; Michalczyk & Kaczmarek 2004). For one hundred years, most species of Minibiotus were hidden under the diagnosis of Macrobiotus intermedius Plate, 1888, or of Minibiotus intermedius after the genus institution (R.O. Schuster, 1980 in Schuster et al. 1980), and the genus remained monotypic until 1988, when Minibiotus maculartus Pilato & Claxton, 1988 was described. Additional species were later described, and Claxton (1998) published a revision of the genus Minibiotus redescribing the type species and describing many new species. Since then, very numerous new species have been described and several have also been transferred to Minibiotus from the genus Macrobiotus C.A.S. Schultze, 1834 based on the characters defined by Claxton (1998) and later supplemented by Guidetti et al. (2007) and Dueñas-Cedillo et al. (2021). Very recently, Kaczmarek et al. (2022) redescribed with the most modern criteria M. intermedius solving several past problems about this species and, consequently, the genus. Currently, the total number of species of Minibiotus has risen to 55 (Guidetti & Bertolani 2005; Degma & Guidetti 2007, 2009–2024). The genus was recorded for the first time in Argentina by Iharos (1963) from Río Negro Province, with M. intermedius. Later, Mihelčič (1972) reported M. acontistus (de Barros, 1942) with no geographic indication but Claps & Rossi (1988) reported this species for Misiones Province; besides, Claps & Rossi (1981) reported M. subintermedius (Ramazzotti, 1962) for Neuquén and Chubut Provinces; Rossi et al. (2009) described M. claxtonae Rossi, Claps & Ardohain, 2009 for Neuquén Province; Claps et al. (2008) reported M. furcatus (Ehrenberg, 1859) for Buenos Aires and Tierra del Fuego Provinces and, more recently, M. pseudostellarus Roszkowska, Stec, Ciobanu & Kaczmarek, 2016 was described for Río Negro Province. ROCHA A. et al., A new species of Minibiotus (Tardigrada) from Salta City (Argentina) 79 In the present paper, we broaden the knowledge on Argentinian tardigrades describing a new species of Minibiotus from Salta Province, from which the genus was up to date unreported. Material and methods The present contribution is part of a tardigradological collection revision work of the Rocha and Doma collection of the Department of Natural Sciences at the National University of La Pampa, Argentina (deposited within 2014 and 2017). Having found the new species (at first identified only morphologically through PCM and morphometry), we carried out resampling in order to obtain new fresh specimens for SEM and molecular analyses. The former sampling had taken place in May of 2014 in Salta Province, Salta City, 24°47′18″ S, 65°24′38″ W, and one sample was collected, producing 49 specimens (of various body sizes) and 6 eggs all mounted on microscopy slides (slide Nos. in “Material Examined”) and deposited in the collection as mentioned above. Resampling took place in June 2022, collecting one sample perfectly corresponding to that of 2014 (same geographic coordinates, tree and position on it, substrate type); this produced only 13 big, dead specimens (no young and no eggs), of which 2 were mounted on a microscopy slide, 10 prepared for SEM analysis, and 1 for DNA analysis. In both cases, the material was extracted from an undetermined lichen and moss sample growing on a sidewalk tree (Handroanthus Mattos); this was about 5 m tall, collecting the sample at chest height on the trunk (1.3 m), on the trunk surface facing the street. The samples were stored in paper bags at room temperature. For processing, they were hydrated for 24 hours each in a plastic sieve (1.1 mm mesh) placed in Petri dishes filled with mineral water. Tardigrades and eggs were sorted using a stereoscopic microscope and extracted with a micropipette. The material intended for light microscopy studies was mounted on microscopic slides with polyvinyllactophenol medium. Tardigrades were mainly identified using a Leica DM500 phase contrast microscope, equipped with a ICC 50 HD digital camera; observations were made also with a Zeiss Axio Scope A1 Differential Interference Contrast (DIC) microscope equipped with a Zeiss AxioCam ICc5CCD digital camera. Specimens prepared for Scanning Electron Microscope (SEM) were processed according to Camarda et al. (2023). The specimens were examined using a Zeiss Crossbeam 340, column Gemini 1 Scanning Electron Microscope in the Institute of Nanoscience and Nanotechnology Bariloche node (CNEA/ CONICET), Bariloche, Argentina. Morphometric data were obtained using AxioVision SE64 software, given in micrometers (μm). Structures were measured only if their orientation was appropriate/suitable. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. Buccal tube length and the level of the stylet support insertion point were measured according to Pilato (1981). Buccal tube width was measured as the external diameter at the level of the stylet support insertion point. Lengths of the claw branches were measured from the base of the claw to the top of the branch including accessory points. The pt ratio is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage (Pilato 1981). Macroplacoid length sequence is given according to Kaczmarek et al. (2014). Morphometric data were handled using the “Parachela” ver. 1.7 template available from the Tardigrada Register (Michalczyk & Kaczmarek 2013). Tardigrade taxonomy follows Bertolani et al. (2014) and Stec et al. (2020b). Student t-tests (one-side tests) for statistical significance of differences between species morphometry (only when ranges of the given characters of the two compared species overlapped) were performed through Microsoft Office Excel software and the results are reported in European Journal of Taxonomy 958: 77–113 (2024) 80 Table 9 (relative