Description of a new genus and species of
Isohypsibioidea (Tardigrada), together with its
mitochondrial genome sequence

Daniele Camarda, Oscar Lisi, Daniel Stec, Matteo Vecchi

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Daniele Camarda, Oscar Lisi, Daniel Stec, Matteo Vecchi. Description of a new genus and species of Isohypsibioidea (Tardigrada), together with its mitochondrial genome sequence. Arthropod Systematics and Phylogeny, 2025, 83, pp.427 - 445. 10.3897/asp.83.e150460. hal-05297753

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# Description of a new genus and species of Isohypsibioidea (Tardigrada), together with its mitochondrial genome sequence

Daniele Camarda¹, Oscar Lisi¹, Daniel Stec², Matteo Vecchi²

1 Department of Biological, Geological and Environmental Sciences, University of Catania, Via Androne 81, 95124, Catania, Italy

2 Institute of Systematics and Evolution of Animals (Polish Academy of Sciences), Slawkowska 17, 31-016, Kraków, Poland

https://zoobank.org/9D8306C7-55F9-4370-B1D1-6F2025D10246

Corresponding author: Matteo Vecchi (matteo vecchi15@gmail.com)

**Received** 19 March 2025

**Accepted** 26 May 2025

**Published** 26 August 2025

**Academic Editors** Brendon E. Boudinot, Klaus-Dieter Klass

**Citation:** Camarda D, Lisi O, Stec D, Vecchi M (2025) Description of a new genus and species of Isohypsibioidea (Tardigrada), together with its mitochondrial genome sequence. Arthropod Systematics & Phylogeny 83: 427–445. https://doi.org/10.3897/asp.83.e150460

## Abstract

A new tardigrade taxon, *Thulyphoribius melitense gen. nov. et sp. nov.* is described from a population of limnic tardigrades collected in the sediment of a temporary pond in Malta. Those habitats, characterized by fluctuating environmental conditions and ephemeral water availability, provide a particularly challenging setting that can drive morphological and genetic diversity in aquatic microfauna. The new genus shows a unique combination of morphological characters, including distinctive morphologies of the peribuccal region, a short and wide ventral lamina and *Pseudobiotus*-type claws. Although it shares certain traits with some extant genera, the presence of unique characters combinations, precludes its assignment to any of the previously established genera. A comprehensive investigation was conducted, including morphological (using Phase-Contrast Light Microscopy, Scanning Electron Microscopy), morphometric, and molecular analysis. In the latter context, the molecular markers 18S, 28S, COI, and ITS2 were sequenced, and the complete mitochondrial genome was obtained and characterized, offering important insights for future molecular studies of tardigrades. Phylogenetic analyses based on Maximum Likelihood and Bayesian methods, incorporating the four aforementioned molecular markers, indicate that the new genus belongs to the superfamily Isohypsibioidea, yet it does not cluster within any currently recognized extant family of tardigrades. Collectively, these findings underscore the relevance of investigating underexplored habitats and highlight the potential for discovering novel evolutionary lineages among limnic microinvertebrates that inhabit temporary ponds.

## Keywords

Parachela, mitogenome, phylogeny, rock pools, systematics, taxonomy

## 1. Introduction

Tardigrades are a phylum of meiofauna belonging to the clade Ecdysozoa Aguinaldo et al., 1997 (molting animals; Schill (2018)) which are also characterized by the ability

to resist extreme environmental stressors (freezing, desiccation, UV; Horikawa et al. 2013; Hengherr and Schill 2018; Schill and Hengherr 2018). Tardigrades need at
least a layer of liquid water to be active (Wełnicz et al. 2011; Nelson et al. 2018), however, thanks to their resistance strategies, they can colonize habitats that experience temporary desiccation events; mosses and lichens have been historically the elective substrate from where tardigrades have been collected (Nelson et al. 2018), but less sampled habitats like soil (Hohberg 2006; Guil et al. 2014), leaf litter (Hinton et al. 2010; Nelson and Bartels 2013; Vecchi et al. 2021), and rock pool sediments (Vecchi et al. 2022a; Vecchi et al. 2023a; Troell and Jönsson 2023) may host abundant and diverse tardigrade communities.

The tardigrade superfamily Isohypsibioidea Sands, McInnes, Marley, Goodall-Copestake, Convey & Linse, 2008 is very diverse in term of ecological strategies, including taxa that thrive in marine habitats (with the only marine eutardigrade genus *Halobiotus* Kristensen, 1982; e.g. Møbjerg et al. 2007), freshwater sediments (e.g. *Pseudobiotus* Nelson, 1980 (in Schuster et al. 1980); e.g. Kathman and Nelson 1987), mosses and lichens (e.g. *Isohypsibius* Thulin, 1928; e.g. Nelson et al. 2019), and sandy soils (e.g. *Eremobiotus* Biserov, 1992, *Apodibius* Dasptych, 1983 and the family Hexapodibiidae Cesari, Vecchi, Palmer, Bertolani, Pilato, Rebecchi & Guidetti, 2016; e.g. Hohberg et al. 2011; Cesari et al. 2016). The diversity in ecological strategies of Isohypsibioidea is mirrored by their morphological diversity, which led to the erection of many genera and families in the last few years (Cesari et al. 2016; Gąsiorek et al. 2019b; Kaczmarek et al. 2020; Tumanov 2022; Tumanov et al. 2024).

In this study we report the description of a new genus and species of an Isohypsibioidea found in a freshwater rock pool on Malta Island (Malta), a territory where the number of known tardigrade taxa is very low (11 according to Camarda et al. 2022), most likely due to the paucity of previous studies. The new taxon exhibits a combination of morphological characters typical for other freshwater Isohypsibioidea, with unique traits not found in any other genus. The phylogenetic analysis places this new genus as a sister group to all extant Isohypsibioidea, suggesting the possibility that this species represents a unique lineage worth of the family level status; however, evident morphological characters to delineate a new family were not identified. Additionally, given the paucity of molecular resources for Isohypsibioidea and to provide data for future phylomitogenomic studies on the phylum Tardigrada, we provide the complete sequence of the mitogenome of the newly described species.

# 2. Materials and methods

## 2.1. Sampling and sample processing

A sediment sample from a dry rock pool located near Qrendi, Malta, was collected on 23 March 2023. The sample was taken from the sediment surface (~2 cm

depth) with a clean plastic spoon. The sample was kept desiccated until processing. Tardigrade extraction was performed by rehydrating the sediment with distilled water and inspecting it under a stereomicroscope. It was possible to keep the specimens in the culture for a limited time: a portion of dry sediment was placed in a Petri dish with ddH₂O and algae (*Chlorococcum* sp.) at a temperature of 16–20°C with weekly water changes and feedings; many animals turned to active state, and the culture lasted approximately 6 months before dying.

## 2.2. Microscopy and imaging

To assess whether the morphological characters appeared consistent, specimens for light microscopy were mounted on microscope slides in a small drop of Hoyer's or Polyvinyl Lactophenol (PVLF) medium, secured with a cover slip, and dried at 60°C for a week. Five exuviae with eggs were extracted from the sample and placed in ddH₂O to obtain hatchlings, which were subsequently mounted on microscope slides as described above. Additional individuals were stained with Orcein (Bertolani 1971) to identify males. Slides were examined under a Leica LM1000 light microscope with phase contrast (PCM), equipped with a Flexacam C3 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 (SEM) were prepared according to protocol A2 from Camarda et al. (2024b) and imaged on a Phenom-XL available at the University of Catania (Catania, Italy). All figures were assembled with GIMP v.2-10 (GIMP Development Team, 2019) and Inkscape (Inkscape Project 2020).

