Mitogenome of a new Ramazzottius species (Tardigrada: Eutardigrada: Ramazzottiidae) discovered in rock pools along with its temperature and desiccation-related proteins repertoire

Vol.:(0123456789) Organisms Diversity & Evolution (2025) 25:119–135 https://doi.org/10.1007/s13127-024-00662-x ORIGINAL ARTICLE Mitogenome ofanew Ramazzottius species (Tardigrada: Eutardigrada: Ramazzottiidae) discovered inrock pools alongwithits temperature anddesiccation‑related proteins repertoire MatteoVecchi1 · DanielStec1 Received: 6 August 2024 / Accepted: 9 October 2024 / Published online: 31 October 2024 © The Author(s) 2024 Abstract Ramazzottius is a widespread genus of tardigrades with extreme cryptobiotic capabilities. Thanks to its ability to survive desiccation and freezing, this genus is usually recorded from harsh habitats such as exposed mosses and lichens and rock pools. In the last years, research focused on both describing Ramazzottius diversity and revealing the molecular mechanisms behind their cryptobiotic capabilities. Despite the research efforts in these fields, much still remains to be discovered. Here we describe a new Ramazzottius species from an Italian rock pool by means of integrative taxonomy (morphology, morphometry, and DNA sequencing) and sequenced its genome with Nanopore technology to provide an assembled mitogenome and annotate its Temperature and Desiccation Resistance Proteins (TDPR) repertoire. The new gonochoric species is phylogenetically close to the parthenogenetic R. varieornatus, a strain of which (YOKOZUNA-1) has been adopted as model organism for the study of cryptobiosis. The mitogenome of the new species shows perfect synteny with R. varieornatus and shares with it most of the TDPR genes. The relative genetic similarity of the new species to the model R. varieornatus, combined with unique biological traits (for example the difference in reproductive mode and the unique habitat it colonizes), makes the new species a potential new addition to the range of model tardigrade species. Keywords Ramazzottius· Anhydrobiosis· Nanopore· New species· Mitogenome Introduction Tardigrades (also known as water bears) are a group of microscopic animals forming a phylum included in the clade Panarthropoda (Schill, 2018). These meiofaunal animals live in a wide variety of habitats: from aquatic to terrestrial environments, from seas to mountains (Nelson etal., 2018), where they can be found in different substrates such as sediments, soil, bryophytes, lichens, and even in cryoconite holes on glaciers (Nelson etal., 2018; Zawierucha etal., 2016). Thanks to their ability to enter into ametabolic life stages (cryptobiosis), some tardigrades can survive extreme conditions like desiccation and freezing (Hengherr & Schill, 2018; Rebecchi etal., 2007; Schill & Hengherr, 2018; Zawierucha etal., 2023). Ramazzottius (Binda & Pilato, 1986) is a genus of limnoterrestrial tardigrades characterized by the presence of apophyses for the insertion of the stylet muscles (AISM) in the shape of blunt hooks, articulated external claws, and sometimes paired elliptical organs on the head. Representatives of Ramazzottius are considered xerophilic, often found in habitats exposed to sunlight, often dwelling in bryophytes and lichens (Bartels etal., 2011; Biserov, 1997; Guidetti etal., 2022), but surprisingly also in more aquatic habitats like rock pools sediment (Vecchi etal., 2022). The genus Ramazzottius includes different species complexes with generally unresolved phylogenetic relationships among and inside them (Dey etal., 2024; Kihm etal., 2023). The most common and widespread group is the polyphyletic oberhaeuseri morpho-group, characterized by eggs with hemispherical processes and rampant cryptic and pseudocryptic speciation (Guidetti etal., 2022; Stec etal., 2018). The monophyletic baumanni and szeptyckii complexes from the tropics are characterized by peculiar ornamented cuticle and * Matteo Vecchi matteo.v[email protected]; m.v[email protected]ow.pl 1 Institute ofSystematics andEvolution ofAnimals, Polish Academy ofSciences, Sławkowska 17, 31-016, Kraków, Poland 120 M.Vecchi, D.Stec their eggs are unknown (Dey etal., 2024). Cryoconicus, recently separated from Ramazzottius and characterized by peculiar AISM and claws morphology and by an intense body pigmentation (Guidetti etal., 2019; Zawierucha etal., 2018), has been suggested to not be