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

Matteo Vecchi, Daniel Stec

► To cite this version:

Matteo Vecchi, Daniel Stec. Mitogenome of a new Ramazzottius species (Tardigrada: Eutardigrada: Ramazzottiidae) discovered in rock pools along with its temperature and desiccation-related proteins repertoire. Organisms Diversity and Evolution, 2024, 25, pp.119 - 135. 10.1007/s13127-024-00662-x.
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# Mitogenome of a new *Ramazzottius* species (Tardigrada: Eutardigrada: Ramazzottiidae) discovered in rock pools along with its temperature and desiccation-related proteins repertoire

Matteo Vecchi<fnref n="1" /> Ⓓ · Daniel Stec<fnref n="1" /> Ⓓ

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, morphology, 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 amebic 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

<footnote>✉ Matteo Vecchi
matteo vecchi 15@gmail.com; m. vecchi@isez.pan.krakow.pl</footnote>

<footnote n="1">¹ Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016, Kraków, Poland</footnote>
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 YOKO-ZUNA-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).

<figure><img src="image_2.png" /></figure>
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).
<table><caption>**Table 1** GenBank accession numbers of the sequences used in the phylogenetic reconstruction</caption><thead><tr><th></th><th>SSU (18S)</th><th>LSU (28S)</th><th>COI</th><th>ITS2</th><th>Source</th></tr></thead><tbody><tr><td>Ramazzottius claudii n.sp. IT.158.1</td><td>PQ108467</td><td>PQ108476</td><td>PQ109084</td><td>PQ110585</td><td>This study</td></tr><tr><td>Ramazzottius claudii n.sp. IT.158.3</td><td>PQ108468</td><td>PQ108477</td><td>PQ109085</td><td>PQ110586</td><td>This study</td></tr><tr><td>Ramazzottius claudii n.sp. S62 1</td><td></td><td></td><td>MW306836</td><td></td><td>Vecchi et al. (2022)</td></tr><tr><td>Ramazzottius claudii n.sp. S62 2</td><td></td><td></td><td>MW306835</td><td></td><td>Vecchi et al. (2022)</td></tr><tr><td>Ramazzottius claudii n.sp. S66 1</td><td>PQ108470</td><td>PQ108475</td><td>MW306833</td><td>PQ110584</td><td>This study; Vecchi et al. (2022)</td></tr><tr><td>Ramazzottius claudii n.sp. S66 2</td><td></td><td></td><td>MW306834</td><td></td><td>Vecchi et al. (2022)</td></tr><tr><td>Ramazzottius claudii n.sp. S67 1</td><td></td><td></td><td>MW306832</td><td></td><td>Vecchi et al. (2022)</td></tr><tr><td>Ramazzottius AT.002 b</td><td></td><td></td><td>MG432811</td><td>MG432814</td><td>Stec et al. (2018)</td></tr><tr><td>Ramazzottius DE.002 a</td><td></td><td></td><td>MG573257</td><td></td><td>Stec et al. (2018)</td></tr><tr><td>Ramazzottius DE.002 b</td><td></td><td>MG432817</td><td>MG432812</td><td>MG432815</td><td>Zawierucha et al. (2018)</td></tr><tr><td>Ramazzottius groenlandensis 1</td><td>OR600266</td><td>OR600265</td><td>OR596527</td><td></td><td>Kihm et al. (2023)</td></tr><tr><td>Ramazzottius groenlandensis 2</td><td></td><td></td><td>MG432810</td><td></td><td>Zawierucha et al. (2018)</td></tr><tr><td>Ramazzottius groenlandensis 3</td><td></td><td></td><td>EU251381</td><td></td><td>Faurby et al. (2008)</td></tr><tr><td>Ramazzottius groenlandensis 4</td><td></td><td></td><td>EU251382</td><td></td><td>Faurby et al. (2008)</td></tr><tr><td>Ramazzottius kretschmanni T1</td><td></td><td></td><td>OM370801</td><td>OM402517</td><td>Guidetti et al. (2022)</td></tr><tr><td>Ramazzottius kretschmanni T2</td><td></td><td></td><td>OM370802</td><td>OM402518</td><td>Guidetti et al. (2022)</td></tr><tr><td>Ramazzottius kretschmanni T3</td><td></td><td></td><td>OM370803</td><td>OM402519</td><td>Guidetti et al. (2022)</td></tr><tr><td>Ramazzottius kretschmanni T4</td><td></td><td></td><td>OM370804</td><td>OM402520</td><td>Guidetti et al. (2022)</td></tr><tr><td>Ramazzottius oberhaeuseri FR.12</td><td>MG573241</td><td>MG573242</td><td>MG573244</td><td>MG573243</td><td>Stec et al. (2018)</td></tr><tr><td>Ramazzottius PL.023</td><td></td><td></td><td>MG573253</td><td></td><td>Stec et al. (2018)</td></tr><tr><td>Ramazzottius PL.140 1</td><td></td><td></td><td>MG573248</td><td></td><td>Stec et al. (2018)</td></tr><tr><td>Ramazzottius PL.140 2</td><td></td><td></td><td>MG573249</td><td></td><td>Stec et al. (2018)</td></tr><tr><td>Ramazzottius PL.141</td><td></td><td></td><td>MG573247</td><td></td><td>Stec et al. (2018)</td></tr><tr><td>Ramazzottius PT.010 1</td><td></td><td></td><td>MG573245</td><td></td><td>Stec et al. (2018)</td></tr><tr><td>Ramazzottius PT.010 2</td><td></td><td></td><td>MG573246</td><td></td><td>Stec et al. (2018)</td></tr><tr><td>Ramazzottius Rama1</td><td></td><td></td><td>EU251380</td><td></td><td>Faurby et al. (2008)</td></tr><tr><td>Ramazzottius Rama2</td><td></td><td></td><td>EU251379</td><td></td><td>Faurby et al. (2008)</td></tr><tr><td>Ramazzottius Ro-Nivaa-1</td><td></td><td></td><td>EF620418</td><td>EF620419</td><td>Møbjerg et al. (2007)</td></tr><tr><td>Ramazzottius S132 1</td><td>PQ108469</td><td>PQ108474</td><td>MW306838</td><td>PQ110583</td><td>This study; Vecchi et al. (2022)</td></tr><tr><td>Ramazzottius S132 2</td><td></td><td></td><td>MW306837</td><td></td><td>Vecchi et al. (2022)</td></tr><tr><td>Ramazzottius S49 1</td><td></td><td></td><td>MW306839</td><td></td><td>Vecchi et al. (2022)</td></tr><tr><td>Ramazzottius S49 2</td><td></td><td></td><td>MW306840</td><td></td><td>Vecchi et al. (2022)</td></tr><tr><td>Ramazzottius S67 2</td><td></td><td></td><td>MW306841</td><td></td><td>Vecchi et al. (2022)</td></tr><tr><td>Ramazzottius subanomalus 1</td><td>MF001997</td><td>MF001998</td><td>MF001999</td><td>MG432819</td><td>Stec et al. (2017)</td></tr><tr><td>Ramazzottius subanomalus 2</td><td></td><td></td><td>KU900021</td><td>KU900019</td><td>Stec et al. (2016)</td></tr><tr><td>Ramazzottius Tar398</td><td>FJ435728</td><td></td><td>FJ435799</td><td></td><td>Guil and Giribet (2012)</td></tr><tr><td>Ramazzottius Tar400</td><td>FJ435727</td><td></td><td>FJ435800</td><td></td><td>Guil and Giribet (2012)</td></tr><tr><td>Ramazzottius varieornatus YOKOZUNA-1</td><td></td><td>MG432818</td><td>MG432813</td><td>MG432816</td><td>Zawierucha et al. (2018)</td></tr><tr><td>Hebesuncus conjugens IT.148.2</td><td>PQ108465</td><td>PQ108478</td><td>PQ109086</td><td>PQ110587</td><td>This study</td></tr><tr><td>Hebesuncus conjugens IT.148.3</td><td>PQ108466</td><td>PQ108479</td><td>PQ109087</td><td>PQ110588</td><td>This study</td></tr></tbody></table>