## 2.3. Morphometrics and morphological nomenclature

All measurements are given in micrometers (µm). Structures were measured only if their orientation and integrity were 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). Nomenclature of peribuccal structures follows (Camarda et al. 2024a), placoid configurations follows Kaczmarek et al. (2014). Claws were measured according to Stec et al. (2018b). Claws *cct* ratio was calculated according to Guidetti et al. (2016) and Vecchi et al. (2023b). The *br* ratio is the ratio of the length of the secondary branch to the length of the primary branch of the claws (Gąsiorek et al. 2019b). Morphometric data were handled using the “Parachela” ver. 1.7 template available from the Tardigrada
da Register (Michalczyk and Kaczmarek 2013). The raw
morphometric data of adults and newborns are provided
separately as Supplementary Material (Files S1, S2). Ab-
breviations for tardigrade genera follow the system pro-
posed by Perry et al. (2019).

**2.4. Genotyping**

DNA was extracted from individual animals extracted
from the sample following a Chelex® 100 resin (BioRad)
extraction method by (Casquet et al. 2012) with modi-
fications described in detail in Stec, Kristensen et al.
(2020). Four DNA fragments, three nuclear (18S rRNA,
28S rRNA, ITS2) and one mitochondrial (COI) were se-
quenced. All fragments were amplified and sequenced
according to the primers and protocols described in Stec
et al. (2020a). Sequencing products were read with the
ABI 3130xl sequencer at the Institute of Systematics and
Evolution of Animals (Polish Academy of Sciences),
Kraków, Poland.

**2.5. Phylogenetic reconstruction**

A phylogenetic reconstruction of the superfamily Isohyp-
sibioidea was produced using the concatenated markers
18S rRNA + 28S rRNA. The same dataset as reported in
Tumanov et al. (2024), which includes sequences from
various research papers (Garey et al. 1999; Jørgensen and
Kristensen 2004; Kiehl et al. 2007; Møbjerg et al. 2007;
Sands et al. 2008; Guil and Giribet 2012; Bartels, et al.
2014; Bertolani et al. 2014b; Dabert et al. 2014; Cesari et
al. 2016; Stec et al. 2018a; Gasiorek et al. 2019a, 2019b;
Stec et al. 2020b; Zawierucha et al. 2020; Mioduchows-
ka et al. 2021; Tumanov 2022; Camarda et al. 2024a;
Tumanov et al. 2024), was used the newly generated
sequences were added. The GenBank accession num-
bers of the sequences and those used in the phylogenetic
reconstruction are presented in File S3. The 18S rRNA
and 28S rRNA sequences were aligned with MAFFT ver.
7 (Katoh 2002; Katoh and Toh 2008) with the G-INS-i
method (thread = 4, threadtb = 5, threadit = 0, reorder,
adjustdirection, anysymbol, maxiterate = 1000, retree 1,
globalpair input). Alignments were visually inspected and
trimmed in MEGA7. Sequences were concatenated with
the R package ‘concatipede’ v1.0.0 (Vecchi and Bruneaux
2021). Model selection and Maximum likelihood recon-
struction were performed with IQTREE (Trifinopoulos et
al. 2016; with parameters -bnni -bb 1000 -mset mrbayes
-merit AICC). Bayesian inference (BI) phylogenetic re-
construction was performed using MrBayes v3.2.6 (Ron-
quist 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 Mr-
Bayes input file with the input alignment and the specified
models is available as File S4, and the IQTREE and Mr-

Bayes output consensus trees are available as File S5. The
phylogenetic tree was visualized with FigTree (Rambaut
2007) and edited with Inkscape (Inkscape Project 2020).

**2.6. Whole Genome Amplification and
sequencing**

One individual of the new species (Iso_MT.008_WGA_1)
was extracted from the sample and starved in sterile dis-
tilled water for 24 hours at 18°C, and then washed twice
in sterile distilled water. The individual was then dissect-
ed in a 0.5 µl drop of sterile distilled water with a steril-
ized 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 1.5 µg of amplified DNA were
debranched for 1 hour with T7 Endonuclease I (Cat. No.
M0302, New England Biolabs) according to the manu-
facturer protocol. The reaction product was again purified
with the GeneMAGNET Kit 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 Technol-
ogies, 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 48h. BaseCALLing, demultiplexing and adaptors
trimming was done with the software MinKNOW (ONT)
using a high accuracy baseCALLing. Sequences of the DNA
Control Sample (DCS) introduced during library prepa-
ration were removed with chopper (De Coster and Rade-
makers 2023).

**2.7. Mitogenome assembly and
annotation**

Clean reads were blasted against a tardigrades mitochon-
drial proteins database (Vecchi and Stec 2024) 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:
--nano-raw -meta -m 1000 -i 5). The assembled se-
quences were visualized with Bandage (Wick et al. 2015)
and the identity of the mitogenome contig confirmed by
blastn against the new species COI sequences produced
with Sanger sequencing (as described above). A final
polishing was done with medaka v 1.11.3 (https://github.
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 Eu-
rope 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-con-
verters/by_program/table2asn). The mitogenome was
visualized with OGDRAW (Greiner et al. 2019). Cover-
age data was extracted from the medaka BAM files us-
ing the R package "gmoviz" (Zeglinski et al. 2021). The
raw reads are available in NCBI SRA (under Bioproject
PRJNA1215272). The annotated mitogenome sequence
is available in GenBank (Accession number PV030524).

3. Results

The phylogenetic reconstruction (Fig. 1) produced a to-
pology consistent with that already reported by (Tuman-
ov et al. 2024), showing a paraphyletic "basal" Isohypsi-

biidae Sands, McInnes, Marley, Goodall-Copestake, Convey & Linse, 2008 and monophyletic Ramajendidae Tumanov, 2022, Halobiotidae Gąsiorek, Stec, Morek & Michalczyk, 2019, Hexapodibiidae Cesari, Vecchi, Palmer, Bertolani, Pilato, Rebecchi & Guidetti, 2016 and Doryphoribiidae Gąsiorek, Stec, Morek & Michalczyk, 2019. The newly sequenced individuals occupy a position "basal" to all other taxa in the superfamily Isohypsibioidea. Due to both morphological and phylogenetic uniqueness of the newly found individuals, a new genus and a new species are erected for them (see Taxonomic account below).

In the Nanopore sequencing run, after the removal
of the DCS sequence, a total of 2.26 Gb of reads (n =
907920, N50 = 4950 bp, average quality = 15.21 Q) were
obtained, of which 0.47% (10.59 Mb) of mitochondrial
origin. The obtained mitochondrial genome (Fig. 1) is
15033 bp in length, with an average coverage of 704X
(min 299X – max 1259X). Two ribosomal RNAs (rrnS
and rrnL), 22 tRNAs (with two trnL and trnS), 2 ATP syn-

<figure><img src="image_2.png" /></figure>

**Figure 1.** Mitogenome and phylogenetic position of *Thulyphoribius melitense* gen. nov. et sp. nov. A Mitogenome visualization: inner circle represents GC content. Arrows indicate direction of transcription. B Bayesian phylogenetic reconstruction of Isohypsibioidea: values above branches represent BI posterior probabilities (pp) / ML bootstrap (BS). Nodes with pp < 0.70 were collapsed. Scale bar indicates substitutions/site.
thase 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 mitochondrial genes order is identical to the one of *Thulinius* sp. DVL-2010 (HM600784; (Rota-Stabelli et al. 2010)) and *Ramazzotius claudii* Vecchi & Stec, 2024 (PP419898; (Vecchi and Stec 2024)), while only tRNA-L1 seems to be positioned in the opposite strand in *Pseudobiotus spinifer* Chang, Kaczmarek, Lee & Michalczyk, 2007 (KF938944; unpublished – however this could represent an annotation error). The mitogenome of *Hypsibius exemplaris* Gąsiorek, Stec, Morek & Michalczyk, 2018 (NC_014848; (Rota-Stabelli et al. 2010)) have instead more differences, of which the most conspicuous is the translocation of *nad1* between *nad2* and *nad4l* in *Hys. exemplaris*.

**3.1. Description of the new genus**

***Thulyphoribius*** ***gen. nov.***

https://zoobank.org/C1DFA251-BA17-4CFB-B7E7-50D27E45C1C2

**Etymology.** A portmanteau of *Thulinius* and *Doryphoribius*, as the new genus possesses some morphological traits similar to those two genera.