valid (Dey etal., 2024), and thus be part of Ramazzottius. Additionally, species that do not fall into any of these groups are present and are generally characterized by body with relatively plain sculpture (compared to the baumanni and szeptyckii complexes) and with egg processes usually in the shape of spines and cones (Bertolani & Kinchin, 1993; Biserov, 1997; Guidetti etal., 2019; Kihm etal., 2023; Stec etal., 2016). The genus Ramazzottius made its way into becoming a model taxon for the study of extreme resistance mechanisms in tardigrades and their biological interactions (see for example Emdee etal., 2024; Horikawa, 2008; Horikawa etal., 2008, 2013); however, at this time, experimental and genomic data are available for only one species of the genus (R. varieornatus Bertolani & Kinchin, 1993 strain YOKOZUNA-1; Horikawa etal., 2008), precluding a comparative approach across related species. Tardigrades from rock pools have been investigated thoroughly only recently (Vecchi etal., 2022); however, two new species from these habitats have been discovered and already described (Vecchi etal., 2023a, 2023b) showing their potential as source of tardigrades biodiversity. Ramazzottius from rock pools have been shown to possess an incredible tolerance to freeze–thaw cycles (Zawierucha etal., 2023), even higher than tardigrades taxa adapted to life on glaciers, highlighting the extreme adaptations encountered in tardigrades from this habitat. Ramazzottius claudii sp. nov. from a rock pool in the Italian Apennines is described here by means of integrative taxonomy using light microscopy (LM), scanning electron microscopy (SEM), and DNA sequencing. To provide the community with more genomic data to help unravel the resistance mechanisms of tardigrades, we also provide for the first time in a tardigrade species description, its annotated whole mitochondrial genome and its repertoire of Temperature and Desiccation-Related Proteins (Fleming etal., 2024) sequenced with long-read third generation sequencing technology (Nanopore). Materials andmethods Sampling andtardigrades extraction A rock pool sediment sample was collected on 27/06/2020 in Corniglio, Parma, Italy (44.3960680 10.00437) by scraping the sediment with a clean plastic spoon into a plastic tube. The sample was kept desiccated and frozen at − 20°C until processing. The sample was processed to extract tardigrades as in (Vecchi etal., 2023a). The sample was collected under sampling permit N.0001671/2020 from Parco Nazionale Appennino Tosco-Emiliano (Italy). Additional material Individuals of Hebesuncus conjungens (Thulin, 1911) were extracted from a moss collected on 15/06/2023 in Corniglio, Parma, Italy (44.3800370 10.0414130) for DNA sequencing (Sample collected under sampling permit N.0003526/2021 from Parco Nazionale Appennino Tosco-Emiliano, Italy). Previously extracted Ramazzottius individuals (S66.01 and S132.02 from Vecchi etal., 2022), and already sequenced for COI, were used for sequencing additional markers (18S, 28S and ITS2). Microscopy andimaging Specimens for light microscopy were mounted on microscope slides in a small drop of Hoyer’s medium, secured with a cover slip and dried at 50°C for a week. Slides were examined under a Leica DMLB light microscope with phase contrast (PCM), associated with a digital camera. For structures that could not be satisfactorily focused on a single light microscope photograph, a stack of 2–5 images were taken with an equidistance of ca. 0.2μm and assembled manually into a single deep-focus image in GIMP v.2–10 (GIMP Development Team, 2019). Specimens for scanning electron microscopy were prepared according to the protocol of (Camarda etal., 2023). Specimens were examined under high vacuum in a Versa 3D Dual Beam Scanning Electron Microscope at the ATOMIN facility of the Jagellonian University, Kraków, Poland. For structures that could not be satisfactorily focused in a single photograph, a stack of 2–6 images were taken with an equidistance of ca 0.2μm and assembled manually into a single deep-focus image. All figures were assembled in Figure J (Mutterer & Zinck, 2013). Morphometrics andmorphological nomenclature All measurements are given in micrometers (μm). Structures were measured only if their orientation was suitable. Body length was measured from the anterior extremity to the posterior 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). The pt index is the ratio of the length of a given structure to the length of the buccal tube (Pilato, 1981). Claws were measured according to (Stec etal., 2018) and ratios are provided according to (Vecchi etal., 2023b). Morphometric data were handled using the “Parachela” ver. 1.7 template available from the Tardigrada Register (Michalczyk & Kaczmarek, 2013). 