to the various differential diagnoses); when ranges did not overlap, they are reported directly in the text of the differential diagnoses. For species identification and differentiation, dichotomous keys reported in Claxton (1998) and papers regarding descriptions and redescriptions of species (Ehrenberg 1859; Ramazzotti 1962; Horning et al. 1978; Dastych 1988, 1990; Binda & Pilato 1992; Claxton 1998; Michalczyk & Kaczmarek 2004; Michalczyk et al. 2005; Miller et al. 2005; Fontoura et al. 2009a, 2009b; Meyer & Hinton 2009; Meyer et al. 2011; Dueñas-Cedillo et al. 2020, 2021; Kaczmarek et al. 2022) were used. For comparison with our material, the following type specimens from the Pilato and Binda Collection (Museum of the Section of Animal Biology, Department of Biological, Geological and Environmental Sciences, University of Catania) were examined: paratypes of Minibiotus eichhorni Michalczyk & Kaczmarek, 2004 (slide No. 5403), holotype and paratypes of Minibiotus sidereus Pilato, Binda & Lisi, 2003 (slides Nos. 4925, 6018, 6021, 6022), one egg of the type series of Minibiotus ethelae (slide No. 4211). Slides from the Zoological Museum of the Jagiellonian University with paratypes of Minibiotus constellatus Michalczyk & Kaczmarek, 2003 (slides Nos. Peru–17, Peru–18, Peru–19) were also examined. Culturing was not possible, preventing exact determination of life stages, since we worked with already mounted slides (first sampling), or with dead specimens (resampling). In the latter case, only big specimens were found, also preventing having SEM material of young specimens and eggs, or a genetic analysis of young specimens. We were only able, depending on body size, cuticular pore pattern and metric characters, to distinguish between smaller specimens (called ‘young’) and larger (called ‘senior’ specimens). Total genomic DNA of the analysed senior specimen was extracted with the QuickExtract™ DNA Extraction Solution (Lucigen, Middleton, WI, USA) following the manufacturer’s protocol. The animal was previously observed in vivo up with LM up to 100 × oil immersion magnification to avoid mistakes in determining the morphology and then photographed, using the method described by Cesari et al. (2011) in order to obtain the voucher specimen. Molecular investigations were carried out using fragments of the mitochondrial cytochrome oxidase 1 (COI) gene, the nuclear internal transcribed spacer ITS2, the small ribosome subunit (18S rRNA) and the large ribosome subunit (28S rRNA) using the primers and protocols described by Cesari et al. (2009), Stec et al. (2018), Bertolani et al. (2014), and Guidetti et al. (2014), respectively. The amplified products were gel purified using the Wizard Gel and PCR Cleaning (Promega) kit, while sequencing reactions were performed using the ABIPRISM® BigDyeTM Terminator ver. 1.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on purified amplicons. Each sequencing reaction contained 0.2 μM of a single PCR primer to initiate the sequencing reaction, 2 μL of BigDyeTM, 70 ng of purified products, 4 μL of 5 × BigDyeTM Terminator ver. 1.1 Sequencing Buffer (Applied Biosystems, Foster City, CA, USA) and bi-distilled H2O for a final volume of 20 μL. Cycling conditions for sequencing reactions consisted of 25 cycles of 96°C for 10 s, 50°C for 5 s and 60°C for 4 min. Both strands were sequenced using an ABI Prism 3100 (Applied Biosystems, Foster City, CA, USA) at UNIMORE. Chromatograms obtained and nucleotide sequences were checked by visual inspection and the sequences were aligned with the MUSCLE algorithm. In order to perform proper molecular comparisons, we included sequences from GenBank of other specimens of Minibiotus in our analysis (Table 1). Pairwise nucleotide sequence divergences between scored haplotypes were calculated by using MEGA 11 (Tamura et al., 2021). The distance-based ASAP species delimitation analysis was performed on COI gene (Table 7) and ITS2 (Table 8) on the ASAP website (https://bioinfo.mnhn.fr/abi/public/asap/, accessed on 29 Feb. 2024, Puillandre et al., 2021). Nucleotide sequences of the newly analyzed specimen ROCHA A. et al., A new species of Minibiotus (Tardigrada) from Salta City (Argentina) 81 Table 1 (continued on next page). Sequences from GenBank of specimens of Minibiotus R.O. Schuster, 1980 used for comparison in the molecular analysis for all analyzed genes. GenBank accession no. Taxon Locality COI ITS2 18S 28S References Minibiotus gumersindoi Madrid, Spain FJ435803 FJ435748 FJ435761 Guil & Giribet 2012 Minibiotus furcatus Madrid, Spain FJ435802 FJ435746 Guil & Giribet 2012 Minibiotus furcatus Portugal JX683828 Vicente et al. 2013 Minibiotus furcatus Portugal JX683829 Vicente et al. 2013 Minibiotus furcatus Madrid, Spain FJ435747 Guil & Giribet 2012 Minibiotus furcatus Madrid, Spain FJ435745 Guil & Giribet 2012 Minibiotus furcatus Gaianello, Italy HQ604977 Bertolani et al. 2014 Minibiotus furcatus Gaianello, Italy HQ604978 Bertolani et al. 2014 Minibiotus ioculator South Africa MT023412 MT024000 MT023998 Stec et al. 2020a Minibiotus pentannulatus Tanzania MT023413 MT024001 MT023999 Stec et al. 2020a Minibiotus pentannulatus Tanzania MT023414 Stec et al. 2020a Minibiotus intermedius Marburg, Germany ON005160 Kaczmarek et al. 2022 Minibiotus intermedius Alaska, USA JX888504 Adams et al. (unpublished) Minibiotus intermedius Alaska, USA JX888505 Adams et al. (unpublished) Minibiotus cf. intermedius Lembolovo Russia OP013287 OP035707 OP035718 Tumanov et al. 2022 Minibiotus cf. intermedius Lembolovo Russia OP013286 OP035708 OP035719 Tumanov et al. 2022 Minibiotus cf. intermedius Lembolovo Russia OP013288 Tumanov et al. 2022 Minibiotus gr. intermedius Andalo, Italy HQ604979 Bertolani et al. 2014 Minibiotus gr. intermedius Andalo, Italy