## 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.

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>
com/nanoporetech/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://usegalaxy.eu/). The annotation was inspected and curated by hand and a GenBank flat file was produced using the scripts mitos2forta.py and aln2tbl.py (https://github.com/IMEDEA/mitogenomics), and table2asn (https://ftp.ncbi.nlm.nih.gov/asn1-converters/by_program/). 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 contigs 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://kumamushi.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.kumamushi.org/data/YOKOZUNA-1.scaffolds.fa). The assembly has a comparable size to *R. varieornatus* YOKOZUNA-1 (Table 2), but it is more fragmented. The

<table><caption>**Table 2** Raw reads and assembly metrics</caption><thead><tr><th></th><th><i>R. claudii</i><br>sp. nov., raw<br>reads</th><th><i>R. claudii</i> sp. nov. assembly</th><th><i>R. varieornatus</i><br>YOKOZUNA-1 assembly</th></tr></thead><tbody><tr><td>Size</td><td>3.69 Gb</td><td>62.89 Mb</td><td>55.82 Mb</td></tr><tr><td>N of sequences</td><td>4,817,658</td><td>1468</td><td>199</td></tr><tr><td>N of sequences > 1 kb</td><td>738,949</td><td>1415</td><td>198</td></tr><tr><td>N of sequences > 10 kb</td><td>28,279</td><td>800</td><td>36</td></tr><tr><td>GC content (%)</td><td>47.4</td><td>47.8</td><td>47.2</td></tr><tr><td>N50</td><td>1399</td><td>157,755</td><td>4,740,345</td></tr><tr><td>L50</td><td>485,725</td><td>109</td><td>4</td></tr><tr><td>N90</td><td>280</td><td>18,551</td><td>1,295,620</td></tr><tr><td>L90</td><td>3,074,768</td><td>549</td><td>15</td></tr><tr><td>BUSCO</td><td></td><td>C: 71.6% [S: 69.4%, D: 2.2%]<br>F: 7.4%<br>M: 21.0%<br>n: 954</td><td>C: 74.4% [S: 73.2%, D: 1.2%]<br>F: 7.0%<br>M: 18.6%<br>n: 954</td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>**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 claudii* 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</figcaption></figure>

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/10.6084/m9 figshare.27061267.

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%.