121 Mitogenome ofanew Ramazzottius species (Tardigrada: Eutardigrada: Ramazzottiidae)… The raw morphometric data are provided as Supplementary Materials (Online resources 01). Genotyping DNA was extracted from individual animals following a Chelex® 100 resin (BioRad) extraction method by Casquet etal. (2012) with modifications described in detail in Stec etal. (2020). Briefly, animals were individually placed in 40μl of a 4% Chelex® solution with the addition of 3μl of proteinase K (20mg/ml, activity ≥ 30 U/mg) and incubated in a thermomixer at 56°C and 750rpm for 30min. The solution was then heated at 70°C for 10min and the supernatant was transferred to a new microcentrifuge tube and used as input for PCR. We sequenced four DNA fragments, three nuclear (18S rRNA, 28S rRNA, and ITS2) and one mitochondrial (COI). All fragments were amplified and sequenced according to the primers and protocols described in Stec etal. (2020). Sequencing products were read with the ABI 3130xl sequencer at the Genomed company (Warsaw, Poland). Phylogenetic reconstruction A phylogenetic reconstruction was made using the concatenated markers 18S rRNA + 28S rRNA + COI + ITS2 from Ramazzottius clade A (sensu Dey etal., 2024), with the addition of sequences from Ramazzottius kretschmanni Guidetti etal., 2022, Ramazzottius groenlandensis Kihm etal., 2023 and the sequences newly generated for this study. Sequences of H. conjugens were used as outgroup. The GenBank accession numbers of the sequences and those used in the phylogenetic reconstruction are presented in Table1. The 18S rRNA, 28S rRNA, and ITS2 sequences were aligned with MAFFT ver. 7 (Katoh, 2002; Katoh & Toh, 2008) with the G-INS-i method (thread = 4, threadtb = 5, threadit = 0, reorder, adjustdirection, anysymbol, maxiterate = 1000, retree 1, globalpair input). The COI sequences were aligned according to their amino acid sequences (translated using the invertebrate mitochondrial code) with the MUSCLE algorithm (Edgar, 2004) in MEGA7 with default settings (all gap penalties = 0, max iterations = 8, clustering method = UPGMB, lambda = 24). Alignments were visually inspected and trimmed in MEGA7. Sequences were concatenated with the R package “concatipede” v1.0.0 (Vecchi & Bruneaux, 2021). Model selection and maximum likelihood (ML) phylogenetic reconstruction were performed with IQTREE (Trifinopulos etal., 2016) on the partitioned dataset on the IQTREE web server (http:// iqtree. cibiv. univie. ac. at/) with default parameters and ultrafast bootstrap with 1000 replicates. The concatenated alignment is available as Supplementary Materials (DATA block of Online resources 02). Model selection results are available as Supplementary Material (Online resources 03). For Bayesian Inference (BI) phylogenetic reconstruction, model selection was performed for each alignment partition (6 in total: 18S rRNA, 28S rRNA, ITS-2 and three COI codons) using PartitionFinder2 (Lanfear etal., 2016). Bayesian inference (BI) phylogenetic reconstruction was performed using MrBayes v3.2.6 (Ronquist etal., 2012). Two runs (one cold chain and three heated chains each) of 20 million generations were used with a burn-in of 2 million generations, sampling a tree every 1000 generations. Posterior distribution sanity was checked using Tracer v1.7 (Rambaut etal., 2018). The MrBayes input file with the input alignment is available as Supplementary Materials (Online resources 02); model selection results are available as Supplementary Material (Online resources 03). Both the IQTREE tree and the MrBayes consensus tree are available as Supplementary Materials (Online resources 04). The phylogenetic trees were visualized with FigTree (Rambaut, 2007) and edited with Inkscape (Inkscape Project, 2020). Whole‑genome amplification andsequencing One individual Ramazzottius of the new species (Ram_ IT.158_WGA_1) was starved in sterile distilled water for 24h at 18°C and then washed twice in sterile distilled water. The individual was then dissected in a 0.5-μl drop of sterile