HQ604980 Bertolani et al. 2014 Minibiotus citlalium Iztaccihuatl, Mexico OP684765 OP696660 Amezcua-Martinez et al. (unpublished) Minibiotus citlalium Iztaccihuatl, Mexico OP684766 OP696661 Amezcua-Martinez et al. (unpublished) Minibiotus citlalium Iztaccihuatl, Mexico OP684767 OP696662 Amezcua-Martinez et al. (unpublished) Minibiotus sidereus Iztaccihuatl, Mexico OP684768 OP696663 Amezcua-Martinez et al. (unpublished) Minibiotus sidereus Iztaccihuatl, Mexico OP684769 OP696664 Amezcua-Martinez et al. (unpublished) European Journal of Taxonomy 958: 77–113 (2024) 82 Table 1 (continued on next page). Sequences from GenBank of specimens of Minibiotus R.O. Schuster, 1980 used for comparison in the molecular analysis for all analyzed genes. GenBank accession no. Taxon Locality COI ITS2 18S 28S References Minibiotus sidereus Iztaccihuatl, Mexico OP684770 OP696665 Amezcua-Martinez et al. (unpublished) Minibiotus sp. Iztaccihuatl, Mexico OP684773 OP696668 Amezcua-Martinez et al. (unpublished) Minibiotus sp Iztaccihuatl, Mexico OP684772 OP696667 Amezcua-Martinez et al. (unpublished) Minibiotus sp Iztaccihuatl, Mexico OP684771 OP696666 Amezcua-Martinez et al. (unpublished) Minibiotus sp.Chile MH079492 Guil et al. 2019 Minibiotus sp. Dronning Maud Land, Antarctica JX865309 Czechowski et al. 2012 Minibiotus sp. Dronning Maud Land, Antarctica JX865313 EU266932 Czechowski et al. 2012; Sands et al. 2008 Minibiotus sp. EU266933 Sands et al. 2008 Minibiotus sp. EU266934 Sands et al. 2008 Minibiotus sp. Tuscan-Emilian Apennine National Park, Italy MW306857 Vecchi et al. 2022a Minibiotus sp. Tuscan-Emilian Apennine National Park, Italy MW306858 Vecchi et al. 2022a Minibiotus sp. Tuscan-Emilian Apennine National Park, Italy MW306859 OK663227 Vecchi et al. 2022a, 2022b Minibiotus sp. Corniglio, Italy OK663216 Vecchi et al. 2022b ROCHA A. et al., A new species of Minibiotus (Tardigrada) from Salta City (Argentina) 83 were submitted to GenBank (accession numbers: PP937153 for COI gene; PP938064 for the ITS2 gene; PP938063 for the 18S; PP938062 for the 28S). Institutional acronyms Specimens from the following institutions and collections were examined (curator in parentheses). MCNS = Museum of Natural Sciences, National University of Salta, Argentina (Ivanna Cruz) UNICT = Università degli Studi di Catania, Italy, Museum of the Department of Animal Biology ‘Marcello La Greca’, Italy, Binda and Pilato collection (Giovanni Pilato and Oscar Lisi) UNLPam = National University of La Pampa, Faculty of Exact and Natural Sciences, Argentina, (Rocha Alejandra Mariana) Results Taxonomic account Phylum Tardigrada Doyère, 1840 Class Eutardigrada Richters, 1926 Order Parachela Schuster, Nelson, Grigarick & Christenberry, 1980 Superfamily Macrobiotoidea Thulin, 1928 in Marley et al. 2011 Family Macrobiotidae Thulin, 1928 Genus Minibiotus R.O. Schuster, 1980 in Schuster et al. 1980 Minibiotus dispositus sp. nov. urn:lsid:zoobank.org:act:780E50E1-48E8-474C-8935-070FCD6B09AE Figs 1–8; Tables 2–6; Supp. files 1–3 Diagnosis Minibiotus with smooth cuticle but with cuticular pores variously sized (0.9–3.1 µm) and shaped; under SEM, most pores are polygonal or multilobate (3–5 angles/lobes/arms); under PCM, pentagonal pores often appear round, and 5-lobate are rarely observable, only caudal or on legs. Dorsal pores arranged in a group of very cephalic and a group of very caudal pores, with in between a series of transverse bands; young specimens with 8 bands of 1–2 rows; senior specimens with 7 bands of about 2–5 less regular rows (band 8 joined to the very caudal pores). Ventral pores arranged in 7 transverse bands, starting posterior to legs I, of a single row each, but partially duplicated medially in senior specimens. Bucco-pharyngeal apparatus typical for the genus; oral cavity armature with three bands of teeth, better visible under SEM, with band I reduced; three macroplacoids (length sequence 3 ≤ 2 < 1) and an evident microplacoid in the pharynx. Robust double claws with short, robust accessory points and small, smooth lunules. Faint leg ‘cuticular bars’, divided on legs I–III, undivided on legs IV; no leg granulation, pulvini present on legs I–III. Moderate allometry regarding buccal tube width, macroplacoid and claw length. Etymology From the Latin word ‘dispositus’ = ‘ordered’, in the meaning of ‘with a pattern’, referred to the cuticular pores forming a pattern. Material examined In total, 51 animals (undetermined sex; 31 senior and 20 young specimens) and 6 eggs mounted on microscope slides in Polivinil lactophenol medium; 10 additional specimens mounted on SEM stubs; one specimen (voucher) used for DNA analysis. European Journal of Taxonomy 958: 77–113 (2024) 84 Holotype ARGENTINA • senior spec.; Salta Province, Salta City; 24°47′18″ S, 65°24′38″ W; 1150 m a.s.l.; 2 May 2014; Rocha and Doma leg.; moss and lichen growing on Handroanthus Mattos; UNLPam 1088(3). Paratypes ARGENTINA • 2 senior specs; same data as for holotype; 2 May 2014; MCNS Tar. 000026(1), Tar. 000026(4) • 1 egg; same data as for holotype; 2 May 2014; MCNS Tar. 000027(1) • 2 senior specs; same data as for holotype; 5 Jun. 2022; UNICT 6010, 6011 • 1 young spec.; same data as for holotype; 2 May 2014; UNICT 6012 • 1 egg; same data as for holotype; 2 May 2014; UNICT 6013 • 26 senior specs; same data as for holotype; 2 May 2014; UNLPam 643(3), 654(1), 655(1), 655(2), 656(1), 659(1), 659(2), 1037(4), 1038(2), 1042(2), 1049(1) to 1049(3), 1050(2), 1050(3), 1056(2) to 1056(4), 1085(1), 1085(3), 1085(4), 1087(3), 1088(4), 1089(3), 1090(1), 1090(2) • 19 young specs; same data as for holotype; 2 May 2014; UNLPam 1033(1), 1034(4), 1035(1), 1035(2), 1036(2), 1038(4), 1040(3), Fig. 1. Minibiotus dispositus sp. nov., habitus. A. Paratype (UNLPam), SEM. B. Paratype (slide No. UNICT 6011), PCM. Scale bars in µm. ROCHA A. et al., A new species of Minibiotus (Tardigrada) from Salta City (Argentina) 85 1041(1), 1041(3), 1044(3), 1046(1) to 1046(4), 1047(1) to 1047(3), 1062(2), 1063(4) • 4 eggs; same data as for holotype; 2 May 2014; UNLPam 348(1), 348(2), 644(1), 1222(3). General morphological description of the animals Body length 97–342 µm (Fig. 1; Tables 3, 5), yellowish before mounting, transparent after mounting. Eyespots, small and very caudal, present (Fig. 1B). Smooth cuticle with pores of different shapes. Under PCM, apparently, the smaller pores (around 1 µm) are usually roundish (though often with irregular margins), or few elliptical, while the larger typically triangular, quadrangular, trilobate or quadrilobate (Fig. 2); the pore size is 0.9–3.1 µm on the dorsum (with the biggest on the head, on the mid-dorsal line along the body, and on the caudal extremity), 1.3–3.4 µm on the legs, 1.0–1.8 µm on the ventral cuticle. Coherently, dorsal pores, on average bigger, appear more often non-roundish, while the ventral ones on average smaller, appear more often roundish. Exceptionally, some caudal or leg pore may appear pentagonalor, extremely rarely, star-shaped (with five lobes/arms; Fig. 2) under PCM, but this occurs only in few specimens and in only 1–2 pores in each of these few specimens. SEM reveals that pores are actually never perfectly rounded; instead, few small pores are truly elliptical, a minority (of any size) is irregular, while the rest are all (both dorsal and ventral, smaller and larger) polygonal or multilobate, from three to five angles/lobes/arms (Fig. 2); pores in an unsuitable position, and/or too small, may give the false impression to be irregularly roundish under SEM also, and, obviously, the lower magnification of PCM gives the impression of seeing a more common shape, especially where pores are smaller. There is no clear distinction between polygonal and multilobate pores, since there are many intermediate shapes (e.g., between triangular and three-lobated/armed, or between quadrangular and four-lobated/ armed), and, very probably, each pore may partially appear more polygonal or multilobate depending on the cuticle distention or contraction. Pentagonal pores are less common but more easily detectable under SEM with respect to PCM (several of them can be seen on each specimen), while properly star-shaped (with five arms/lobes) are quite rare: some of the specimens mounted for SEM apparently lack them, while the others may show one or few of them (Fig. 2). Pores are arranged in transverse bands made of one or more transverse rows (more regular in young specimens). The cuticle along the body, as visible in most eutardigrades, forms transverse folds marking the division into the five body segments (head plus four segments of the trunk), and, additionally, each segment is subdivided into 2 ‘subsegments’, one more anterior, and one more posterior, by an additional transverse cuticular fold. In this way, there are ten cuticular subsegments of the body clearly followed by the dorsal (Tables 2, 4), and partially ventral, pore arrangement. Smaller (97–156 µm, called young) and larger (180–342 µm, called senior) specimens show differences in the pore number and arrangement on each subsegment, but we concluded they must belong to the same species (additionally to having been found in the same sample, first sampling) for the following reasons: 1) only one egg type was found; 2) most morphological characters were the same; 3) regarding the more detailed characters for which they differed, the two groups had clear body size distinction (97–156 µm vs 180–342 µm) with no exceptions; 4) the main difference regarded the pore number and pattern, but this was consistent with ontogenetic changes documented in other species (e.g., Minibiotus pentannulatus Londoño, Daza, Lisi & Quiroga, 2017), and the pattern of the bigger specimens was still perfectly comparable with that of the smaller, just more complicated due to the appearance of more pores; 5) the other differences were metric, but they all appeared consistent with allometric growing (buccal tube becoming wider, and placoids and claws becoming longer) already known in eutardigrades, European Journal of Taxonomy 958: 77–113 (2024) 92 Table 3. Measurements (in µm) and pt values of selected morphological structures of the young paratypes of Minibiotus dispositus sp. nov. N = number of specimen/structures measured; Range = refers to the smallest and the largest structure among all measured specimens; SD = standard deviation. Character N Range Mean SD µm pt µm pt µm pt Body length 19 97 – 156 433 – 705 126 550 17 71 Buccopharyngeal tube Buccal tube length 19 21.5 – 25.0 –22.9 –0.8 – Stylet support insertion point 17 13.7 – 16.8 62.9 – 72.9 15.1 66.3 0.9 2.1 Buccal tube external width 19 1.4 – 2.1 6.4 – 8.7 1.8 7.9 0.2 0.6 Buccal tube internal width 19 0.9 – 1.2 3.8 – 5.2 1.0 4.5 0.1 0.4 Ventral lamina length 10 9.9 – 11.4 45.4 – 49.5 10.7 47.0 0.5 1.6 Placoid lengths Macroplacoid 1 18 1.9 – 2.3 8.0 – 9.7 2.0 8.8 0.1 0.5 Macroplacoid 2 18 1.6 – 2.0 7.2 – 8.5 1.8 7.8 0.1 0.4 Macroplacoid 3 18 1.6 – 1.9 6.8 – 8.0 1.7 7.5 0.1 0.3 Microplacoid 16 0.8 – 1.0 3.2 – 4.2 0.8 3.7 0.1 0.3 Macroplacoid row 18 5.9 – 7.3 26.0 – 30.9 6.3 27.4 0.4 1.1 Placoid row 16 7.0 – 8.6 30.1 – 36.3 7.3 32.2 0.4 1.3 Claw 1 heights External primary branch 13 6.5 – 7.3 28.1 – 31.5 6.8 30.0 0.2 1.0 External secondary branch 5 4.3 – 5.3 18.7 – 22.4 4.7 20.2 0.4 1.5 Internal primary branch 10 6.3 – 7.1 27.6 – 31.1 6.7 29.2 0.3 1.2 Internal secondary branch 6 4.0 – 5.0 17.1 – 21.3 4.2 18.4 0.4 1.5 Claw 2 heights External primary branch 11 6.7 – 7.5 27.8 – 31.8 6.9 30.3 0.2 1.3 External secondary branch 9 4.0 – 5.3 17.3 – 22.3 