**Abbreviation.** The genus name, should be abbreviated as “*Thp*”, following the abbreviations adopted by (Perry et al. 2019).

<figure><img src="image_3.png" /><figcaption>**Figure 2.** *Thulyphoribius melitense* gen. nov. et sp. nov. – habitus seen (A) under Phase Contrast Microscopy in Hoyer’s medium (PCM) and (B) Scanning Electron Microscopy (SEM). Scale bars = A 100 µm; B: 50 µm.</figcaption></figure>
**Type species.** *Thulyphoribius melitense gen. nov.* et *sp. nov.* by original designation and monotypy (Articles 68.2 and 68.3 of the ICZN, 1999).

**Diagnosis.** Peribuccal lamellae or papulae absent, although in PCM, sometimes, internal septa in the buccal ring are visible giving the appearance of the presence of lamellae or papular lamellae. Buccal cone with 6 papular peribuccal lobes visible only under SEM and only when the buccal cone is fully extended (Fig. 3A–D). When observed laterally, the buccal tube appears to have

two bends: the first, more pronounced, at the beginning of the anterior portion of the buccal tube, in correspondence with the area bearing the ventral lamina; the second, less pronounced, approximately halfway along the tube (Fig. 4A). A very short, protruding ventral strengthening lamina (similar to a large, well protruding crest) is present. The lamina has a modest notch approximately one-third along its length: the first third is nearly straight, with a smooth margin, the central portion (from 1/3rd to 2/3rd of the lamina length) has a convex, more thickened, and slightly serrated (more visible in bigger specimens)

<figure><img src="image_4.png" /><figcaption>**Figure 3.** *Thulyphoribius melitense gen. nov.* et *sp. nov.*, head and details of the oral cavity armature (OCA) and cuticle ornamentation. **A** Head with protruded buccal cone, showing the papular peribuccal lobes. **B** Detail of the mouth opening, showing the continuous buccal ring and the second band of teeth of the OCA. **C–D** Details of the buccal opening, showing the continuous buccal ring, dorsal (**C**) and ventral (**D**) teeth of the third band of the OCA. **E** Ornamented cuticle covered with small tubercles approximately 0.2 microns in size. Arrowheads indicate the second band of teeth, indented arrowheads indicate the third band of teeth, and asterisks indicate the papular peribuccal lobes. Scale bars = A: 10 µm; B: 2 µm; C–E: 5 µm.</figcaption></figure>
margin; the final portion of the posterior segment appears straight and smooth (see Fig. 4A, D). Three macroplacoids (length sequence 2<3<1), with the first being very long and rod-shaped (more than twice the length of the second), showing a slight median incision; the second macroplacoid has granular shape; the third macroplacoid is elongated and nearly twice the length of the second (Fig. 4E).

*Pseudobiotus* morphotype of the *Isohypibius*-type claws (Gąsiorek et al. 2019b), resembling those found in the genus *Thulinius* and *Pseudobiotus*, i.e., claws elongated, with a clear hump on the primary branch (Fig. 5) and with relatively elongated secondary branches br > 70% (Gąsiorek et al. 2019b). Lunulae or pseudolunulae absent, but claw bases in all legs with internal septa clearly visible under LM (see Fig. 5C), giving the impression of a “duck’s foot” shape; this particular structure showed to be more visible in specimens mounted in Hoyer’s than in specimens mounted in Polyvinyl Lactophenol, probably due to a stronger clearing effect of the former mounting medium.

*Thulyphoribius gen. nov. differential diagnosis.* The new genus is morphologically and molecularly (Fig. 1) well separated from all other extant genera of Isohypibius-

oidea, and in particular it is distinct from known members of the family Doryphoribiidae, with which the new genus shares the most morphological characters (primary and secondary branches of claws of similar size and presence of a ventral lamina in the buccal apparatus in some genera; Gąsiorek et al. 2019b).

Among Doryphoribiidae, based on the presence of a rigid buccal tube, three macroplacoids and no microplacoid in the pharynx and a cuticle devoid of dorsal gibbosities, *Thulyphoribius gen. nov.* can be compared to *Doryphoribius* Pilato, 1969 (species in the *zappalai* group sensu Michalczyk and Kaczmarek 2010), *Thulinius* Bertolani, 2003, *Pseudobiotus* (only some species have 3+0 placoids configuration) and *Grevenius* Gąsiorek, Stec, Morek & Michalczyk, 2019 (belonging to asper group sensu Massa et al. 2024). Characters differentiating *Thulyphoribius gen. nov.* from the above-mentioned genera/groups are presented in Table 1.

In particular, *Thulyphoribius gen. nov.* can be considered particularly similar to *Doryphoribius* (for the presence of a ventral lamina in the buccal apparatus) and *Thulinius* (by the shape of the placoids, claw structure and shape and presence of cuticular bars on legs). Therefore, we compare it below with these two genera.

<figure><img src="image_5.png" /><figcaption>**Figure 4.** *Thulyphoribius melitense* gen. nov. et sp. nov. bucco-pharyngeal apparatus under PCM. A Bucco-pharyngeal apparatus in toto, lateral view. **B** Detail of the buccal opening, showing the apparent lamellae. **C** Stylet furcae. **D** Detail of the first portion of the buccal tube, highlighting the distinctive ventral lamina. **E.** Placoids. Arrowheads indicate the notch in the middle portion of the ventral lamina. Indented arrowheads indicate a septum that, under PCM, gives the impression of peribuccal lamellae surrounding the mouth opening. Scale bars = A, E: 20 µm; B, C: 10 µm.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 5.** *Thulyphoribius melitense gen. nov. et sp. nov.* – claws under phase contrast microscopy (PCM) and scanning electron microscopy (SEM). **A, B** Second pair of claws shown under PCM in Hoyer’s medium (**A**) and SEM (**B**). **C** Detail of the base of the external claw of the second pair of legs, shown under PCM, highlighting the internal septa. **D, E** Third pair of claws shown under PCM in Hoyer’s medium (**D**) and SEM (**E**). **F, G** Fourth pair of claws shown under PCM in Polyvinil Lactophenol medium (**F**) and SEM (**G**). Black asterisks indicate the base equipped with internal septa. White asterisks indicate the “hump” in the internal and anterior claws. Indented empty arrowheads indicate the cuticular bar at the base of the claws. Indented arrowheads indicate the small accessory points. Scale bars = A, B, D, E, G: 10 µm; C: 2 µm; F: 20 µm.</figcaption></figure>
<table><caption>**Table 1.** Diagnostic characters of *Thulyphoribius gen. nov.* compared with Doryphoribiidae genera with developed claws, 3+0 placoids configuration and without gibbosities in the cuticle. Y = character present; N = character absent; AISM = Apophyses of the stylet muscle insertion.</caption><thead><tr><th></th><th>Thulyphoribius<br>gen. nov.</th><th>Doryphoribius<br>(zappalai group)<sup>1</sup></th><th>Thulinius</th><th>Pseudobiotus<sup>2</sup></th><th>Grevenius<br>(asper group)<sup>3</sup></th></tr></thead><tbody><tr><td>Cuticle gibbosities</td><td>N</td><td>Y/N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>Peribuccal structures</td><td>6 papular peribuccal lobes</td><td>6 peribuccal lobes</td><td>6 peribuccal lobes</td><td>6 peribuccal lobes</td><td>Peribuccal lobes absent<sup>5</sup></td></tr><tr><td>AISM</td><td>Short and thick ventral lamina (pt 35–45)</td><td>Long ventral lamina (pt >50)</td><td>No ventral lamina, symmetrical and ridge-like</td><td>No ventral lamina, symmetrical and ridge-like</td><td>No ventral lamina, symmetrical and ridge-like</td></tr><tr><td>Placoids number and configuration</td><td>3+0 (1&gt;3&gt;2)</td><td>3+0 (various configurations)</td><td>3+0 (1&gt;3&gt;2)</td><td>3+0 (3&gt;1&gt;2 or 1&gt;3&gt;2)</td><td>3+0 (various configurations)</td></tr><tr><td>Placoids shape</td><td>1° and 3° elongated, 2° granular</td><td>Granular or almost granular</td><td>1° and 3° elongated, 2° granular</td><td>Elongated</td><td>1° elongated, 2° granular</td></tr><tr><td>Basal portion of the claw (PCM)</td><td>Indented, resembling a “duck-foot” shape</td><td>Smooth</td><td>Smooth</td><td>Smooth</td><td>Smooth</td></tr><tr><td>Pseudolunulae</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Y<sup>4</sup></td></tr><tr><td>Cuticular bars on legs</td><td>Long, present under both claws</td><td>N</td><td>Long, present under both claws</td><td>N</td><td>Single cuticular bar or no cuticular bars</td></tr></tbody></table>