distilled water with a sterilized entomological needle and used as starting material for a whole-genomic amplification (WGA) reaction using the REPLI-g Mini Kit (Cat. No. 150023, Qiagen) according to the manufacturer protocol. The reaction product was purified with a GeneMAGNET PCR/DNA Clean-Up Purification Kit (Cat. No. E3420, EURx) and the dsDNA was quantified using a Qubit Fluorometric assay. Approximately 2μg of amplified DNA were debranched for 1h with T7 Endonuclease I (Cat. No. M0302, New England Biolabs) according to the manufacturer protocol. The reaction product was again purified with GeneMAGNET and quantified with Qubit. The dsDNA was used as input for library preparation with the Oxford Nanopore Native Barcoding Kit 24v14 (Cat. No. SQK-NBD114.24, Oxford Nanopore Technologies—ONT) following the ONT community protocol NBE_9169_V114_REVP_15SEP2022 and sequenced on part of a FLO-MIN114 R10 flow cell using a MinION Mk1B for 48h. Basecalling, demultiplexing, and adaptors trimming were done with the software MinKNOW (ONT) using a fast basecalling strategy. Sequences of the DNA control sample (DCS) introduced during library preparation were removed with chopper (De Coster & Rademakers, 2023). The raw reads were submitted to NCBI SRA (BioProject PRJNA1082523, accession number SRR28173874). 122 M.Vecchi, D.Stec Mitogenome assembly andannotation The clean reads were blasted against a tardigrades mitochondrial proteins database (compiled from tardigrade mitochondrial protein sequences present in GenBank; Online resources 05) using DIAMOND (Buchfink etal., 2015; options: –sensitive –max-target-seqs 1) and the matching query sequences were extracted with seqtk (https:// github. com/ lh3/ seqtk) and assembled with Flye v 2.9.3 (Kolmogorov etal., 2019; Lin etal., 2016) with 5 polishing round (options: -i 5). The assembled sequences were visualized with Bandage (Wick etal., 2015) and the identity of the mitogenome contig confirmed by blastn against the Ramazzottius varieornatus YOKOZUNA-1 mitogenome (GenBank NC_031407). A final polishing of the retrieved mitogenome contig was done with medaka v 1.11.3 (https:// github. Table 1 GenBank accession numbers of the sequences used in the phylogenetic reconstruction SSU (18S) LSU (28S) COI ITS2 Source Ramazzottius claudii n.sp. IT.158.1 PQ108467 PQ108476 PQ109084 PQ110585 This study Ramazzottius claudii n.sp. IT.158.3 PQ108468 PQ108477 PQ109085 PQ110586 This study Ramazzottius claudii n.sp. S62 1 MW306836 Vecchi etal. (2022) Ramazzottius claudii n.sp. S62 2 MW306835 Vecchi etal. (2022) Ramazzottius claudii n.sp. S66 1 PQ108470 PQ108475 MW306833 PQ110584 This study; Vecchi etal. (2022) Ramazzottius claudii n.sp. S66 2 MW306834 Vecchi etal. (2022) Ramazzottius claudii n.sp. S67 1 MW306832 Vecchi etal. (2022) Ramazzottius AT.002 b MG432811 MG432814 Stec etal. (2018) Ramazzottius DE.002 a MG573257 Stec etal. (2018) Ramazzottius DE.002 b MG432817 MG432812 MG432815 Zawierucha etal. (2018) Ramazzottius groenlandensis 1 OR600266 OR600265 OR596527 Kihm etal. (2023) Ramazzottius groenlandensis 2 MG432810 Zawierucha etal. (2018) Ramazzottius groenlandensis 3 EU251381 Faurby etal. (2008) Ramazzottius groenlandensis 4 EU251382 Faurby etal. (2008) Ramazzottius kretschmanni T1 OM370801 OM402517 Guidetti etal. (2022) Ramazzottius kretschmanni T2 OM370802 OM402518 Guidetti etal. (2022) Ramazzottius kretschmanni T3 OM370803 OM402519 Guidetti etal. (2022) Ramazzottius kretschmanni T4 OM370804 OM402520 Guidetti etal. (2022) Ramazzottius oberhaeuseri FR.12 MG573241 MG573242 MG573244 MG573243 Stec etal. (2018) Ramazzottius PL.023 MG573253 Stec etal. (2018) Ramazzottius PL.140 1 MG573248 Stec etal. (2018) Ramazzottius PL.140 2 MG573249 Stec etal. (2018) Ramazzottius PL.141 MG573247 Stec etal. (2018) Ramazzottius PT.010 1 MG573245 Stec etal. (2018) Ramazzottius PT.010 2 MG573246 Stec etal. (2018) Ramazzottius Rama1 EU251380 Faurby etal. (2008) Ramazzottius Rama2 EU251379 Faurby etal. (2008) Ramazzottius Ro-Nivaa-1 EF620418 EF620419 Møbjerg etal. (2007) Ramazzottius S132 1 PQ108469 PQ108474 MW306838 PQ110583 This study; Vecchi etal. (2022) Ramazzottius S132 2 MW306837 Vecchi etal. (2022) Ramazzottius S49 1 MW306839 Vecchi etal. (2022) Ramazzottius S49 2 MW306840 Vecchi etal. (2022) Ramazzottius S67 2 MW306841 Vecchi etal. (2022) Ramazzottius subanomalus 1 MF001997 MF001998 MF001999 MG432819 Stec etal. (2017) Ramazzottius