4.3 18.7 0.5 1.6 Internal primary branch 7 6.4 – 7.3 29.1 – 30.6 6.7 29.8 0.3 0.6 Internal secondary branch 4 4.0 – 4.9 18.0 – 20.5 4.3 18.8 0.4 1.2 Claw 3 heights External primary branch 10 6.6 – 7.0 28.1 – 32.2 6.8 29.6 0.1 1.1 External secondary branch 7 4.0 – 5.7 17.5 – 23.8 4.5 19.2 0.6 2.2 Internal primary branch 10 6.4 – 7.5 27.9 – 32.0 6.8 29.5 0.3 1.4 Internal secondary branch 5 4.0 – 5.4 17.4 – 22.6 4.3 18.9 0.6 2.2 Claw 4 heights Anterior primary branch 6 6.9 – 8.4 28.9 – 35.4 7.4 31.5 0.6 2.4 Anterior secondary branch 5 4.2 – 4.5 18.2 – 19.2 4.4 18.8 0.1 0.5 Posterior primary branch 8 6.7 – 7.9 29.6 – 34.9 7.2 32.0 0.4 1.9 Posterior secondary branch 6 3.8 – 4.8 16.5 – 20.1 4.1 18.2 0.3 1.3 ROCHA A. et al., A new species of Minibiotus (Tardigrada) from Salta City (Argentina) 93 are arranged in 8 bands (one per subsegment) made of transverse rows, and, lastly, subsegment 10 has pores not arranged in proper rows and continuing onto the dorsal part of legs IV. Describing textually in detail the complete dorsal/dorso-lateral pore arrangement would be long and complicated, with continuous necessity to compare the text with Figures and Tables; for this reason, we refer to these latter (Fig. 6; Table 2) for the description of the dorsal/dorso-lateral pore arrangement. Leg pores (Fig. 6, Table 2): on the external side of each leg (I–IV) there is a big, usually lobate (three or four lobes) pore, but sometimes just triangular/quadrangular (usually triangular or three-armed on legs I–II, while usually quadrangular or four-armed on legs III–IV); legs III show few additional, smaller and more caudal pores, while legs IV show 3–4 additional, dorsal pores. Fig. 7. Minibiotus dispositus sp. nov. Schematic drawing of the arrangement of the pores of the senior specimens. A. Dorsal cuticle. B. Ventral cuticle. European Journal of Taxonomy 958: 77–113 (2024) 94 Arrangement of ventral pores (Fig. 6): ventral cuticle may show 1–2 medial pores, aligned longitudinally, on the caudal portion of the head (subsegment 2), and, normally, seven rows (each representing also a band) each on subsegments 3–9 (subsegment 10 without ventral pores), organized as follows: two rows are present on each of the first three segments of the trunk (subsegments 3–8), while only one on the hind segment (subsegment 9); four medial pores are present in all seven rows, but the three rows just behind each of legs I–III (subsegments 3, 5 and 7) show some (usually three) additional, smaller, pores lying more laterally just at the base of the legs; instead, the four rows consisting of only the four medial, bigger pores, are the interlegs 4, 6, 8 and 9. Morphometry is reported in Table 3. Senior specimens (Figs 1, 7; Tables 4–5; Supp. file 2) In the senior specimens, the dorsal and dorso-lateral pore pattern (Figs 1, 7), and, partially, the ventral one, is basically a complication of that of the young, with an increase of the pore number (and their rows) and also introducing more variability and less ‘order’ especially in those that should be the pore transverse rows. Arrangement of dorsal and dorso-lateral pores: the bigger pores are kept rather similar in shape and size from the earlier life stage(s), with rather good correspondence especially on the head and on the legs, while additional pores appear, usually smaller, resulting in the above-mentioned increase in row number and decrease in row clearness especially on the rest of the dorsum. As stated for the young, we refer to Figures and Tables (Figs 1, 7; Table 4) for the detailed description of the dorsal/dorso-lateral pore arrangement of the senior specimens, with all the more reason since the pattern is indeed more complicated. In general, there is a group of cephalic pores (subsegment 1), 7 bands of pores (subsegments 2–8) more or less organized in transverse rows, while the eighth band that was recognisable in the smaller specimens (subsegment 9), here is joined to the group of caudal pores (subsegment 10 plus dorsum of legs IV), so that the dorsal and dorso-lateral cuticle of the whole hind segment (plus legs IV) show a unique, large, caudal group made of many pores with no clear rows recognisable. Arrangement of leg pores: on the external side of legs I–III there are some large, usually lobate (with 3–4 lobes) pores, but sometimes just triangular/quadrangular; legs IV show several triangular, quadrangular and lobate/star-shaped (with 3–5 lobes/arms) pores. Arrangement of ventral pores: ventrally, there is instead a good correspondence with the pattern of the young specimens; thus, to avoid repetition, we stress here only the difference, consisting of a tendency to complicate only the medial part of the transverse rows, which are partially multiplied with 4–12 pores forming in the centre a patch of pores sometimes similar to some geometric figure such as a rhombus, a circle, a square or a pentagon. Morphometry is reported in Table 5. These senior specimens, in comparison to the young, have a slightly wider buccal tube (e.g., pt of external width [8.3–11.5] vs [6.4–8.7] in young) and longer macroplacoids (e.g., pt of macroplacoid row [32.7–39.4] vs [26.0–30.9] in young) and claws (e.g., pt of claw I external primary branches [28.7–35.9] and of claw IV posterior primary branches [38.0–46.0] vs [28.1–31.5] and [29.6–34.9] respectively in young). Eggs (Fig. 8; Table 6; Supp. file 3) Eggs are light orange in colour before mounting, spherical and laid freely. Processes in the shape of elongated cones, rarely bifurcate distally, usually ending at the tip in a filament (Fig. 8A–E). In some processes, single bubble-like structures can be seen inside the distal half portion of the processes ROCHA A. et al., A new species of Minibiotus (Tardigrada) from Salta City (Argentina) 95 