**The new genus differs from *Doryphoribius* by:** The ventral lamina which has a peculiar shape, being very short and thick (pt 35–45 in the new genus vs pt > 50 in *Doryphoribius*, except for *D. smokiensis* which, however, possesses only two macroplacoids), resulting in a buccal tube morphology with a sharply curved proximal section (Figs 4A, 6B).

First and third macroplacoid have a bar-like shape, while the second has a granular shape, whereas in *Doryphoribius* (in species exhibiting three macroplacoids), all macroplacoids are granular (or almost granular) in shape.

Claws of the *Pseudobiotus*-type in the new genus according to Gąsiorek et al. (2019b): “with secondary and primary branches similar in height (br typically >70%), elongated basal tracts, and typically prominent humps on primary branches of internal and anterior claws”, while in *Doryphoribius*, claws are of *Isohypsibius*-type in (i.e. “without the hump on the primary branch and with a considerable difference in primary and secondary branch height (br ≤ 70%)” according to (Gąsiorek et al. 2019b)).

Claw bases provided with internal septa in the new genus while in *Doryphoribius* the septa in claw bases are absent.

A long cuticular bar under both internal and external bases of claws I–III is present, while cuticular bars on legs are absent in *Doryphoribius*.

**The new genus differs from *Thulinius* by:** The peribuccal cone surrounded externally to the buccal ring by 6 papular peribuccal lobes in the new genus, while ordinary peribuccal lobes are present in *Thulinius*.

Apophyses of the stylet muscle insertion (AISM) represented only by a single, large, ventral apophysis (i.e.,

the ventral lamina) in the new genus, whereas both ventral and dorsal crests are present, smaller and similar in shape, in *Thulinius*.

Claw bases provided with internal septa in the new genus whereas in *Thulinius* the septa in claw bases are absent.

## 3.2. Description of the new species

### *Thulyphoribius melitense* sp. nov.

https://zoobank.org/3872F2DC-F8AC-4F31-86EB-D437902628AE

Figures 2–8; Tables 2, 3; Files S1, S2

**Etymology.** The specific epithet refers to the location where the new species has been found (*melitense* meaning from Malta).

**Type locality.** Malta • Qrendi, sediment collected in a dried freshwater rock pool; approximately 35°49'54"N; 14°26'29"E, elev. ~50 m, 23 March 2023; M.V. leg.

**Material examined.** 58 animals, and 11 cysts mounted on microscope slides in Hoyer's medium; 12 animals mounted on microscope slides in Polyvinyl Lactophenol mounting medium (PVLF); 12 hatchlings mounted on microscope slides in PVLF, 4 exuviae on microscope slides in PVL. 7 animals prepared for SEM. 2 individuals used for DNA sequencing, 1 individual used for WGA and mitogenome sequencing.

<footnote n="1">¹ species group according to (Michalczyk and Kaczmarek, 2010)</footnote>

<footnote n="2">² excluding *P. megalonyx* which has 2 macroplacoids and *P. longiunguis*, considered as species dubia according to (Pilato et al. 2010)</footnote>

<footnote n="3">³ species group according to (Massa et al. 2024)</footnote>

<footnote n="4">⁴ according to (Gąsiorek et al. 2019b)</footnote>

<footnote n="5">⁵ absent according to Gąsiorek (2024), however most species in genus should be re-investigated</footnote>
**Type depositories.** Holotype with 5 paratypes (slide CT.6087 in Hoyer's); 31 paratypes (slides: CT.6088-6095 in Hoyer's); 20 paratypes (slides CT.6093-6099 in PVL); 7 animals mounted for SEM analysis (Stub No. UNICT-39) deposited at the University of Catania, Via Santa Sofia 102, Catania, Italy. 7 paratypes (slides: MT.008.01 – 02 in PVL), 32 paratypes (MT.008.03-08 in Hoyer's) deposited at the Institute of Systematics and Evolution of Animals (Polish Academy of Sciences), Sławkowska 17, Kraków, Poland

**Animals.** Body size up to approximately 500 µm (Table 2), with hatchlings ranging from about 120 to 180 µm (Table 3). Hatchlings exhibit the same morphological

characters as adults (Fig. 6), although morphometric values differ slightly, the *pt* values being higher in adults for some measurement of placoids and claws (Tables 2, 3). Before mounting, the animals appeared whitish/ transparent and became transparent after mounting in PVLF or Hoyer's medium. Eyes are present (Fig. 4A), and the entire cuticle appears finely dotted under SEM (Fig. 3E). The buccal ring appears divided under PCM (Fig. 4B), giving the impression of the presence of papular lamellae, but under SEM it appears smooth (Fig. 3A–D). Five-six papular peribuccal lobes are present (in correspondence to peribuccal lobes), visible only under SEM and only when the buccal cone is fully extended (Fig. 3A, C).

<figure><img src="image_7.png" /><figcaption>**Figure 6.** *Thulyphoribius melitense* gen. nov. et sp. nov. - hatchling under Phase Contrast Microscope (PCM) in Polyvinil Lacto-phenol medium. **A** Habitus. **B** Bucco-pharyngeal apparatus in toto, lateral view. **C** Third pair of claws with barely visible cuticular bar under their base. **D** Fourth pair of claws. Scale bars = A: 50 µm; B–D: 10 µm.</figcaption></figure>
The oral cavity armature (OCA) is visible only in SEM and consists of two bands: the first band (probably homologous to the second band of teeth of the Macrobiotoidea) comprises small, rounded teeth (gradually decreasing in density toward the second band of teeth) (Fig. 3B), second band composed by a single line of small round teeth, slightly bigger than those of the first band (Fig. 3C, D). Buccal tube with two bends: the first, more pronounced, at the beginning of the anterior portion of the buccal tube,

in correspondence with the area bearing the ventral lamina; the second, less pronounced, approximately halfway along the tube (Fig. 4A). Ventral lamina present (Fig. 4A, D), with characters described for the genus.

Typically-shaped stylet furcae (according to Pilato and Binda (2010b)), each possessing a pair of condyles (symmetrical with respect to the medial axis of the furca) located between the stylet base and the two swellings at the base of the furca (Fig. 4C).

<figure><img src="image_8.png" /><figcaption>**Figure 7.** *Thulyphoribius melitense gen. nov. et sp. nov.* – habitus and details of the legs under SEM (Stub No. UNICT.39). A habitus, ventral view. B First pair of legs. C Second pair of legs. D Third pair of legs. Arrowheads indicate the botton-like structures in the dorsal portion of the leg. Indented arrowhead indicates the small accessory points. Scale bars = A: 100 µm; B–D: 10 µm.</figcaption></figure>
Three macroplacoids (length sequence: 2<3<1) are present: the first is very long and rod-shaped (more than twice the length of the second), with a slight median constriction; the second, located almost adherent to the first macroplacoid, is granular; and the third is rod-shaped and nearly twice the length of the second (Fig. 4E).