## 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

## 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).

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>**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</figcaption></figure>

<table><thead><tr><th>Gene in R. varieornatus YOKOZUNA-1</th><th>R. claudii sp. nov.</th><th>Gene in R. varieornatus YOKOZUNA-1</th><th>R. claudii sp. nov.</th><th>Gene in R. varieornatus YOKOZUNA-1</th><th>R. claudii sp. nov.</th></tr></thead><tbody><tr><td><strong>CAHS</strong></td><td></td><td><strong>SAHS Subfamily 1</strong></td><td></td><td><strong>MAHS</strong></td><td></td></tr><tr><td>CAHS 1a</td><td>PQ117601</td><td>SAHS 2</td><td>PQ117620</td><td>MAHS 1</td><td>PQ117614</td></tr><tr><td>CAHS 1b</td><td>PQ117608</td><td>SAHS 7</td><td>PQ117618</td><td><strong>MRE11</strong></td><td></td></tr><tr><td>CAHS 2a</td><td>PQ117603</td><td>SAHS 12</td><td></td><td>MRE11 A</td><td>PQ117615</td></tr><tr><td>CAHS 3a</td><td>PQ117609</td><td>SAHS 8</td><td>PQ117619</td><td>MRE11 B</td><td></td></tr><tr><td>CAHS 3b</td><td>PQ117599</td><td>SAHS 9</td><td>Not found</td><td>MRE11 C</td><td></td></tr><tr><td>CAHS 3c</td><td>PQ117607</td><td>SAHS 10</td><td>Not found</td><td>MRE11 D</td><td></td></tr><tr><td>CAHS 4a</td><td>PQ117602</td><td>SAHS 11</td><td>PQ117615</td><td><strong>FABP</strong></td><td></td></tr><tr><td>CAHS 5a</td><td>PQ117610</td><td><strong>SAHS Subfamily 2</strong></td><td></td><td>FABP 1</td><td>Not found</td></tr><tr><td>CAHS 5b</td><td>PQ117604</td><td>SAHS 1</td><td>PQ117617</td><td>FABP 2</td><td>PQ117628</td></tr><tr><td>CAHS 5c</td><td>PQ117606</td><td>SAHS 3</td><td>PQ117621</td><td>FABP 3</td><td>PQ117625</td></tr><tr><td>CAHS 6a</td><td>PQ117612</td><td>SAHS 4</td><td>PQ117616</td><td>FABP 4</td><td>PQ117627</td></tr><tr><td>CAHS 6b</td><td>PQ117613</td><td>SAHS 5</td><td>PQ117623</td><td>FABP 5</td><td>PQ117626</td></tr><tr><td>CAHS 6c</td><td>Not found</td><td>SAHS 6</td><td>PQ117622</td><td>FABP 6</td><td>PQ117624</td></tr><tr><td>CAHS 6d</td><td>PQ117611</td><td>SAHS 13</td><td>Not found</td><td>FABP 7</td><td>Not found</td></tr><tr><td>CAHS 7a</td><td>PQ117605</td><td><strong>Dsup</strong></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td>Dsup</td><td>PQ356341</td><td></td><td></td></tr></tbody></table>

### SAHS-FABP

A total of 8 SAHS genes were identified (Table 3, Online resources 07). The Macrobiotidae SAHS and the SAHS sub-family 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).
## 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:0B3D4075-0C0B-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:

<figure><img src="image_9.png" /></figure>
<table><caption>**Table 4** Summary of animals morphometrics of *Ramazzottius claudii* sp. nov. *cbt* and *pr* ratios according to Vecchi et al. (2023b)</caption><thead><tr><th rowspan="2">Character</th><th rowspan="2">N</th><th colspan="2">Range</th><th colspan="2">Mean</th><th colspan="2">SD</th><th colspan="2">Holotype</th></tr><tr><th>μm</th><th>pt</th><th>μm</th><th>pt</th><th>μm</th><th>pt</th><th>μm</th><th>pt</th></tr></thead><tbody><tr><td>Body length</td><td>20</td><td>292</td><td>488</td><td>934</td><td>1205</td><td>391</td><td>1061</td><td>55</td><td>67</td><td>379</td><td>1120</td></tr><tr><td>Buccal tube</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Buccal tube length</td><td>20</td><td>29.7</td><td>43.9</td><td></td><td></td><td>36.7</td><td></td><td>3.9</td><td></td><td>33.8</td><td></td></tr><tr><td>Stylet support insertion point</td><td>20</td><td>19.9</td><td>30.0</td><td>65.9</td><td>70.4</td><td>25.0</td><td>68.1</td><td>2.8</td><td>1.3</td><td>22.9</td><td>67.8</td></tr><tr><td>Buccal tube external width</td><td>20</td><td>2.5</td><td>3.6</td><td>7.1</td><td>9.8</td><td>3.0</td><td>8.3</td><td>0.3</td><td>0.6</td><td>3.3</td><td>9.8</td></tr><tr><td>Buccal tube internal width</td><td>18</td><td>1.0</td><td>2.0</td><td>3.0</td><td>6.0</td><td>1.4</td><td>3.9</td><td>0.2</td><td>0.7</td><td>2.0</td><td>6.0</td></tr><tr><td>Placoid lengths</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Macroplacoid 1</td><td>19</td><td>3.7</td><td>6.7</td><td>11.2</td><td>16.6</td><td>4.9</td><td>13.2</td><td>0.9</td><td>1.5</td><td>3.8</td><td>11.3</td></tr><tr><td>Macroplacoid 2</td><td>19</td><td>3.0</td><td>5.9</td><td>9.6</td><td>14.6</td><td>4.3</td><td>11.5</td><td>0.8</td><td>1.2</td><td>3.6</td><td>10.7</td></tr><tr><td>Macroplacoid row</td><td>19</td><td>7.0</td><td>12.9</td><td>22.9</td><td>32.0</td><td>9.9</td><td>26.6</td><td>1.8</td><td>2.5</td><td>8.2</td><td>24.3</td></tr><tr><td>Claw 1 lengths</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>External base</td><td>19</td><td>8.0</td><td>13.1</td><td>22.9</td><td>32.2</td><td>10.0</td><td>27.6</td><td>1.3</td><td>2.6</td><td>9.4</td><td>27.7</td></tr><tr><td>External primary branch</td><td>18</td><td>12.5</td><td>19.1</td><td>38.9</td><td>48.9</td><td>16.0</td><td>43.9</td><td>2.1</td><td>2.6</td><td>15.1</td><td>44.8</td></tr><tr><td>External secondary branch</td><td>17</td><td>6.9</td><td>11.4</td><td>23.4</td><td>32.6</td><td>9.4</td><td>26.1</td><td>1.3</td><td>2.2</td><td>9.4</td><td>27.7</td></tr><tr><td>External cbt ratio</td><td>18</td><td>49.0</td><td>73.8</td><td></td><td></td><td>63.1</td><td></td><td>6.0</td><td></td><td>61.9</td><td></td></tr><tr><td>External br ratio</td><td>16</td><td>49.8</td><td>67.4</td><td></td><td></td><td>59.1</td><td></td><td>3.9</td><td></td><td></td><td></td></tr><tr><td>External total</td><td>17</td><td>18.8</td><td>31.0</td><td>60.6</td><td>78.0</td><td>24.8</td><td>68.5</td><td>3.3</td><td>5.2</td><td>24.3</td><td>71.8</td></tr><tr><td>Internal base</td><td>13</td><td>4.5</td><td>8.5</td><td>13.8</td><td>22.6</td><td>7.2</td><td>19.6</td><td>1.2</td><td>2.6</td><td>7.5</td><td>22.2</td></tr><tr><td>Internal primary branch</td><td>12</td><td>6.7</td><td>12.5</td><td>20.1</td><td>31.1</td><td>9.8</td><td>26.7</td><td>1.7</td><td>3.0</td><td>8.8</td><td>25.9</td></tr><tr><td>Internal secondary branch</td><td>13</td><td>4.4</td><td>10.3</td><td>13.3</td><td>25.5</td><td>7.5</td><td>20.7</td><td>1.4</td><td>3.2</td><td>7.3</td><td>21.7</td></tr><tr><td>Internal cbt ratio</td><td>12</td><td>47.0</td><td>93.4</td><td></td><td></td><td>74.2</td><td></td><td>13.4</td><td></td><td>85.5</td><td></td></tr><tr><td>Internal br ratio</td><td>12</td><td>63.4</td><td>93.9</td><td></td><td></td><td>76.9</td><td></td><td>8.4</td><td></td><td></td><td></td></tr><tr><td>Internal total</td><td>10</td><td>10.8</td><td>16.6</td><td>32.4</td><td>41.0</td><td>13.2</td><td>36.4</td><td>1.7</td><td>2.6</td><td>?</td><td>?</td></tr><tr><td>Claw 2 lengths</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>External base</td><td>19</td><td>8.3</td><td>13.5</td><td>24.8</td><td>35.2</td><td>10.8</td><td>29.5</td><td>1.5</td><td>2.6</td><td>10.4</td><td>30.8</td></tr><tr><td>External primary branch</td><td>18</td><td>11.1</td><td>21.4</td><td>37.2</td><td>52.8</td><td>16.4</td><td>45.0</td><td>2.3</td><td>3.5</td><td>16.5</td><td>48.7</td></tr><tr><td>External secondary branch</td><td>19</td><td>7.5</td><td>11.7</td><td>24.4</td><td>30.7</td><td>10.0</td><td>27.4</td><td>1.1</td><td>1.8</td><td>9.6</td><td>28.4</td></tr><tr><td>External cbt ratio</td><td>18</td><td>56.9</td><td>77.3</td><td></td><td></td><td>65.9</td><td></td><td>7.5</td><td></td><td>63.3</td><td></td></tr><tr><td>External br ratio</td><td>18</td><td>51.2</td><td>75.1</td><td></td><td></td><td>61.4</td><td></td><td>5.7</td><td></td><td></td><td></td></tr><tr><td>External total</td><td>14</td><td>22.2</td><td>33.1</td><td>61.8</td><td>82.3</td><td>26.8</td><td>73.5</td><td>3.5</td><td>5.4</td><td>25.0</td><td>73.8</td></tr><tr><td>Internal base</td><td>15</td><td>3.6</td><td>9.5</td><td>10.3</td><td>22.2</td><td>7.1</td><td>19.5</td><td>1.4</td><td>2.9</td><td>7.5</td><td>22.2</td></tr><tr><td>Internal primary branch</td><td>15</td><td>7.7</td><td>14.4</td><td>24.0</td><td>37.7</td><td>10.4</td><td>28.6</td><td>1.7</td><td>3.4</td><td>8.8</td><td>25.9</td></tr><tr><td>Internal