Fig. 8. Minibiotus dispositus sp. nov. Eggs seen in PCM. A. Paratype (UNLPam 1222(3)), midsection. B, F. Paratype (UNICT 6013). C–E. Paratype (UNLPam 348(1)). B. Surface. C–F. Details of egg. Black filled flat arrowheads indicate bifurcated process, white empty flat arrowhead indicates singular bubble-like structure, white filled indented arrowhead indicates filament tip, white filled flat arrowhead indicates process base whereas black filled indented arrowhead indicates granulation of the chorion. Scale bars in µm. European Journal of Taxonomy 958: 77–113 (2024) 96 Table 4 (continued on next page). Schematization of the dorsal pore pattern of senior specimens of Minibiotus dispositus sp. nov. with reference to the precise body districts. Body regionsBody segments Subsegments and legs Number of rows Number of pores Pore shape Head Head 1 4 2 4 2 6 triangular quadrilobated triangular various 2 2 8 10 various Trunk Second segment (segment I of the trunk), with legs I 3 (with legs I) 4 plus lateral patch 6–10 6–10 6–10 6–10 lateral patch of about 7–12 various Legs I 1 big Three – or four –armed 4 2 8–10 8–10 various Third segment (segment II of the trunk), with legs II 5 (with legs II) 4 plus lateral patch 6–10 6–10 6–10 6–10 lateral patch of about 7–12 various Legs II 1 big Three – or four –armed pore 6 2 8–10 8–10 various Fourth segment (segment III of the trunk), with legs III 7 (with legs III) 5 plus lateral patch 4–10 4–10 4–10 4–10 4–10 lateral patch of about 7–12 various ROCHA A. et al., A new species of Minibiotus (Tardigrada) from Salta City (Argentina) 97 Table 4 (continued). Schematization of the dorsal pore pattern of senior specimens of Minibiotus dispositus sp. nov. with reference to the precise body districts. Body regionsBody segments Subsegments and legs Number of rows Number of pores Pore shape Trunk Fourth segment (segment III of the trunk), with legs III 7 (with legs III) 5 plus lateral patch 4–10 4–10 4–10 4–10 4–10 lateral patch of about 7–12 various Legs III 1 big more anterior some caudal smaller Four – armed various 8 3 4–10 4–10 4–10 Fifth segment (segment VI of the trunk), with legs VI 9 continuous, big, caudal group made of many pores with no clear rows recognisable (also on the dorsum of legs IV) various 10 (with legs IV) Legs IV One big, four – or five – armed, others of various shape European Journal of Taxonomy 958: 77–113 (2024) 98 Table 5. Measurements (in µm) and pt values of selected morphological structures of senior types (including the holotype) of Minibiotus dispositus sp. nov. (first sampling specimens). N = number of specimen/structures measured; Range = refers to the smallest and the largest structure among all measured specimens; SD = standard deviation. Character N Range Mean SD Holotype µm pt µm pt µm pt µm pt Body length 29 180 – 342 656 – 1047 258 883 47 112 300 989 Buccopharyngeal tube Buccal tube length 29 24.6 – 33.6 29.0 –2.2 –30.3 – Stylet support insertion point 28 17.2 – 23.2 65.0 – 71.4 19.9 68.2 1.5 1.7 20.1 66.2 Buccal tube external width28 2.2 – 3.6 8.3 – 11.5 2.8 9.7 0.4 0.8 3.5 11.5 Buccal tube internal width 28 1.1 – 2.3 4.2 – 7.4 1.6 5.5 0.4 1.0 2.2 7.4 Ventral lamina length16 13.0 – 16.0 45.1 – 53.7 14.6 50.6 1.0 2.3 16.0 52.8 Placoid lengths Macroplacoid 1 29 2.6 – 4.0 9.8 – 13.4 3.3 11.3 0.4 0.9 3.6 11.7 Macroplacoid 2 29 2.1 – 3.5 8.1 – 11.6 2.7 9.2 0.3 0.7 2.9 9.6 Macroplacoid 3 29 1.9 – 3.3 7.5 – 11.1 2.6 8.9 0.3 0.7 2.9 9.6 Microplacoid 29 0.9 – 1.8 3.5 – 5.9 1.4 4.9 0.3 0.8 1.7 5.7 Macroplacoid row 28 8.5 – 12.5 32.7 – 39.4 10.5 36.1 1.2 2.1 11.9 39.2 Placoid row 28 9.7 – 14.9 39.4 – 47.8 12.5 43.0 1.4 2.8 14.5 47.8 Claw 1 heights External primary branch 27 7.8 – 10.6 28.7 – 35.9 9.4 32.2 0.8 1.7 9.5 31.3 External secondary branch 26 5.4 – 8.6 20.6 – 26.6 6.9 23.5 0.8 1.5 7.5 24.7 Internal primary branch 28 7.2 – 10.1 27.1 – 34.9 9.0 30.8 0.9 2.1 9.1 29.9 Internal secondary branch 27 5.1 – 7.8 19.9 – 25.6 6.5 22.3 0.8 1.7 7.0 23.1 Claw 2 heights External primary branch 28 8.0 – 11.2 30.5 – 38.5 9.9 34.3 1.0 1.8 10.4 34.4 External secondary branch 25 5.7 – 8.7 22.1 – 28.8 7.4 25.4 0.9 1.8 7.7 25.2 Internal primary branch 28 7.8 – 10.9 29.7 – 37.7 9.6 33.0 1.0 2.1 10.0 32.9 Internal secondary branch 27 5.0 – 8.7 19.6 – 26.6 6.9 23.8 0.9 1.9 7.1 23.2 Claw 3 heights External primary branch 29 8.4 – 11.9 32.4 – 39.7 10.3 35.6 0.9 1.7 10.7 35.3 External secondary branch 29 5.9 – 9.1 23.1 – 29.4 7.6 26.2 0.9 1.8 8.3 27.3 Internal primary branch 27 7.9 – 11.1 30.5 – 37.9 9.7 33.5 1.0 2.0 9.8 32.4 Internal secondary branch 26 5.5 – 8.2 21.5 – 28.1 7.2 24.7 0.8 1.8 7.3 24.1 Claw 4 heights Anterior primary branch 27 8.7 – 13.7 35.3 – 43.6 11.5 39.6 1.4 2.6 12.6 41.4 Anterior secondary branch 26 6.3 – 10.8 25.1 – 32.9 8.5 29.0 1.2 2.3 8.6 28.2 Posterior primary branch 27 9.7 – 14.1 38.0 – 46.0 12.2 41.7 1.2 2.6 12.7 41.8 Posterior secondary branch 27 6.8 – 10.7 26.0 – 34.2 8.7 29.6 1.1 2.5 8.9 29.5 ROCHA A. et al., A new species of Minibiotus (Tardigrada) from Salta City (Argentina) 99 (Fig. 8C). On the egg circumference 29–34 processes are present and about 135–177 in the hemisphere depending also on the egg size. Process bases without projections on the chorion (Fig. 8F), but this latter, between the process, has evident granulation (Fig. 8A–B, F). Quantitative data are reported in Table 6. DNA sequences The sequenced senior specimen (V4) of Minibiotus dispositus sp. nov. is differentiated from all the other sequenced species belonging to the genus Minibiotus, as indicated by the ranges of genetic p-distances: COI (523 bp dataset): 21.4 to 26.5% (Supp. file 4), with the most similar being unpublished sequences of M. citlalium (OP684766, OP684767) from Mexico; ITS2 (531 bp dataset): 12.3 to 27.8% (Supp. file 5), with the most similar