Claws are large and slender, similar to those of *Thulinius*, with humps on the primary branches of the internal claws (Fig. 4E) and with Br ratio higher in internal claws (mean external/posterior claws = 68.2%; mean internal/ anterior claws = 73.2%; mean general = 70.1%. For detailed information see File S1). Accessory points appear absent under PCM analysis; however, SEM reveals their presence adjacent to either side of the primary branch in all claws (Fig. 5B, E, G). This overlap with the main branch prevents their observation under light microscopy. Only occasionally, they appear elevated above the primary branch on claws IV under PCM.

The claw bases on all claws appear internally septate (Fig. 5C); such septa are internal structures and thus invisible under SEM (Fig. 5B, G, E).

Lunulae and pseudolunulae are absent; a long, thin and smooth cuticular bar is present under the claw bases of claws I–III (visible under both SEM and PCM, as observed in Fig. 5A–E), well-spaced from the claws and not originating from the internal claw (e.g., similar to the cuticular bar observed in the genus *Thulinius*).

Moreover, a wrinkled button-like cuticular fold is present above the claws of the I–III pair of legs (Fig. 7B–D) this structure is well visible under SEM and only sometimes slightly visible with PCM.

**Cysts.** The animals form cysts that are dark in colour, ranging from dark green to nearly black (Fig. 8). The legs are barely visible under PCM within the external cuticle. The surface of the cyst is covered with numerous folds