subanomalus 2 KU900021 KU900019 Stec etal. (2016) Ramazzottius Tar398 FJ435728 FJ435799 Guil and Giribet (2012) Ramazzottius Tar400 FJ435727 FJ435800 Guil and Giribet (2012) Ramazzottius varieornatus YOKOZUNA-1 MG432818 MG432813 MG432816 Zawierucha etal. (2018) Hebesuncus conjugens IT.148.2 PQ108465 PQ108478 PQ109086 PQ110587 This study Hebesuncus conjugens IT.148.3 PQ108466 PQ108479 PQ109087 PQ110588 This study 123 Mitogenome ofanew Ramazzottius species (Tardigrada: Eutardigrada: Ramazzottiidae)… com/ nanop orete ch/ medaka) using all the original reads. The mitochondrial genome was annotated with MITOS2 (Bernt etal., 2013; Donath etal., 2019) on the Galaxy Europe web server (https:// usega laxy. eu/). The annotation was inspected and curated by hand and a GenBank flat file was produced using the scripts mitos2fasta.py and aln2tbl. py (https:// git hub. com/ IMEDEA/ mitog enomi cs), and table2asn (https:// ftp. ncbi. nlm. nih. gov/ asn1conve rters/ by_ progr am/). The mitogenome was visualized with OGDRAW (Greiner etal., 2019). Coverage data was extracted from the medaka BAM files using the R package “gmoviz” (Zeglinski etal., 2021). Divergence and synteny of the mitochondrial genomes of the new species and R. varieornatus YOKOZUNA-1 (NC_031407) were visualized using the R packages “ggplot2” (Wickham, 2016), “genoPlotR” (Guy etal., 2010), “gggenes” (Wilkins, 2024), and “patchwork” (Pedersen, 2023). The newly assembled mitogenome sequence is available in GenBank (Accession number PP419898). Temperature anddesiccation‑related protein genes annotation Genes in the new Ramazzottius species were identified from a genome assembly of all clean reads obtained with Flye and Medaka as explained above. The completeness of the assembly was evaluated with BUSCO (Simão etal., 2015) (lineage metazoa_odb10) on the gVolante webserver (Nishimura etal., 2017). Contigs containing potential Temperature and Desiccation-Related Proteins (TDRPs) sequences were extracted with DIAMOND (options: –sensitive) using the eutardigrade TDRPs (CAHS, MAHS, MRE11, SAHS) sequences provided by Fleming etal. (2024), with the addition of Dsup (GenBank LC050827). The identified contings were annotated abinitio on the AUGUSTUS webserver (Hoff & Stanke, 2013; Stanke etal., 2008) trained on the genome and proteome of R. varieornatus YOKOZUNA-1 (http:// kumam ushi. org/). TDRPs were identified among the predicted proteins with BLASTp (e-value = 10e − 10) against the TDRPs protein sequences from Fleming etal., (2024). The identified protein sequences (and their relative coding sequences) were manually curated to confirm their identity. Maximum Likelihood phylogenies of the main TDRPs groups were obtained as in Fleming etal. (2024), with the addition of the sequences produced in this study. Results Phylogenetic reconstruction The phylogenetic reconstruction was performed with both Bayesian inference (BI) and maximum likelihood (ML) methods with the models provided in Online Resources 03. Both methods retrieved congruent topologies, even though ML provided a generally less resolved tree (Fig.1; Online Resource 03). In both reconstruction topologies, the new Ramazzottius species was found in a clade (pp = 1, BS = 98%) with R. varieornatus YOKOZUNA-1 and Ramazzottius aff. varieornatus from Italy. Genome sequencing andassembly The sequencing run resulted in 3.69Gb of sequences. The summary statistics of the raw reads are presented in Table2. The assembly metrics and the BUSCO score were compared to those of the R. varieornatus YOKOZUNA-1 assembly generated by Hashimoto etal. (2016) (available at http:// www. kumam ushi. org/ data/ YOKOZ UNA-1. scaff olds. fa). The assembly has a comparable size to R. varieornatus YOKOZUNA-1 (Table2), but it is more fragmented. The Table 2 Raw reads and assembly metrics R. claudii sp. nov.. raw reads R. claudii sp. nov. assembly R. varieornatus YOKOZUNA-1 assembly Size 3.69Gb 62.89Mb 55.82Mb N of sequences 4,817,658 1468 199 N of sequences > 1kb 738,949 1415 198 N of sequences > 10kb 28,279 800 36 GC content (%) 47.4 47.8 47.2 N50 1399 157,755 4,740,345 L50 485,725 109 4 N90 280 18,551 1,295,620 L90 3,074,768 549 15 BUSCO C: 71.6% [S: 69.4%, D: 2.2%] F: 7.4% M: 21.0% n: 954 C: 74.4% [S: 73.2%, D: 1.2%] F: 7.0% M: 18.6% n: 954 124 M.Vecchi, D.Stec BUSCO score of the new assembly (71.6%) is comparable to the one of R. varieornatus YOKOZUNA-1 (74.4%). The assembly of R. claudii sp. nov. genome generated in this study can be found at https:// doi. org/https:// doi. org/ 10. 