secondary branch</td><td>11</td><td>6.1</td><td>9.8</td><td>18.2</td><td>25.6</td><td>8.2</td><td>22.7</td><td>1.1</td><td>2.4</td><td>8.7</td><td>25.6</td></tr><tr><td>Internal cbt ratio</td><td>15</td><td>42.9</td><td>85.6</td><td></td><td></td><td>68.6</td><td></td><td>11.7</td><td></td><td>85.6</td><td></td></tr><tr><td>Internal br ratio</td><td>11</td><td>59.0</td><td>98.9</td><td></td><td></td><td>80.0</td><td></td><td>12.1</td><td></td><td></td><td></td></tr><tr><td>Internal total</td><td>14</td><td>10.1</td><td>17.7</td><td>29.9</td><td>44.2</td><td>14.0</td><td>38.3</td><td>2.4</td><td>3.6</td><td>10.1</td><td>29.9</td></tr><tr><td>Claw 3 lengths</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>External base</td><td>19</td><td>7.6</td><td>13.9</td><td>25.4</td><td>34.3</td><td>10.8</td><td>29.2</td><td>1.7</td><td>2.3</td><td>9.3</td><td>27.5</td></tr><tr><td>External primary branch</td><td>20</td><td>13.6</td><td>20.0</td><td>44.3</td><td>53.2</td><td>17.6</td><td>47.8</td><td>1.9</td><td>2.3</td><td>17.4</td><td>51.6</td></tr><tr><td>External secondary branch</td><td>18</td><td>6.8</td><td>12.1</td><td>22.8</td><td>29.9</td><td>9.8</td><td>26.8</td><td>1.4</td><td>1.8</td><td>8.7</td><td>25.8</td></tr><tr><td>External cbt ratio</td><td>19</td><td>52.9</td><td>73.3</td><td></td><td></td><td>61.2</td><td></td><td>5.7</td><td></td><td>53.4</td><td></td></tr><tr><td>External br ratio</td><td>18</td><td>49.3</td><td>62.8</td><td></td><td></td><td>56.0</td><td></td><td>4.2</td><td></td><td></td><td></td></tr><tr><td>External total</td><td>15</td><td>20.6</td><td>34.8</td><td>64.7</td><td>85.8</td><td>26.6</td><td>72.6</td><td>4.2</td><td>6.0</td><td>25.5</td><td>75.5</td></tr><tr><td>Internal base</td><td>17</td><td>5.8</td><td>11.8</td><td>17.6</td><td>28.2</td><td>7.9</td><td>21.6</td><td>1.3</td><td>2.6</td><td>6.8</td><td>20.2</td></tr><tr><td>Internal primary branch</td><td>18</td><td>7.3</td><td>12.9</td><td>21.2</td><td>33.6</td><td>10.3</td><td>28.6</td><td>1.8</td><td>3.6</td><td>9.6</td><td>28.4</td></tr><tr><td>Internal secondary branch</td><td>17</td><td>5.9</td><td>10.9</td><td>16.2</td><td>30.1</td><td>8.5</td><td>23.3</td><td>1.5</td><td>3.3</td><td>8.3</td><td>24.4</td></tr><tr><td>Internal cbt ratio</td><td>17</td><td>59.1</td><td>106.1</td><td></td><td></td><td>75.8</td><td></td><td>12.9</td><td></td><td>71.1</td><td></td></tr><tr><td>Internal br ratio</td><td>17</td><td>58.3</td><td>141.6</td><td></td><td></td><td>82.1</td><td></td><td>18.1</td><td></td><td></td><td></td></tr><tr><td>Internal total</td><td>15</td><td>12.5</td><td>17.3</td><td>35.0</td><td>43.0</td><td>14.5</td><td>39.6</td><td>1.5</td><td>2.6</td><td>13.8</td><td>40.8</td></tr></tbody></table>
<table><caption>**Table 4 (continued)**</caption><thead><tr><th rowspan="2">Character</th><th rowspan="2">N</th><th colspan="2">Range</th><th colspan="2">Mean</th><th colspan="2">SD</th><th colspan="2">Holotype</th></tr><tr><th>µm</th><th>pt</th><th>µm</th><th>pt</th><th>µm</th><th>pt</th><th>µm</th><th>pt</th></tr></thead><tbody><tr><td colspan="10"><strong>Claw 4 lengths</strong></td></tr><tr><td>Anterior base</td><td>13</td><td>6.3</td><td>-</td><td>10.0</td><td>15.3</td><td>-</td><td>26.5</td><td>8.2</td><td>22.2</td><td>1.3</td><td>3.1</td><td>8.5</td><td>25.2</td></tr><tr><td>Anterior primary branch</td><td>13</td><td>9.0</td><td>-</td><td>13.6</td><td>26.1</td><td>-</td><td>35.8</td><td>11.4</td><td>30.7</td><td>1.4</td><td>2.9</td><td>12.1</td><td>35.8</td></tr><tr><td>Anterior secondary branch</td><td>9</td><td>6.2</td><td>-</td><td>12.8</td><td>18.7</td><td>-</td><td>31.5</td><td>9.7</td><td>25.9</td><td>2.1</td><td>4.2</td><td>9.5</td><td>28.2</td></tr><tr><td>Anterior <i>cbt</i> ratio</td><td>12</td><td>58.8</td><td>-</td><td>82.5</td><td>-</td><td>-</td><td>-</td><td>71.6</td><td>-</td><td>7.6</td><td>-</td><td>70.3</td><td>-</td></tr><tr><td>Anterior <i>br</i> ratio</td><td>9</td><td>61.3</td><td>-</td><td>99.1</td><td>-</td><td>-</td><td>-</td><td>81.9</td><td>-</td><td>12.1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Anterior total</td><td>12</td><td>13.5</td><td>-</td><td>17.9</td><td>37.1</td><td>-</td><td>45.3</td><td>15.5</td><td>41.1</td><td>1.6</td><td>2.7</td><td>15.1</td><td>44.8</td></tr><tr><td>Posterior base</td><td>16</td><td>0.4</td><td>-</td><td>14.6</td><td>1.1</td><td>-</td><td>36.1</td><td>10.3</td><td>27.8</td><td>3.1</td><td>7.7</td><td>10.2</td><td>30.0</td></tr><tr><td>Posterior primary branch</td><td>19</td><td>13.3</td><td>-</td><td>22.2</td><td>44.0</td><td>-</td><td>56.9</td><td>18.5</td><td>50.3</td><td>2.5</td><td>3.7</td><td>17.2</td><td>50.8</td></tr><tr><td>Posterior secondary branch</td><td>12</td><td>6.5</td><td>-</td><td>10.6</td><td>19.7</td><td>-</td><td>27.9</td><td>9.0</td><td>23.9</td><td>1.3</td><td>2.8</td><td>8.1</td><td>24.1</td></tr><tr><td>Posterior <i>cbt</i> ratio</td><td>15</td><td>47.7</td><td>-</td><td>70.2</td><td>-</td><td>-</td><td>-</td><td>58.7</td><td>-</td><td>6.6</td><td>-</td><td>59.0</td><td>-</td></tr><tr><td>Posterior <i>br</i> ratio</td><td>11</td><td>41.8</td><td>-</td><td>56.9</td><td>-</td><td>-</td><td>-</td><td>48.1</td><td>-</td><td>5.1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Posterior total</td><td>14</td><td>24.2</td><td>-</td><td>37.3</td><td>72.6</td><td>-</td><td>92.1</td><td>29.4</td><td>79.1</td><td>4.3</td><td>5.9</td><td>24.9</td><td>73.5</td></tr></tbody></table>