<figure><img src="image_9.png" /><figcaption>**Figure 8.** *Thulyphoribius melitense* gen. nov. et sp. nov. – cysts seen under Phase Contrast Microscope (PCM). A specimen outside its cyst. B Cyst with specimen inside, where buccal apparatus is barely visible at the centre of the cyst. Scale bars 100 µm.</figcaption></figure>
<table><caption>**Table 2.** Measurements [in µm] and *pt* values of selected morphological structures of non-hatchlings specimens of *Thulyphoribius melitense gen. nov. et sp. nov.*; specimens mounted in Hoyer's or Polyvinil Lactophenol medium (see File S1 for more details); N: number of specimen/structures measured; range: refers to the smallest and the largest structure among all measured specimens; SD: standard deviation.</caption><thead><tr><th rowspan="2">Character</th><th rowspan="2">N</th><th colspan="6">Range</th><th colspan="2">Mean</th><th colspan="2">SD</th><th colspan="2">Holotype</th></tr><tr><th colspan="3">µm</th><th colspan="3">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>16</td><td>281</td><td>—</td><td>492</td><td>881</td><td>—</td><td>1312</td><td>381</td><td>1080</td><td>63</td><td>110</td><td>402</td><td>1220</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><td></td><td></td></tr><tr><td>Buccal tube length</td><td>21</td><td>23.5</td><td>—</td><td>40.9</td><td>—</td><td>—</td><td>—</td><td>34.0</td><td>—</td><td>5.2</td><td>—</td><td>32.9</td><td>—</td></tr><tr><td>Stylet support insertion point</td><td>21</td><td>17.2</td><td>—</td><td>29.5</td><td>67.7</td><td>—</td><td>73.4</td><td>23.7</td><td>69.8</td><td>3.8</td><td>1.6</td><td>22.9</td><td>69.4</td></tr><tr><td>Buccal tube external width</td><td>19</td><td>3.0</td><td>—</td><td>5.9</td><td>10.9</td><td>—</td><td>14.3</td><td>4.2</td><td>12.3</td><td>0.8</td><td>1.1</td><td>4.3</td><td>13.0</td></tr><tr><td>Buccal tube internal width</td><td>19</td><td>2.0</td><td>—</td><td>4.6</td><td>7.2</td><td>—</td><td>11.6</td><td>2.9</td><td>8.6</td><td>0.6</td><td>1.1</td><td>2.8</td><td>8.6</td></tr><tr><td>Ventral lamina length</td><td>19</td><td>10.0</td><td>—</td><td>15.5</td><td>35.0</td><td>—</td><td>43.9</td><td>13.2</td><td>39.0</td><td>1.7</td><td>2.3</td><td>13.2</td><td>40.0</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><td></td><td></td></tr><tr><td>Macroplacoid 1</td><td>19</td><td>3.9</td><td>—</td><td>8.6</td><td>15.5</td><td>—</td><td>21.1</td><td>6.1</td><td>17.8</td><td>1.4</td><td>1.7</td><td>5.7</td><td>17.4</td></tr><tr><td>Macroplacoid 2</td><td>19</td><td>2.3</td><td>—</td><td>3.9</td><td>6.9</td><td>—</td><td>9.8</td><td>2.9</td><td>8.6</td><td>0.5</td><td>0.9</td><td>2.5</td><td>7.5</td></tr><tr><td>Macroplacoid 3</td><td>19</td><td>3.0</td><td>—</td><td>6.5</td><td>11.4</td><td>—</td><td>15.8</td><td>4.7</td><td>13.8</td><td>1.0</td><td>1.2</td><td>4.4</td><td>13.4</td></tr><tr><td>Macroplacoid row</td><td>20</td><td>10.0</td><td>—</td><td>20.2</td><td>39.4</td><td>—</td><td>49.6</td><td>15.2</td><td>44.3</td><td>3.0</td><td>2.9</td><td>14.9</td><td>45.3</td></tr><tr><td>Claw I heights</td><td></td><td></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>9</td><td>5.2</td><td>—</td><td>10.5</td><td>16.4</td><td>—</td><td>25.9</td><td>7.5</td><td>21.4</td><td>2.0</td><td>3.3</td><td>7.3</td><td>22.1</td></tr><tr><td>External primary branch</td><td>6</td><td>15.1</td><td>—</td><td>24.7</td><td>50.3</td><td>—</td><td>60.7</td><td>20.1</td><td>56.3</td><td>3.7</td><td>4.3</td><td>19.5</td><td>59.3</td></tr><tr><td>External secondary branch</td><td>11</td><td>9.1</td><td>—</td><td>16.2</td><td>29.6</td><td>—</td><td>42.5</td><td>12.4</td><td>35.3</td><td>2.5</td><td>4.1</td><td>12.6</td><td>38.1</td></tr><tr><td>External base/primary branch (cct)</td><td>5</td><td>37.2</td><td>—</td><td>44.6</td><td>—</td><td>—</td><td>—</td><td>40.6</td><td>—</td><td>3.2</td><td>—</td><td>37.2</td><td>—</td></tr><tr><td>Internal base</td><td>8</td><td>5.7</td><td>—</td><td>8.8</td><td>18.2</td><td>—</td><td>21.6</td><td>6.8</td><td>20.0</td><td>1.1</td><td>1.2</td><td>7.1</td><td>21.6</td></tr><tr><td>Internal primary branch</td><td>8</td><td>9.8</td><td>—</td><td>18.1</td><td>35.7</td><td>—</td><td>45.7</td><td>12.8</td><td>39.6</td><td>2.5</td><td>3.1</td><td>13.7</td><td>41.4</td></tr><tr><td>Internal secondary branch</td><td>11</td><td>7.0</td><td>—</td><td>13.1</td><td>25.9</td><td>—</td><td>32.1</td><td>9.7</td><td>28.9</td><td>1.6</td><td>2.3</td><td>10.6</td><td>32.1</td></tr><tr><td>Internal base/primary branch (cct)</td><td>5</td><td>48.7</td><td>—</td><td>52.1</td><td>—</td><td>—</td><td>—</td><td>50.0</td><td>—</td><td>1.4</td><td>—</td><td>52.1</td><td>—</td></tr><tr><td>Claw II heights</td><td></td><td></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>14</td><td>5.6</td><td>—</td><td>10.0</td><td>17.9</td><td>—</td><td>26.4</td><td>7.3</td><td>21.3</td><td>1.5</td><td>2.3</td><td>7.4</td><td>22.3</td></tr><tr><td>External primary branch</td><td>9</td><td>14.6</td><td>—</td><td>24.9</td><td>46.0</td><td>—</td><td>62.8</td><td>18.2</td><td>54.2</td><td>3.3</td><td>5.4</td><td>20.7</td><td>62.8</td></tr><tr><td>External secondary branch</td><td>16</td><td>8.1</td><td>—</td><td>18.9</td><td>30.0</td><td>—</td><td>46.1</td><td>12.1</td><td>36.3</td><td>2.7</td><td>3.9</td><td>13.3</td><td>40.4</td></tr><tr><td>External base/primary branch (cct)</td><td>8</td><td>35.6</td><td>—</td><td>46.4</td><td>—</td><td>—</td><td>—</td><td>40.1</td><td>—</td><td>4.0</td><td>—</td><td>35.6</td><td>—</td></tr><tr><td>Internal base</td><td>17</td><td>4.5</td><td>—</td><td>9.4</td><td>17.8</td><td>—</td><td>26.1</td><td>7.1</td><td>21.1</td><td>1.4</td><td>2.1</td><td>7.7</td><td>23.4</td></tr><tr><td>Internal primary branch</td><td>12</td><td>9.5</td><td>—</td><td>18.7</td><td>35.6</td><td>—</td><td>47.5</td><td>13.5</td><td>41.3</td><td>2.6</td><td>3.2</td><td>14.1</td><td>42.7</td></tr><tr><td>Internal secondary branch</td><td>17</td><td>7.0</td><td>—</td><td>13.0</td><td>26.1</td><td>—</td><td>34.8</td><td>10.1</td><td>30.3</td><td>1.6</td><td>2.2</td><td>11.5</td><td>34.8</td></tr><tr><td>Internal base/primary branch (cct)</td><td>12</td><td>44.5</td><td>—</td><td>57.8</td><td>—</td><td>—</td><td>—</td><td>51.5</td><td>—</td><td>3.7</td><td>—</td><td>54.8</td><td>—</td></tr><tr><td>Claw III heights</td><td></td><td></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>9</td><td>4.8</td><td>—</td><td>9.3</td><td>17.8</td><td>—</td><td>22.8</td><td>6.6</td><td>19.8</td><td>1.5</td><td>1.6</td><td>?</td><td>?</td></tr><tr><td>External primary branch</td><td>8</td><td>11.4</td><td>—</td><td>23.9</td><td>43.5</td><td>—</td><td>59.4</td><td>16.5</td><td>50.8</td><td>4.9</td><td>5.6</td><td>?</td><td>?</td></tr><tr><td>External secondary branch</td><td>14</td><td>7.7</td><td>—</td><td>17.9</td><td>28.6</td><td>—</td><td>43.8</td><td>11.5</td><td>34.9</td><td>3.0</td><td>4.3</td><td>?</td><td>?</td></tr><tr><td>External base/primary branch (cct)</td><td>7</td><td>34.3</td><td>—</td><td>44.3</td><td>—</td><td>—</td><td>—</td><td>39.6</td><td>—</td><td>3.4</td><td>—</td><td>?</td><td>—</td></tr><tr><td>Internal base</td><td>13</td><td>4.7</td><td>—</td><td>7.9</td><td>17.3</td><td>—</td><td>22.7</td><td>6.2</td><td>19.7</td><td>1.1</td><td>1.7</td><td>7.5</td><td>22.7</td></tr><tr><td>Internal primary branch</td><td>11</td><td>9.3</td><td>—</td><td>14.8</td><td>34.6</td><td>—</td><td>43.7</td><td>11.9</td><td>38.7</td><td>1.7</td><td>3.0</td><td>14.1</td><td>42.9</td></tr><tr><td>Internal secondary branch</td><td>14</td><td>6.6</td><td>—</td><td>11.0</td><td>24.5</td><td>—</td><td>32.8</td><td>9.1</td><td>29.1</td><td>1.3</td><td>2.0</td><td>10.8</td><td>32.8</td></tr><tr><td>Internal base/primary branch (cct)</td><td>11</td><td>43.6</td><td>—</td><td>57.0</td><td>—</td><td>—</td><td>—</td><td>50.4</td><td>—</td><td>3.8</td><td>—</td><td>52.9</td><td>—</td></tr><tr><td>Claw IV heights</td><td></td><td></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>Anterior base</td><td>13</td><td>4.7</td><td>—</td><td>10.9</td><td>17.4</td><td>—</td><td>29.7</td><td>7.0</td><td>21.1</td><td>1.9</td><td>3.1</td><td>7.5</td><td>22.8</td></tr><tr><td>Anterior primary branch</td><td>12</td><td>10.1</td><td>—</td><td>20.4</td><td>37.7</td><td>—</td><td>50.0</td><td>14.3</td><td>42.7</td><td>3.0</td><td>3.5</td><td>15.1</td><td>45.8</td></tr><tr><td>Anterior secondary branch</td><td>13</td><td>7.3</td><td>—</td><td>13.0</td><td>27.3</td><td>—</td><td>33.3</td><td>9.9</td><td>30.1</td><td>1.8</td><td>1.9</td><td>10.5</td><td>31.8</td></tr><tr><td>Anterior base/primary branch (cct)</td><td>10</td><td>42.6</td><td>—</td><td>59.1</td><td>—</td><td>—</td><td>—</td><td>47.7</td><td>—</td><td>4.7</td><td>—</td><td>49.8</td><td>—</td></tr><tr><td>Posterior base</td><td>5</td><td>5.5</td><td>—</td><td>8.9</td><td>17.3</td><td>—</td><td>23.9</td><td>7.1</td><td>19.9</td><td>1.4</td><td>2.9</td><td>7.9</td><td>23.9</td></tr><tr><td>Posterior primary branch</td><td>7</td><td>14.0</td><td>—</td><td>22.6</td><td>44.8</td><td>—</td><td>57.0</td><td>18.1</td><td>52.9</td><td>3.1</td><td>4.6</td><td>18.0</td><td>54.8</td></tr><tr><td>Posterior secondary branch</td><td>12</td><td>8.4</td><td>—</td><td>14.9</td><td>28.7</td><td>—</td><td>42.7</td><td>11.6</td><td>34.9</td><td>2.2</td><td>4.3</td><td>12.5</td><td>37.8</td></tr><tr><td>Posterior base/primary branch (cct)</td><td>4</td><td>35.8</td><td>—</td><td>43.7</td><td>—</td><td>—</td><td>—</td><td>40.4</td><td>—</td><td>3.6</td><td>—</td><td>43.7</td><td>—</td></tr></tbody></table>