6084/ m9. figsh are. 27061 267. Mitogenome sequencing Of all the raw reads, about 1.75% were of mitochondrial origin. The mitogenome assembly is 14,437bp in length (Fig.1) with an average coverage of 4486 × (min 1506 × – max 6785 ×). Two ribosomal RNAs (rrnS an rrnL), 22 tRNAs (with two trnL and trnS), 2 ATP synthase genes, and the genes for the complexes I, III, and IV are present. A putative unannotated control region is present between rrnL and trnL. The order of the genes is identical to the mitogenome of Ramazzottius varieornatus YOKOZUNA-1 (Fig.2), with an average nucleotidic divergence of 20.6%. TDRP identification CAHS A total of 15 CAHS genes were identified (Table3, Online resources 06). The phylogenetic reconstructions based of the CAHS protein sequences are similar to the ones found by (Fleming etal., 2024), with the exceptions of the CAHS families 2, 3, and 5 which have been found paraphyletic (Online resources 06). 0.03 Ramazzottius sp. DE.002 2 Ramazzottius groenlandensis 2 Ramazzottius groenlandensis 1 Ramazzottius sp. Tar398 Ramazzottius claudii n. sp. S66 2 Ramazzottius claudii n. sp. S66 1 Ramazzottius sp. Rama1 Ramazzottius subanomalus 1 Ramazzottius varieornatus YOKOZUNA-1 Ramazzottius sp. PL.141 Hebesuncus conjugens IT.148.3 Ramazzottius sp. Ro-Nivaa-1 Ramazzottius claudii n. sp. IT.158.1 Ramazzottius groenlandensis 4 Ramazzottius sp. PL.140 2 Ramazzottius sp. DE.002 1 Ramazzottius sp. Tar400 Ramazzottius sp. AT.002 Ramazzottius aff. varieornatus S132 1 Ramazzottius sp. PT.010 1 Ramazzottius aff. varieornatus S132 2 Ramazzottius kretschmanni T4 Ramazzottius aff. varieornatus S49 1 Ramazzottius groenlandensis 3 Ramazzottus sp. PL.023 Ramazzottius kretschmanni T1 Ramazzottius aff. varieornatus S49 2 Hebesuncus conjugens IT.148.2 Ramazzottius sp. Rama2 Ramazzottius claudii n. sp. S62 2 Ramazzottius sp. PL.140 1 Ramazzottius claudii n. sp. S67 2 Ramazzottius kretschmanni T2 Ramazzottius kretschmanni T3 Ramazzottius claudii n. sp. IT.158.3 Ramazzottius aff. varieornatus S67 1 Ramazzottius subanomalus 2 Ramazzottius claudii n. sp. S62 1 Ramazzottius oberhaeuseri FR.124 Ramazzottius sp. PT.010 2 0.95/- 0.99/98 1/91 1/98 1/100 0.87/- 1/100 1/100 0.94/98 0.99/98 0.99/99 1/100 0.95/99 1 1/100 1/100 1/100 1/100 0.8/- 1/98 0.98/96 0.71/98 0.91/- 1/100 Ramazzous claudii n. sp. mitochondrial genome 14 437 bp AB Fig. 1 Ramazzottius claudii sp. nov. mitogenome and phylogenetic position. A Mitogenome visualization: inner circle represents GC content. Direction of transcription is clockwise for genes in on the inner side, whereas it is anticlockwise for the genes on the external side. B Topology of the Bayesian Inference (BI) phylogenetic reconstruction of Ramazzottius clade A (sensu (Dey etal., 2024)): values above branches represent BI posterior probabilities/ML bootstrap (“-” indicated bootstrap value < 70% or node not present in ML phylogenetic reconstruction), scale bar indicates substitutions/site 125 Mitogenome ofanew Ramazzottius species (Tardigrada: Eutardigrada: Ramazzottiidae)… SAHS–FABP A total of 8 SAHS genes were identified (Table3, Online resources 07). The Macrobiotidae SAHS and the SAHS subfamily 1 were recovered as monophyletic (Online resources 07), while SAHS subfamily 2 was recovered as paraphyletic. 5 FABP (fatty acid binding proteins, see (Fleming etal., 2024) for their phylogenetic relation with SAHS) genes were also recovered and included in the phylogeny. MAHS A single MAHS gene was found (Table3, Online resources 09). MRE11 A single MRE11 gene was identified (Table3, Online resources 09). Fig. 2 Divergence and synteny of Ramazzottius claudii sp. nov. and R. varieornatus YOKOZUNA mitogenomes. In black: nucleotidic divergence; in red: indel divergence. Divergence was calculated on 250bp sliding windows; Arrow directions indicate transcription direction Divergence Ramaz zo us claudii n.sp. Ramaz zo us varieornatus YOKOZUNA Table 3 GenBank accession numbers of the TDRPs genes identified in Ramazzottius claudii sp. nov. classified according to their homology with the ones from R. varieornatus YOKOZUNA-1 Gene in R. varieornatus YOKOZUNA-1 R. claudii sp. nov. Gene in R. varieornatus YOKOZUNA-1 R. claudii sp. nov. Gene in R. varieornatus YOKOZUNA-1 R. claudii sp. nov. CAHS SAHS Subfamily 1 