being M. ioculator (MT024000) from South Africa; 18S (778 bp dataset): 0.2 to 13.4% (Supp. file 6), with the most similar being M. furcatus (FJ435745) from Spain; 28S (817 bp dataset): 1.8 to 3.1% (Supp. file 7), with the most similar being Minibiotus sp. (MH079492) from Chile. The ASAP analysis for both COI and ITS2 genes (Tables 7–8) further confirms the status of Minibiotus dispositus sp. nov. specimen V4 as a clearly distinct species from the other sequenced taxa of the genus. The integration of the present molecular data with the morphological ones, therefore points to the validity of the erection of Minibiotus dispositus sp. nov. Morphological differential diagnosis Cuticular pores arranged in transverse bands are reported in many species of the genus, but the following are excluded from comparison for the reasons indicated in brackets: Minibiotus formosus Zawierucha, Dziamięcki, Jakubowska, Michalczyk & Kaczmarek, 2014, M. granatai (Pardi, 1941) and M. gumersindoi Guil & Guidetti, 2005 (they have only round/elliptical pores, lacking lobated/starshaped ones); M. jonesorum Meyer, Lyons, Nelson & Hinton, 2011 (it lacks microplacoid and has polygonal pores, that also are very large and very densely distributed); M. pseudofurcatus (Pilato, 1972) (pores have at maximum 3 lobes/arms). Table 6. Measurements of selected morphological structures, and other metric traits, of eggs of Minibiotus dispositus sp. nov. mounted in polivinil lactofenol medium. N = number of eggs/structures measured; Range = refers to smallest and largest structure or value among all measured eggs/structures; SD = standard deviation. CharacterN Range Mean SD Diameter of egg without processes (in μm) 6 55.6–82.1 66.9 9.6 Diameter of egg with processes (in μm) 6 77.2–110 92.2 11.6 Process height (in μm) 6 13.0–17.0 14.8 1.4 Process base width (in μm) 6 2.0–4.5 3.2 0.9 Distance between processes 6 1.3–3.4 2.3 0.8 Number of processes on the egg circumference 6 29–34 31.3 0.9 Number of processes on the egg hemisphere 6 135–177 149.4 4.9 European Journal of Taxonomy 958: 77–113 (2024) 100 For differential diagnosis, we here compare M. dispositus sp. nov. with the species sharing the presence of smooth cuticle, multilobated pores (3–5 arms/lobes) and three macroplacoids plus evident microplacoid. However, considering the paucity of 5-armed pores recognisable under PCM (only 0–2 per specimen, and only caudally or on legs) in the new species, we excluded from comparison the species with very numerous, evident star-shaped pores (5 arms or more) clearly observable under PCM on all the body (thus excluding: M. citlalium Dueñas-Cedillo & García-Román, 2020, M. claxtonae Rossi, Claps & Ardohain, 2009, M. constellatus Michalczyk & Kaczmarek, 2003, M. pentannulatus Londoño, Daza, Lisi & Quiroga, 2017, M. pseudostellarus Roszkowska, Stec, Ciobanu & Kaczmarek, 2016, M. sidereus Pilato, Binda & Lisi, 2003). Table 7. Results of species delimitation analysis of the genus Minibiotus R.O. Schuster, 1980 from GenBank by automatic partitioning (ASAP) on COI gene (lower ASAP-score = 1.50; threshold p-distance = 7.86%). Specimen Species partition FJ435802 Minibiotus furcatus Tar527 Madrid Spain 1 FJ435803 Minibiotus gumersindoi Tar710 Madrid Spain 2 JX683828 Minibiotus furcatus 2003 C3042 V10 Portugal 2 JX683829 Minibiotus furcatus 2007 C3039 Mini 1 Portugal 2 JX865309 Minibiotus sp. Mini_06_138 3 JX865313 Minibiotus sp. Mini_07_120 4 MT023412 Minibiotus ioculator ZA.274 South Africa 5 MT023413 Minibiotus pentannulatus TZ.027 haplotype 1 Tanzania 6 MT023414 Minibiotus pentannulatus TZ.027 haplotype 2 Tanzania 6 MW306857 Minibiotus sp. A S.297 Min Italy 7 MW306858 Minibiotus sp. A S.297 Min 2 Italy 7 MW306859 Minibiotus sp. A S.69 Min Italy 7 ON005160 Minibiotus intermedius Min4 GR Marburg Germany 8 OP013286 Minibiotus cf. intermedius DT274 Lembolovo Russia 9 OP013287 Minibiotus cf. intermedius DT270 Lembolovo Russia 9 OP013288 Minibiotus cf. intermedius DT279 Lembolovo Russia 9 OP684765 Minibiotus citlalium AI04 Iztaccihuatl Mexico 10 OP684766 Minibiotus citlalium AI08 Iztaccihuatl Mexico 10 OP684767 Minibiotus citlalium AI19 Iztaccihuatl Mexico 10 OP684770 Minibiotus sidereus AI12 Iztaccihuatl Mexico 10 OP684768 Minibiotus sidereus AI09 Iztaccihuatl Mexico 10 OP684769 Minibiotus sidereus AI11 Iztaccihuatl Mexico 10 OP684773 Minibiotus sp. AI33 Iztaccihuatl Mexico 10 OP684772 Minibiotus sp. AI31 Iztaccihuatl Mexico 10 OP684771 Minibiotus sp. AI13 Iztaccihuatl Mexico 10 V4 Minibiotus sp. nov. 11 ROCHA A. et al., A new species of Minibiotus (Tardigrada) from Salta City (Argentina) 101 Lastly, we decided not to limit the comparison with species clearly having a pore pattern (described or at least well visible in the description illustrations), because such a character may not have been noticed or reported in some past descriptions; in this way, M. aculeatus Murray, 1910 and M. vinciguerrae Binda & Pilato, 1992 are included, also considering that the old illustrations available may not be perfectly accurate. All that considered, Minibiotus dispositus sp. nov. is to be compared with: M. aculeatus (Murray, 1910); M. bernhardi Schuster, 2021; M. bisoctus (Horning, Schuster & Gregarick, 1978); M. eichhorni Michalczyk & Kaczmarek, 2004; M. ethelae Claxton, 1998; M. furcatus (Ehrenberg, 1859); M. harrylewisi Meyer & Hinton, 2009; M. lazzaroi (Maucci, 1986); M. orthofasciatus Fontoura, Pilato, Lisi & Morais, 2009; M. pustulatus (Ramazzotti, 1959); M. vinciguerrae Binda & Pilato, 1992; M. weglarskae Michalczyk, Kaczmarek & Claxton, 2005; M. xavieri Fontoura, Pilato, Morais & Lisi, 2009. For correct morphometric comparisons, considering the allometry of some characters of the new species, we compared the morphometric characters in the present differential