<table><caption>**Table 3.** Measurements [in µm] and *pt* values of selected morphological structures of hatchling specimens of *Thoriphorybius meli-tense gen. nov.* et *sp. nov.*; specimens mounted in Polyvinil Lactophenol medium; N: number of specimen/structures measured; range: refers to the smallest and the largest structure among all measured specimens; SD: standard deviation.</caption><thead><tr><th rowspan="2">Character</th><th rowspan="2">N</th><th colspan="6">Range</th><th colspan="2">Mean</th><th colspan="2">SD</th></tr><tr><th colspan="3">µm</th><th colspan="3">pt</th><th>µm</th><th>pt</th><th>µm</th><th>pt</th></tr></thead><tbody><tr><td>Body length</td><td>8</td><td>123</td><td>-</td><td>178</td><td>659</td><td>-</td><td>910</td><td>154</td><td>794</td><td>18</td><td>83</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>8</td><td>18.7</td><td>-</td><td>19.8</td><td></td><td>-</td><td></td><td>19.3</td><td>-</td><td>0.4</td><td>-</td></tr><tr><td>Stylet support insertion point</td><td>8</td><td>12.8</td><td>-</td><td>14.3</td><td>67.6</td><td>-</td><td>72.0</td><td>13.6</td><td>70.1</td><td>0.5</td><td>1.6</td></tr><tr><td>Buccal tube external width</td><td>7</td><td>2.4</td><td>-</td><td>3.1</td><td>12.5</td><td>-</td><td>15.8</td><td>2.7</td><td>14.1</td><td>0.3</td><td>1.2</td></tr><tr><td>Buccal tube internal width</td><td>7</td><td>1.6</td><td>-</td><td>2.6</td><td>8.5</td><td>-</td><td>12.9</td><td>2.1</td><td>11.0</td><td>0.4</td><td>1.8</td></tr><tr><td>Ventral lamina length</td><td>8</td><td>7.0</td><td>-</td><td>9.0</td><td>37.5</td><td>-</td><td>45.5</td><td>8.0</td><td>41.4</td><td>0.7</td><td>2.8</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>8</td><td>2.5</td><td>-</td><td>3.2</td><td>12.5</td><td>-</td><td>16.4</td><td>2.7</td><td>13.8</td><td>0.2</td><td>1.2</td></tr><tr><td>Macroplacoid 2</td><td>8</td><td>1.3</td><td>-</td><td>1.8</td><td>7.0</td><td>-</td><td>9.4</td><td>1.6</td><td>8.4</td><td>0.2</td><td>0.8</td></tr><tr><td>Macroplacoid 3</td><td>8</td><td>1.8</td><td>-</td><td>2.5</td><td>9.4</td><td>-</td><td>12.9</td><td>2.2</td><td>11.3</td><td>0.2</td><td>1.1</td></tr><tr><td>Macroplacoid row</td><td>8</td><td>7.0</td><td>-</td><td>8.6</td><td>37.2</td><td>-</td><td>43.7</td><td>7.7</td><td>39.8</td><td>0.5</td><td>2.0</td></tr><tr><td>Claw I heights</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>2</td><td>4.4</td><td>-</td><td>4.7</td><td>22.9</td><td>-</td><td>24.1</td><td>4.6</td><td>23.5</td><td>0.2</td><td>0.9</td></tr><tr><td>External primary branch</td><td>1</td><td>7.2</td><td>-</td><td>7.2</td><td>37.2</td><td>-</td><td>37.2</td><td>7.2</td><td>37.2</td><td>?</td><td>?</td></tr><tr><td>External secondary branch</td><td>4</td><td>5.4</td><td>-</td><td>6.8</td><td>27.6</td><td>-</td><td>35.7</td><td>6.2</td><td>31.8</td><td>0.7</td><td>3.8</td></tr><tr><td>External base/primary branch (cct)</td><td>1</td><td>61.5</td><td>-</td><td>61.5</td><td></td><td>-</td><td></td><td>61.5</td><td>-</td><td>?</td><td>-</td></tr><tr><td>Internal base</td><td>4</td><td>2.9</td><td>-</td><td>4.0</td><td>15.4</td><td>-</td><td>20.8</td><td>3.6</td><td>18.8</td><td>0.5</td><td>2.5</td></tr><tr><td>Internal primary branch</td><td>2</td><td>7.6</td><td>-</td><td>7.8</td><td>39.9</td><td>-</td><td>40.6</td><td>7.7</td><td>40.2</td><td>0.1</td><td>0.5</td></tr><tr><td>Internal secondary branch</td><td>5</td><td>5.3</td><td>-</td><td>6.8</td><td>28.1</td><td>-</td><td>34.4</td><td>5.8</td><td>30.1</td><td>0.6</td><td>2.7</td></tr><tr><td>Internal base/primary branch (cct)</td><td>2</td><td>38.0</td><td>-</td><td>51.7</td><td></td><td>-</td><td></td><td>44.9</td><td>-</td><td>9.7</td><td>-</td></tr><tr><td>Claw II heights</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>2</td><td>4.6</td><td>-</td><td>4.7</td><td>23.9</td><td>-</td><td>24.1</td><td>4.6</td><td>24.0</td><td>0.0</td><td>0.2</td></tr><tr><td>External primary branch</td><td>0</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 secondary branch</td><td>3</td><td>5.6</td><td>-</td><td>6.6</td><td>28.2</td><td>-</td><td>33.7</td><td>6.2</td><td>31.7</td><td>0.5</td><td>3.0</td></tr><tr><td>External base/primary branch (cct)</td><td>0</td><td>-</td><td>?</td><td>-</td><td>-</td><td>-</td><td>-</td><td>?</td><td>-</td><td>?</td><td>-</td></tr><tr><td>Internal base</td><td>3</td><td>4.1</td><td>-</td><td>4.3</td><td>21.4</td><td>-</td><td>21.7</td><td>4.2</td><td>21.5</td><td>0.1</td><td>0.2</td></tr><tr><td>Internal primary branch</td><td>1</td><td>7.5</td><td>-</td><td>7.5</td><td>39.5</td><td>-</td><td>39.5</td><td>7.5</td><td>39.5</td><td>?</td><td>?</td></tr><tr><td>Internal secondary branch</td><td>3</td><td>5.3</td><td>-</td><td>5.9</td><td>27.8</td><td>-</td><td>29.5</td><td>5.6</td><td>28.7</td><td>0.3</td><td>0.9</td></tr><tr><td>Internal base/primary branch (cct)</td><td>1</td><td>54.2</td><td>-</td><td>54.2</td><td></td><td>-</td><td></td><td>54.2</td><td>-</td><td>?</td><td>-</td></tr><tr><td>Claw III heights</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>3</td><td>4.3</td><td>-</td><td>4.9</td><td>21.8</td><td>-</td><td>25.4</td><td>4.6</td><td>23.6</td><td>0.3</td><td>1.8</td></tr><tr><td>External primary branch</td><td>2</td><td>8.1</td><td>-</td><td>9.1</td><td>41.1</td><td>-</td><td>48.5</td><td>8.6</td><td>44.8</td><td>0.7</td><td>5.3</td></tr><tr><td>External secondary branch</td><td>4</td><td>5.8</td><td>-</td><td>6.6</td><td>29.5</td><td>-</td><td>33.9</td><td>6.2</td><td>32.2</td><td>0.3</td><td>1.9</td></tr><tr><td>External base/primary branch (cct)</td><td>1</td><td>4.2</td><td>-</td><td>4.2</td><td></td><td>-</td><td>-</td><td>4.2</td><td>-</td><td>?</td><td>-</td></tr><tr><td>Internal base</td><td>2</td><td>4.1</td><td>-</td><td>4.2</td><td>21.5</td><td>-</td><td>21.7</td><td>4.2</td><td>21.6</td><td>0.1</td><td>0.2</td></tr><tr><td>Internal primary branch</td><td>2</td><td>5.7</td><td>-</td><td>7.1</td><td>28.6</td><td>-</td><td>38.1</td><td>6.4</td><td>33.4</td><td>1.0</td><td>6.7</td></tr><tr><td>Internal secondary branch</td><td>3</td><td>5.0</td><td>-</td><td>5.7</td><td>26.9</td><td>-</td><td>29.1</td><td>5.4</td><td>28.1</td><td>0.3</td><td>1.1</td></tr><tr><td>Internal base/primary branch (cct)</td><td>0</td><td>-</td><td>?</td><td>-</td><td>-</td><td>-</td><td>-</td><td>?</td><td>-</td><td>?</td><td>-</td></tr><tr><td>Claw IV heights</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>Anterior base</td><td>4</td><td>3.8</td><td>-</td><td>4.1</td><td>19.3</td><td>-</td><td>20.9</td><td>4.0</td><td>20.2</td><td>0.1</td><td>0.7</td></tr><tr><td>Anterior primary branch</td><td>3</td><td>7.2</td><td>-</td><td>7.9</td><td>36.4</td><td>-</td><td>41.2</td><td>7.5</td><td>38.8</td><td>0.4</td><td>2.4</td></tr><tr><td>Anterior secondary branch</td><td>5</td><td>5.2</td><td>-</td><td>5.9</td><td>26.7</td><td>-</td><td>30.0</td><td>5.7</td><td>29.0</td><td>0.3</td><td>1.3</td></tr><tr><td>Anterior base/primary branch (cct)</td><td>2</td><td>49.8</td><td>-</td><td>56.4</td><td></td><td>-</td><td>-</td><td>53.1</td><td>-</td><td>4.7</td><td>-</td></tr><tr><td>Posterior base</td><td>3</td><td>3.6</td><td>-</td><td>5.2</td><td>18.5</td><td>-</td><td>26.5</td><td>4.5</td><td>22.8</td><td>0.8</td><td>4.1</td></tr><tr><td>Posterior primary branch</td><td>4</td><td>8.6</td><td>-</td><td>9.8</td><td>45.1</td><td>-</td><td>50.1</td><td>9.4</td><td>48.1</td><td>0.5</td><td>2.2</td></tr><tr><td>Posterior secondary branch</td><td>5</td><td>5.9</td><td>-</td><td>6.8</td><td>31.6</td><td>-</td><td>34.8</td><td>6.5</td><td>33.4</td><td>0.4</td><td>1.4</td></tr><tr><td>Posterior base/primary branch (cct)</td><td>3</td><td>37.5</td><td>-</td><td>55.3</td><td></td><td>-</td><td>-</td><td>46.5</td><td>-</td><td>8.9</td><td>-</td></tr></tbody></table>
and wrinkles, which trap a significant amount of sediment (Fig. 8).