MAHS CAHS 1a PQ117601 SAHS 2 PQ117620 MAHS 1 PQ117614 CAHS 1b PQ117608 SAHS 7 PQ117618 MRE11 CAHS 2a PQ117603 SAHS 12 MRE11 A PQ117615 CAHS 3a PQ117609 SAHS 8 PQ117619 MRE11 B CAHS 3b PQ117599 SAHS 9 Not found MRE11 C CAHS 3c PQ117607 SAHS 10 Not found MRE11 D CAHS 4a PQ117602 SAHS 11 PQ117615 FABP CAHS 5a PQ117610 SAHS Subfamily 2 FABP 1 Not found CAHS 5b PQ117604 SAHS 1 PQ117617 FABP 2 PQ117628 CAHS 5c PQ117606 SAHS 3 PQ117621 FABP 3 PQ117625 CAHS 6a PQ117612 SAHS 4 PQ117616 FABP 4 PQ117627 CAHS 6b PQ117613 SAHS 5 PQ117623 FABP 5 PQ117626 CAHS 6c Not found SAHS 6 PQ117622 FABP 6 PQ117624 CAHS 6d PQ117611 SAHS 13 Not found FABP 7 Not found CAHS 7a PQ117605 Dsup Dsup PQ356341 126 M.Vecchi, D.Stec Dsup A single Dsup gene was identified (Table3, Online resources 10). Taxonomic account Ramazzottius claudii sp. nov. Vecchi & Stec, 2024 Zoobank registration: urn:lsid:zoobank.org:act:0B3D40750C0B-4552-B7B6-73EC10711ED4 Ramazzottius sp. A in (Vecchi etal., 2022) Ramazzottius sp. Italy in (Zawierucha etal., 2023) Type locality. Rock pool sediment from vicinity of Sella del Marmagna, Corniglio, Parma, Italy (44°23′45.8″N 10°00′15.7″E). Material examined. Holotype (Slide IT.158.7), 23 paratypes (Slides IT.158.4 – IT.158.10) and 20 eggs (Slides IT.158.01 – IT.158.03) mounted on slides in Hoyer’s medium. 4 animals (Stub TAR.2.06) and 5 eggs (Stub TAR.2.07) mounted on stubs for SEM. 3 animals used for DNA sequencing (Ram. sp.IT.158.01, Ram.sp.IT.158.03 and Ram_IT.158_WGA_1). Material repository. Tardigrada collection of the Institute of Systematics and Evolution of Animals (Polish Academy of Sciences), Sławkowska 17, 31–016, Kraków, Poland. Etymology. This species is dedicated to Claudio Ferrari (University of Parma, Italy), to acknowledge his constant support and help in exploring the Italian Northern Apennines in search of rock pools. (Tables4 and 5, Figs.3, 4, 5, 6, 7, 8, and 9, Online resources 01). Description. Animals (morphometrics in Table4, raw measurements in Online resources 01): Eyes absent in live individuals. Pigmentation of the cuticle red-brown, distributed in one ring around the mouth, a dorso-caudal continuous band, and 8 lateral-ventral bands (Fig.3). Cuticle with faint sculpture, more visible in the dorso-caudal part of the body (Figs.4 and 5). A pair of sensory organs present on the head (Fig.5A). The sculpture is composed by rounded polygonal granules; however, in some individuals (in particular in the caudal portion of bigger individuals), the granules have an elongated “spindle” shape (Figs.4 and 5). Bucco-pharyngeal apparatus of the Ramazzottius-type (Fig.6). Mouth opening antero-ventral. Oral cavity armature visible under PCM (Fig.6B and C). The armature is composed of one band of teeth, located in the posterior oral cavity (Fig.6B and C). The band composed of a single row of large and regularly spaced granular teeth. In ventral view, the teeth are less visible and seem connected to the posterior edge of the oral cavity (Fig.6C). Under PCM, other structures within oral cavity not visible (Fig.6B and C). Apophyses for the insertion of stylet muscles (AISM) in the shape of blunt hooks and asymmetrical in size and shape with respect to the frontal plane (Fig.6D). Stylet furcae with rounded ends. Buccal tube with a posterior bend and thickened walls posteriorly from the stylet support insertion point. Pharyngeal bulb (bulbus) almost oval, with apophyses and two clearly separated macroplacoids. Pharyngeal apophyses triangular, smaller than macroplacoids (Fig.6E). Macroplacoid length configuration 2 < 1. Microplacoid and septulum absent (Fig.6E). Macroplacoids are usually roundish; however, in bigger specimens, they look slightly more elongated. Constrictions in the macroplacoids are present. In the first macroplacoid a central constriction is present, whereas in the second macroplacoids a constriction in central-posterior position is present. Claws of the Ramazzottius-type. Primary branches of external and posterior claws long and thin. A non-sclerotized light refracting unit (LRU) is present between the claw base and the primary branch, which are connected by a couple of thin cuticular filaments (Fig.7A, B). Internal and anterior claws much smaller and of a different shape than external claws (Fig.7). Claws with smooth pseudolunules (Fig.7B). Accessory points on primary branches of all claws present (Fig.7D-E). Bars and other