diagnosis taking into account the body size (available from the literature) of each compared species. Our young specimens had body sizes of up to about 156 µm while our senior specimens had body lengths starting from about 180 µm; most of the compared species had a body length starting from at least 200 µm; thus, we compared the morphometry of these species with that of our senior specimens; only two species, M. orthofasciatus and M. weglarskae, had body lengths starting from less than 180 µm (from 138 µm and 166 µm, respectively) but exceeding 200 µm in the maximum value: in this case we joined together our morphometric ranges of young and senior specimens for comparison (no compared species had a body length range compatible only with our young specimens). Table 8. Results of species delimitation analysis of the genus Minibiotus R.O. Schuster, 1980 from GenBank by automatic partitioning (ASAP) on ITS2 gene (lower ASAP-score = 2.00; threshold p-distance = 6.07%). Specimen Species partition MT024000 Minibiotus ioculator ZA.274 South Africa 1 MT024001 Minibiotus pentannulatus TZ.027 Tanzania 2 OP696660 Minibiotus citlalium AI04 Iztaccihuatl Mexico 3 OP696661 Minibiotus citlalium AI08 Iztaccihuatl Mexico 3 OP696662 Minibiotus citlalium AI19 Iztaccihuatl Mexico 3 OP696663 Minibiotus sidereus AI09 Iztaccihuatl Mexico 3 OP696664 Minibiotus sidereus AI11 Iztaccihuatl Mexico 3 OP696666 Minibiotus sp. AI13 Iztaccihuatl Mexico 3 OP696667 Minibiotus sp. AI31 Iztaccihuatl Mexico 3 OP696665 Minibiotus sidereus AI12 Iztaccihuatl Mexico 3 OP696668 Minibiotus sp. AI33 Iztaccihuatl Mexico 3 OK663216 Minibiotus sp. S69 01 S69 Min 1 Italy 4 OP035707 Minibiotus cf. intermedius DT270 Lembolovo Russia 5 OP035708 Minibiotus cf. intermedius DT274 Lembolovo Russia 5 V4 Minibiotus sp. nov. 6 European Journal of Taxonomy 958: 77–113 (2024) 108 Claxton S.K. 1998. A revision of the genus Minibiotus Tardigrada: Macrobiotidae with descriptions of new species from Australia. 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The impact of fire on terrestrial tardigrade biodiversity: a first case-study from Portugal. Journal of Limnology 72: 152–159. https://doi.org/10.4081/jlimnol.2013.s1.e19 Zawierucha K., Dziamięcki J., Jakubowska N., Michalczyk Ł. & Kaczmarek Ł. 2014. New tardigrade records for the Baltic states with a description of Minibiotus formosus sp. n. (Eutardigrada, Macrobiotidae). ZooKeys 408: 81–105. https://doi.org/10.3897/zookeys.408.6612 Manuscript received: 17 December 2023 Manuscript accepted: 6 May 2024 Published on: 20 September 2024 Topic editor: Magalie Castelin Section editor: Daniel Stec Desk editor: Pepe Fernández Printed versions of all papers are deposited in the libraries of four of the institutes that are members of the EJT consortium: Muséum national dʼHistoire naturelle, Paris, France; Meise Botanic Garden, Belgium; Royal Museum for Central Africa, Tervuren, Belgium; Royal Belgian Institute of Natural Sciences, Brussels, Belgium. The other members of the consortium are: Natural History Museum of Denmark, Copenhagen, Denmark; Naturalis Biodiversity Center, Leiden, the Netherlands; Museo Nacional de Ciencias Naturales-CSIC, Madrid, Spain; Leibniz Institute for the Analysis of Biodiversity Change, Bonn – Hamburg, Germany; National Museum of the Czech Republic, Prague, Czech Republic; The Steinhardt Museum of Natural History, Tel Aviv, Israël. Supplementary files For the Excel files (1–2), the Excel template “Parachela” (ver. 1.7) from the tardigrade Register (Michalczyk & Kaczmarek 2013) was used. Supp. file 1. Complete Excel version of measurements [in µm] and pt values of selected morphological structures of the young paratypes of Minibiotus dispositus sp. nov. https://doi.org/10.5852/ejt.2024.958.2663.12321 Supp. file 2. Complete Excel version of measurements [in µm] and pt values of selected morphological structures of the senior types of Minibiotus dispositus sp. nov. https://doi.org/10.5852/ejt.2024.958.2663.12323 Supp. file 3. Complete Excel version of the quantitative characters of the eggs of Minibiotus dispositus sp. nov. https://doi.org/10.5852/ejt.2024.958.2663.12325 Supp. file 4. Genetic distance values (p-distance) for the COI gene among all available Minibiotus species. The analysis was carried out on a dataset of 523 bp. Newly analyzed specimen is given in bold. https://doi.org/10.5852/ejt.2024.958.2663.12327 ROCHA A. et al., A new species of Minibiotus (Tardigrada) from Salta City (Argentina) 113 Supp. file 5. Genetic distance values (p-distance) for the ITS2 gene among all available Minibiotus species. The analysis was carried out on a dataset of 531 bp. Newly analyzed specimen is given in bold. https://doi.org/10.5852/ejt.2024.958.2663.12329 Supp. file 6. Genetic distance values (p-distance) for the 18S gene among all available Minibiotus species. The analysis was carried out on a dataset of 778 bp. Newly analyzed specimen is given in bold. https://doi.org/10.5852/ejt.2024.958.2663.12331 Supp. file 7. Genetic distance values (p-distance) for the 28S gene among all available Minibiotus species. The analysis was carried out on a dataset of 817 bp. Newly analyzed specimen is given in bold. https://doi.org/10.5852/ejt.2024.958.2663.12333 Supp. file 8. Complete Excel version of statistically significant differences (through one-side Student t-tests) of overlapping pt ranges of selected metric characters between specimens of Minibiotus dispositus sp. nov. and similar species. https://doi.org/10.5852/ejt.2024.958.2663.12335 Supp. file 9. Template from Tables 2–4; schematization for the indication of the disposition of cuticular structures (pores, pseudoplates, gibbosities etc.) with reference to the precise body districts. https://doi.org/10.5852/ejt.2024.958.2663.12337