**Eggs.** Laid in the exuvia, with smooth chorion. Whitish/ transparent in colour, round in shape and in number of 4–8 per exuvia.

**Additional observations.** Both sexes (see File S6 for males stained with orcein) are reported for this species, with no evident secondary sexual dimorphism. Multiple instances of a behaviour in which a single female is surrounded by 2–5 males that anchor themselves to her body using their mouth have been observed.

**DNA.** The following DNA sequences are associated with the type population of the new species: **18S**: PV016871–2. — **28S**: PV016873–4. — **COI**: PV017087–8. — **ITS2**: PV019027–8. — **Mitogenome**: PV030524. — **Genomic reads**: Bioproject PRJNA1215272.

**4. Discussion and conclusion**

This study allowed for the identification of a new genus and species of tardigrade from superfamily Isohypsibioidea, which due to its unique combination of morphological characters and basal phylogenetic position in Isohypsibioidea that, if confirmed, may provide new insights into evolutionary hypotheses regarding limno-terrestrial tardigrades.

The new genus exhibits traits which for their evolutionary significance or peculiar morphological should be discussed: The presence of a ventral strengthening structure in the buccal apparatus (ventral lamina) is considered a constant character at generic level (Pilato and Binda, 2010a), but it shows variability at higher taxonomic levels: whereas it is present in all Macrobiotoidea Thulin, 1928 {in Marley et al. 2011} (Bertolani et al. 2014b), its presence in Isohypsibioidea is limited to some unrelated generation (Gąsiorek et al. 2019b). The fact that Macrobiotoidea and Isohypsibioidea are not closely related (Bertolani et al. 2014b), lead Gąsiorek et al. (2019b) to hypothesize that the ventral lamina is an example of parallel evolution. The situation inside Isohypsibioidea is however more complex, with four unrelated lineages having it (Fig. 1). Gąsiorek et al. (2019b) hypothesized that the ventral lamina was present in the ancestor of Hexapodibiidae + Doryphoribiidae, and was then lost in *Thulinius*, *Pseudobiotus* and *Grevenius*. If the basal position of *Thulyphoribius gen. nov.* among Isohypsibioidea is confirmed, it would provide an even more complex scenario involving an acquisition of ventral lamina in the Isohypsibioidea ancestor followed by multiple losses, or by multiple independent evolutions and losses of this character inside Isohypsibioidea. The ventral lamina observed in *Thulyphoribius gen. nov.* is shorter, more protruding, and displays a series of incisions never reported for the ventral lamina of *Doryphoribius* or Hexapodibiidae (Fig. 3A). Those differences could pro-

vide a hint that the ventral lamina of *Thulyphoribius gen. nov.* represent an independent evolution of this structure compared to the ventral lamina in *Doryphoribius* + Hexapodibiidae (where it was then lost in some lineages).

Around the buccal cone, peribuccal papular lobes visible only under SEM are present in the new genus (Fig. 3C). Up to now, those structures were only observed in *Calohypsibius* Thulin, 1928 (Gąsiorek et al. 2019a), and this new finding of those structures in an unrelated taxon open to the possibility that similar structures may be present in other eutardigrade genera, but could have been overlooked due to insufficient investigation; an example of similar structures (button-like structures on the buccal cone), which warrants further study is shown in the genus *Ursulinius* Gąsiorek, Stec, Morek & Michalczyk, 2019 (Figure 5B in Gąsiorek et al. 2019b).

"Button-like cuticular folds" are present on the frontal surface of legs I–III (Fig. 7 B–D), and they are visible only under SEM; these structures could potentially be present in other genera but may have not been reported, considering that they are only detectable using SEM. Cuticular structures on the legs (other than claws) are present in some xerophilous tardigrades (Gąsiorek et al. 2019b; Camarda et al. 2024a; Stec, 2024; Vecchi et al. 2022b) where they are hypothesized to aid in the locomotion by providing more adherence on the substrate, and they also present in non-xerophilous species (Stec and Morek, 2022). However the new species has been found in a mostly aquatic habitat, so probably they are not aiding in locomotion. These "button-like cuticular folds" in *Thulyphoribius gen. nov.* could instead be related to mating to increase male adherence on female bodies, as males have been observed to cling on females and structures used by males tardigrades to cling to females are already known to exist (Rebecchi and Nelson, 1998).

Internal septa at the base of claws I–IV (Fig. 5C), detectable only under light microscopy are present. It is also important to note that, in the new species, the septa in the claw bases are difficult to observe under light microscopy in specimens mounted in PVLF medium. Therefore, Isohypsibioidea species without indented claw bases (and lacking lunulae/pseudolunulae) that have been studied exclusively using PVLF mounting medium should be re-investigated using alternative mounting media to determine whether internal septa are really absent.

The genetic analyses clearly indicated the distinctiveness of the new genus supporting its validity. *Thulyphoribius gen. nov.* has been recovered as a sister lineage of all the other sequenced Isohypsibioidea indicating the distinctiveness that could be potentially elevated to the family level. However, due to absence of clear traits differentiating the new genus compared to Doryphoribiidae, and a general lack of strongly distinctive traits separating Isohypsibioidea families, we preferred for the moment a more conservative and cautious choice, waiting for more data (e.g., more than one species to be analysed with more morphological and genetic traits) before instituting a new family. The mitogenome sequence of the new species shows an almost perfect synteny with other Isohypsibioidea taxa and notably with the distantly related *Ram*.
*claudii*, indicating a general conserver order of mitochondrial genes in Parachela. Due to the low number of available tardigrades mitogenomes, we could not leverage the newly generated one to produce a whole-mitogenome based phylogeny and confirm the unique phylogenetic position of the new species.

*Thulophoribius melitense n.sp.* has been found in freshwater rock pools, which have been only recently explored in terms of tardigrade diversity that might be present in this ephemeral habitat (Troell and Jönsson 2023; Vecchi et al. 2022a; Vecchi et al. 2023a; Vecchi and Stec 2024). Rock pools often support unique and specialized organisms adapted to withstand challenging environmental conditions, such as desiccation and temperature fluctuations (Jocque et al. 2010). Despite their transient nature, freshwater rock pools act as biodiversity hotspots, harbouring even endemic species (Brendonck et al. 2016; Jocque et al. 2010), and the finding of a new tardigrade genus in this environment confirms its potential of undiscovered biodiversity.

## 5. Acknowledgments

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].

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# Supplementary Material 1

## Files S1–S6

**Authors:** Camarda D, Lisi O, Stec D, Vecchi M (2025)

**Data type:** .zip

**Explanation notes:** File S1. Raw morphometric measurements – juveniles and adults. — File S2. Raw morphometric measurements – newborns. — File S3. GenBank sequences used in phylogenetic reconstructions. — File S4. mrbayes input with alignment used for phylogenetic reconstructions (.nexus file). — File S5. iqtree and mrbayes output trees (.nmk file). — File S6. Photographs of males stained with Orcein.

**Copyright notice:** This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

**Link:** https://doi.org/10.3897/asp.83.e150460.suppl1