<table><caption>**Table 5 Summary of eggs morphometrics of *Ramazzottius claudii* sp. nov**</caption><thead><tr><th>Character</th><th>N</th><th>Range</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td>Egg bare diameter</td><td>18</td><td>55.4</td><td>-</td><td>65.7</td><td>61.2</td><td>2.6</td></tr><tr><td>Egg full diameter</td><td>18</td><td>70.7</td><td>-</td><td>80.7</td><td>75.2</td><td>2.8</td></tr><tr><td>Process height</td><td>54</td><td>4.6</td><td>-</td><td>27.9</td><td>7.7</td><td>3.3</td></tr><tr><td>Process base width</td><td>54</td><td>0.6</td><td>-</td><td>2.9</td><td>1.3</td><td>0.4</td></tr><tr><td>Process base/height ratio</td><td>54</td><td>3%</td><td>-</td><td>43%</td><td>19%</td><td>7%</td></tr><tr><td>Inter-process distance</td><td>54</td><td>1.0</td><td>-</td><td>6.1</td><td>2.8</td><td>1.0</td></tr><tr><td>Number of processes on the egg circumference</td><td>12</td><td>40</td><td>-</td><td>52</td><td>46.3</td><td>3.5</td></tr></tbody></table>

* *Ramazzottius anomalus* (Ramazzotti, 1962): by having a thinner buccal tube [external diameter pt 11.5 in *R. anomalus* vs. 7.1 – 9.8 µm in *R. claudii* sp. nov.] and by the shape of the egg processes [long cone/aculeus in *R. anomalus* spines/filaments in *R. claudii* sp. nov.].

* *Ramazzottius groenlandensis* Kihm et al., 2023: by having smaller eggs [bare diameter 80.6 3 µm in *R. groenlandensis* vs. 55.4 – 65.7 µm in *R. claudii* sp. nov.] and by the processes shape [mostly cone shaped in *R. groenlandensis* vs. elongated spikes and filamentous in *R. claudii* sp. nov.].

* *Ramazzottius horningi* Binda & Pilato, 1994: by having longer external I-III claws [52.26 – 55.73 pt in *R. horningi* vs. 58.3 – 85.8 pt in *R. claudii* sp. nov.], and by the absence of a crown of dots around the base of the egg processes [crown of dots presents in *R. horningi* vs. absent in *R. claudii* sp. nov.].

* *Ramazzottius theroni* Dastych, 1993: by the absence of eyes [eyes present in *R. theroni* vs. absent in *R. claudii* sp. nov.] and a weakly visible cuticle sculpturing [very evident in *R. theroni* vs. weakly visible in *R. claudii* sp. nov.]