cuticular thickenings on legs, absent. A papilla is present on the external side of legs IV, but not always visible depending on the animal positioning (Fig.7C). Eggs (morphometrics in Table5, raw measurements in Online resources 01): Laid freely, white and spherical, covered in processes with shape ranging from spike-like to filamentous (Fig.8). The processes exhibit extreme diversity in size, with most of them being within the range of 4.6–14.4μm, but with some of them reaching up to 27.9μm (Fig.8C). Egg processes and surface between processes dotted under PCM, smooth under SEM (Fig.8I). Males Individuals with gonad filled with developing spermatids were found, indicating the presence of males (Fig.9). DNA sequences. SSU (18S): PQ108467 – 8, PQ108470; LSU (28S): PQ108475 – PQ108477; COI: MW306832 – MW306836, PQ109084 – 5; ITS-2: PQ110584 – 6. Differential diagnosis By having eggs with dotted chorion and elongated processes, R. claudii sp. nov. is similar to six other species, however it differs from: 127 Mitogenome ofanew Ramazzottius species (Tardigrada: Eutardigrada: Ramazzottiidae)… Table 4 Summary of animals morphometrics of Ramazzottius claudii sp. nov. cbt and pr ratios according to Vecchi etal. (2023b) Character NRange Mean SD Holotype μm pt μm pt μm pt μm pt Body length 20 292 – 488 934 – 1205 391 1061 55 67 379 1120 Buccal tube  Buccal tube length 20 29.7 – 43.9 – 36.7 –3.9 –33.8 –  Stylet support insertion point 20 19.9 – 30.0 65.9 – 70.4 25.0 68.1 2.8 1.3 22.9 67.8  Buccal tube external width 20 2.5 – 3.6 7.1 – 9.8 3.0 8.3 0.3 0.6 3.3 9.8  Buccal tube internal width 18 1.0 – 2.0 3.0 – 6.0 1.4 3.9 0.2 0.7 2.0 6.0 Placoid lengths  Macroplacoid 1 19 3.7 – 6.7 11.2 – 16.6 4.9 13.2 0.9 1.5 3.8 11.3  Macroplacoid 2 19 3.0 – 5.9 9.6 – 14.6 4.3 11.5 0.8 1.2 3.6 10.7  Macroplacoid row 19 7.0 – 12.9 22.9 – 32.0 9.9 26.6 1.8 2.5 8.2 24.3 Claw 1 lengths  External base 19 8.0 – 13.1 22.9 – 32.2 10.0 27.6 1.3 2.6 9.4 27.7  External primary branch 18 12.5 – 19.1 38.9 – 48.9 16.0 43.9 2.1 2.6 15.1 44.8  External secondary branch 17 6.9 – 11.4 23.4 – 32.6 9.4 26.1 1.3 2.2 9.4 27.7  External cbt ratio 18 49.0 – 73.8 – 63.1 –6.0 –61.9 –  External br ratio 16 49.8 – 67.4 59.1 3.9  External total 17 18.8 – 31.0 60.6 – 78.0 24.8 68.5 3.3 5.2 24.3 71.8  Internal base 13 4.5 – 8.5 13.8 – 22.6 7.2 19.6 1.2 2.6 7.5 22.2  Internal primary branch 12 6.7 – 12.5 20.1 – 31.1 9.8 26.7 1.7 3.0 8.8 25.9  Internal secondary branch 13 4.4 – 10.3 13.3 – 25.5 7.5 20.7 1.4 3.2 7.3 21.7  Internal cbt ratio 12 47.0 – 93.4 – 74.2 –13.4 –85.5 –  Internal br ratio 12 63.4 – 93.9 76.9 8.4  Internal total 10 10.8 – 16.6 32.4 – 41.0 13.2 36.4 1.7 2.6 ?? Claw 2 lengths  External base 19 8.3 – 13.5 24.8 – 35.2 10.8 29.5 1.5 2.6 10.4 30.8  External primary branch 18 11.1 – 21.4 37.2 – 52.8 16.4 45.0 2.3 3.5 16.5 48.7  External secondary branch 19 7.5 – 11.7 24.4 – 30.7 10.0 27.4 1.1 1.8 9.6 28.4  External cbt ratio 18 56.9 – 77.3 – 65.9 –7.5 –63.3 –  External br ratio 18 51.2 – 75.1 61.4 5.7  External total 14 22.2 – 33.1 61.8 – 82.3 26.8 73.5 3.5 5.4 25.0 73.8  Internal base 15 3.6 – 9.5 10.3 – 22.2 7.1 19.5 1.4 2.9 7.5 22.2  Internal primary branch 15 7.7 – 14.4 24.0 – 37.7 10.4 28.6 1.7 3.4 8.8 25.9 I nternal secondary branch 11 6.1 – 9.8 18.2 – 25.6 8.2 22.7 1.1 2.4 8.7 25.6  Internal cbt ratio 15 42.9 – 85.6 – 68.6 –11.7 –85.6 –  Internal br ratio 11 59.0 – 98.9 80.0 12.1  Internal total 14 10.1 – 17.7 29.9 – 44.2 14.0 38.3 2.4 3.6 10.1 29.9 Claw 3 lengths  External base 19 7.6 – 13.9 25.4 – 34.3 10.8 29.2 1.7 2.3 9.3 27.5  External primary branch 20 13.6 – 20.0 44.3 – 53.2 17.6 47.8 1.9 2.3 17.4 51.6  External secondary branch 18 6.8 – 12.1 22.8 – 29.9 9.8 26.8 1.4 1.8 8.7 25.8  External cbt ratio 19 52.9 – 73.3 – 61.2 –5.7 –53.4 –  External br ratio 18 49.3 – 62.8 56.0 4.2  External total 15 20.6 – 34.8 64.7 – 85.8 26.6 72.6 4.2 6.0 25.5 75.5  Internal base 17 5.8 – 11.8 17.6 – 28.2 7.9 21.6 1.3 2.6 6.8 20.2  Internal primary branch 18 7.3 – 12.9 21.2 – 33.6 10.3 28.6 1.8 3.6 9.6 28.4  Internal secondary branch 17 5.9 – 10.9 16.2 – 30.1 8.5 23.3 1.5 3.3 8.3 24.4  Internal cbt ratio 17 59.1 – 106.1 – 75.8 –12.9 –71.1 –  Internal br ratio 17 58.3 – 141.6 82.1 18.1  Internal total 15 12.5 – 17.3 35.0 – 43.0 14.5 39.6 1.5 2.6 13.8 40.8 134 M.Vecchi, D.Stec of the family Ramazzottiidae (Eutardigrada: Hypsibioidea). 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