* *Ramazzottius valaamis* Biserov & Tumanov, 1993: by the shape of the egg processes [very thin filaments in *R. valaamis* vs. spines/filaments in *R. claudii* sp. nov.]

<figure><img src="image_10.png" /><figcaption>**Fig. 3** *Ramazzottius claudii* sp. nov. in toto. **A** In vivo animal under transmitted light showing pigmentation bands. **B** Holotype under PCM. Indented arrowheads: dorso-lateral pigmentation bands. Indented arrowhead: pigmentation ring around the mouth. Scale bars 100 µm</figcaption></figure>

<figure><img src="image_11.png" /></figure>
**Fig. 4** *Ramazzottius claudii*
sp. nov. cuticle under PCM.
**A** Anterior dorsal cuticle. **B** Median dorsal cuticle. **C** Caudal dorsal cuticle. **D** Anterior dorsal cuticle in cross section. **E** Median dorsal cuticle in cross section. **F** Caudal dorsal cuticle in cross section. **G** Cuticle of a large individual showing “spindle” shaped granulation, more pronounced on the caudal side (on the right side of the image). White arrowhead: spindle-shaped granulation. Scale bars 20 µm

<figure><img src="image_12.png" /></figure>

**Fig. 5** *Ramazzottius claudii*
sp. nov. cuticle under SEM. **A** Dorsal cephalic region showing sensory organs. **B** Dorsal caudal cuticle showing granular sculpture. **C** Dorsal caudal cuticle showing “spindle” like granulation. Arrowheads: cephalic sensory organs. White arrowhead: spindle-shaped granulation. Scale bars **A**, **C** 10 µm; **B** 2 µm

<figure><img src="image_13.png" /></figure>
**Fig. 6** *Ramazzottius claudii* sp. nov. buccal apparatus under PCM. **A** Buccal apparatus in *toto* in dorsal view. **B** OCA in dorsal view. **C** Oral cavity armature (OCA) in ventral view. **D** AISM in lateral view. **E** Placoids in ventral view. Arrowheads: third band of OCA. Indented arrowhead: dorsal hook of AISM. Empty indented arrowhead: ventral hook of AISM. Empty arrow: pharyngeal apophyses. White arrows: macroplacoids constric-tions. Scale bars 10 µm

<figure><img src="image_14.png" /></figure>

* *Ramazzottius varieornatus* Bertolani & Kinchin, 1993: by having smaller eggs [bare diameter 69.9 – 73.3 µm in *R. varieornatus* vs. 55.4 – 65.7 µm in *R. claudii* sp. nov.], with generally longer processes [up to 10.2 µm in *R. varieornatus* vs. up to 27.9 µm in *R. claudii* sp. nov.], a shorter external claws II-III [58.2 – 68.6 *pt* in *R. varieornatus*

vs. 61.8 – 85.8 *pt* in *R. claudii* sp. nov.], and by the absence of males [absent in *R. varieornatus* vs. present in *R. claudii* sp. nov.].

Additionally, for five other *Ramazzottius* species, the eggs are unknown; however, the new species differs from them by:

**Fig. 7** *Ramazzottius claudii* sp. nov. legs associated structures. **A** Claws III under PCM. **B** Claws IV under PCM. **C** Papilla on leg IV under PCM. **D** Claws III under SEM. **E** Claws IV under SEM. Black arrow-heads: pseudolunules. White arrowheads: claws accessory points. White arrows: LRU with cuticular filaments. Indented arrowhead: Papilla on leg IV. Scale bars 10 µm

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>
* *Ramazzottius belubellus* Bartels et al., 2011: by the absence of spines on the dorsal cuticle [present in *R. belubellus* vs. absent in *R. claudii* sp. nov.].

* *Ramazzottius baumanni* (Ramazzotti, 1962): by the absence of stronged sculptured cuticle with elevated polygons [present in *R. baumanni* vs. absent in *R. claudii* sp. nov.].

* *Ramazzottius montivagus* (Dastych, 1983): by having a sculptured cuticle [smooth in *R. montivagus* vs. sculptured in *R. claudii* sp. nov.] and by more slender primary branch of external claws (morphometric data for R. montivagus not available, but see Figs. 5, 6, and 7 in Dastych, 1983).

* *Ramazzottius saltensis* (Claps & Rossi, 1984): by the absence of stronged sculptured cuticle with elevated polygons [present in *R. saltensis* vs. absent in *R. claudii* sp. nov.].

* *Ramazzottius szeptyckii* (Dastych, 1980): by the absence of dorsolateral gibbosities [present in *R. szeptycki* vs. absent in *R. claudii* sp. nov.].

## Discussion

Despite being charismatic organisms, tardigrade biodiversity is still unexplored. In particular, some habitats have historically received less attention, and rock pools is one of them. Recently, tardigrades have been found and described from rock pools (Troell & Jönsson, 2023; Vecchi et al., 2022, 2023a, 2023b), showing the potential of this habitat for identifying a novel component of tardigrade diversity. Tardigrades from rock pools (and in particular the newly described *Ramazzottius*) are adapted to their extremely harsh conditions (multiple desiccation and freezing cycles) and show higher performances in surviving freeze-thaw cycles respect to congeneric taxa from other habitats (Zawierucha et al., 2023). The identification and description of this new tardigrade species with extreme resistance capabilities will be useful in finding new model taxa for the study of the molecular processes behind their incredible capabilities.

While one *Ramazzottius* species (*R. varieornatus* YOKO-ZUNA-1) recently became a popular model organism for

<figure><img src="image_17.png" /><figcaption>**Fig. 8** *Ramazzottius claudii* sp. nov. eggs. **A–H** Eggs under PCM. **I** Egg under SEM. Scale bars 20 µm</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>**Fig. 9** *Ramazzottius claudii* sp. nov. males under PCM. **A–B** Males posterior part. Arrow-heads: gonads with developing spermatids. Scale bars 100 µm</figcaption></figure>

the study of the molecular mechanisms of stress resistance in tardigrades (e.g., Emdee et al., 2024; Horikawa et al., 2008, 2013; Neves et al., 2020), a comparative approach with multiple species would provide a more detailed knowledge of their molecular repertoire and evolution (Fleming et al., 2024). The newly described species is closely related to *R. varieornatus* YOKOZUNA-1 (Fig. 1), however they differ by their reproductive mode (parthenogenesis in *R. varieornatus* YOKOZUNA-1; Bertolani & Kinchin, 1993; Horikawa et al., 2008 vs. gonochorism in *R. claudii* sp. nov.) and probably by their cryptobiotic performances (*R. varieornatus* YOKOZUNA-1 was collected from a moss sample in Sapporo (Japan) at sea level, whereas the new species was collected in a shallow rock pool at ~1600 m a.s.l.; thus, they experience and are probably adapted to a different amount of environmental stress). Some anhydrobiosis related genes have been found to be differently expressed in the sexes in *Paramacrobiotus metropolitanus* Sugiura, Matsumoto & Kunieda, 2022 (Sugiura et al., 2024), and a similar phenomenon could occur in *Ramazzottius* too. Given its characteristic, the new species could be a potential candidate to

complement *R. varieornatus* YOKOZUNA-1 as model for the study of the molecular mechanisms behind cryptobiosis.

As the first step to provide resources on this new species, we sequenced its mitogenome and annotated its TDRPs (Temperature and Desiccation Resistance Proteins) genes. As expected, due to their phylogenetic proximity (Fig. 1), *R. claudii* sp. nov. and *R. varieornatus* YOKOZUNA-1 show a perfect synteny of their mitochondrial genes. However, the two genomes show a discrete nucleotidic divergence, in particular in the regions of the ATP synthase and nad2 genes (Fig. 2) which can reach a divergence higher than 30%. In context, the divergence in the cox1 gene (traditionally used in species delimitation and population genetic studies) in the two species examined here is about 17%. This suggests that another region of the mitochondrial genome could provide a more fine-scaled signal, which could be exploited in tardigrade species delimitation of closely related species and population genetics.

In addition to the mitogenome sequence, we identified TDRPs genes studied by Fleming et al. (2024) in different tardigrade taxa. Our results are congruent with what

<figure><img src="image_19.png" /></figure>
found by Fleming et al. (2024) for *R. varieornatus* YOKO-ZUNA-1, with most of the TDRPs found in *R. claudii* sp. nov. In particular, the 15 identified CAHS genes show a close correspondence with the CAHS of *R. varieornatus* YOKOZUNA-1 (Table 2, Online resources 06) (Fleming et al., 2024), with the exception of CAHS6c that was not found in the new species. A less congruent situation was found for SAHS, where in the new species the SASH 9, 10, 11, and 13 were not identified and for one it was not possible to determine its homology with SAHS7 or 12 (Table 2, Online resources 06). Additionally, for the single copy of the MRE11 found in the new species, it was not possible to determine its homology to any of the 4 MRE11 identified in *R. varieornatus* YOKOZUNA-1, which form a monophyletic clade (Online resources 09) in sister group with the new *R. claudii* sp. nov. MRE11. This situation could suggest a possible expansion of the MRE11 genes in *R. varieornatus* YOKOZUNA-1; however, a more in-depth sequencing of the new species is required to confirm this hypothesis. Lastly, coherently to what was found in *R. varieornatus* YOKOZUNA-1, a single Dsup gene was also identified. Nevertheless, we showed that the TDRPs repertoire is conserved across closely related species even with contrasting reproductive modes and that the genome sequencing protocol used in this study is a viable solution for this task.

In conclusion, we described a new *Ramazzottius* species and provided novel genetic resources for the study of cryptobiosis molecular mechanisms.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s13127-024-00662-x.

**Acknowledgements** This study was founded by the Polonez Bis grant No. 2022/45/P/NZ8/01512 to MV co-funded by the National Science Centre and the European Union framework Programme for Research and Innovation Horizon 2020 under the Marie Skłodowska-Curie grant agreement [No. 945339]. We wish to thank Dott. Izabela Potocka and Yelyzaveta Matsko (University of Silesia, Katowice, Poland) for help with the SEM stubs coating, and Dott. Claudio Ferrari (University of Parma, Italy) for his assistance during sampling.

**Data availability** The datasets generated during and analysed during the current study are available in the GenBank repository (see main text for accession numbers) or included in this published article [and its supplementary information files].

## Declarations

**Competing interests** The authors declare no competing interests.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in

the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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