An integrative description of Mesobiotus mandalori sp. nov. (Eutardigrada, Macrobiotoidea) from Poland

Abstract In this paper we provide integrative description (morphological and genetic) of the new eutardigrade species Mesobiotus mandalori sp. nov. from central Poland, found during research on the vertical distribution of Tardigrada in the Czerniejewskie Forests. Mesobiotus mandalori sp. nov. belongs to the harmsworthi group, but it differs from the members of this group in some morphometric characters of the bucco-pharyngeal apparatus, claws and/or eggs. Morphologically, Meb. mandalori sp. nov. is most similar to Meb. pseudocoronatus, but it differs from it by a larger bare egg diameter and smaller number of egg processes on egg circumference. Morphometrical data were supported by results of comparative genetic analysis of four molecular markers: 18S rRNA, 28S rRNA, ITS2 and COI. According to COI the new species is most similar to Meb. skorackii with the genetic distance of 21.1%.https://zoobank.org/urn:lsid:zoobank.org:act:0222E328-CF9E-4D04-8F8C-69C80515FEE2


Introduction
Tardigrada is a phylum of microscopic multicellular animals widely distributed in most aquatic and terrestrial ecosystems throughout the world (Nelson et al. 2015).Currently, approximately 1400 species and subspecies of tardigrades have been described (Degma & Guidetti 2009-2024).However, it is very likely that the actual number of taxa is much higher (Bartels et al. 2016).
The tardigrade fauna of Poland has been studied since the early of twentieth century (Jakubski 1915), and so far, 114 species have been reported from this area.However, most of the species were identified many years ago and the current state of knowledge on Polish tardigrades is far from complete (see discussion below).
During research on the vertical distribution of Tardigrada on trees in the complex of Czerniejewskie Forests in central Poland (30 km E of Poznań, near Kostrzyn Wielkopolski), the results of which will soon be published in a separate paper, a new species of the genus Mesobiotus (Vecchi et al. 2016b) was found in a few of collected samples.
Currently, in the genus Mesobiotus, 75 species (76 with newly described one) have been described which makes it the second richest genus in the family Macrobiotidae (Degma & Guidetti 2009-2024).What is more, this richness is still growing very fast: only since the beginning of 2020, nine new species from all around the world have been described (Degma & Guidetti 2009-2024).The genus Mesobiotus is morphologically divided into two main species groups that differ in egg shell morphology: i) the Meb.harmsworthi group, characterized by eggs with conical, or hemispherical, reticulated processes; ii) the Meb.furciger group which is characterized by branched egg processes (see e.g.Degma & Guidetti 2009-2024;Kaczmarek et al. 2020a).
In this paper we provide description of the new Mesobiotus species (harmsworthi group) prepared by means of an integrative taxonomy approach combining morphological, morphometric and genetic data.

Study area, sampling and specimens extraction
Samples were collected in the Czerniejewskie Forests near Wagowo in the Wielkopolska Province (52°26′N, 17°20′E).The Czerniejewskie Forests are a small complex of managed forests with an area of approx.15 km 2 .The character of the forests is mixed: mostly pines (Pinus silvestris) with a great admixture of deciduous trees, such as the Quercus robur, Q. petraea, Betula pendula, Populus nigra and Populus tremula, and is representative of the typical mixed coniferous forests of west and central Poland.
Moss samples were collected in autumn from an entire tree trunk of Q. robur (samples were taken from the base of the tree to the crown, at intervals of 50 cm) height of a trunk of examined tree reached 10 m.Then, samples were packed into paper envelopes and labelled with the number of the tree and a numerical designation of the height.Later, they were delivered to the laboratory at the Faculty of Biology, Adam Mickiewicz University in Poznań, where they were slowly air-dried.
All samples were examined according to standard methods (Stec et al. 2016) with minor modifications for ecological studies.A cut-off piece of a sample (no bigger than 0.25 g) was weighed and placed in a beaker filled with 200 ml of H 2 O for eight hours.Afterwards, the moss was vigorously stirred within the beaker with the use of a glass rod.The supernatant (containing tardigrades, their eggs, and other animals inhabiting the moss alongside moss particles) was transferred into a 250 ml cylinder for 30 minutes, to allow all the particles to fall to the bottom of the cylinder.Then, the top 120 ml of water was discarded and the remaining 80 ml was stirred and poured onto 10 cm Ø glass Petri dishes.Tardigrades and their eggs were extracted using an Olympus SZ61 stereomicroscope (Olympus Corporation, Shinjuku-ku, Japan).All the remaining sediments were placed on four layers of paper towel, where they were left for five days for drying.
The dried material was weighed and packed into envelopes.
A total of 50 animals and 22 eggs of the new species were extracted from the samples CF.A 2.20 (CF.A -Czerniejewskie Forests Autumn) at a height of 9.5 m (40 animals and 22 eggs), CF.A 2.19 at a height of 9.0 m (four specimens) and CF.A 2.18 at a height of 8.5 m (six specimens) from tree (52°25′ 29.46″N, 17°21′16.22″E).Of these, 39 specimens and 20 eggs were fixed on microscope slides in Hoyer's medium, one specimen and two eggs were prepared for Scanning Electron Microscopy (SEM) analysis, and the remaining 10 individuals were used for DNA extraction and sequencing.Animals used for SEM and molecular analyses were not included in the type series.

Microscopy and imaging
Fixed tardigrades and their eggs were examined under Phase Contrast Microscopy (PCM) (Olympus BX41 associated with an Olympus SC50 digital camera, Olympus Corporation, Shinjuku-ku, Japan).Specimens (animals and eggs) for SEM imagining were prepared according to the protocol described in Roszkowska et al. (2018) and examined under a high vacuum in a Hitachi S3000N Scanning Electron Microscope (Olympus Corporation, Shinjuku-ku, Japan).
All the figures were assembled in Corel Photo-Paint X6.For deep structures that could not be fully focused in a single photograph, a series of 2-8 images were taken every approximately 0.5 µm and then manually assembled into a single deep-focus image in Corel Photo-Paint X6.

Morphometry and morphological nomenclature
The sample size for morphometrics was chosen according to Stec et al. (2016).All measurements are given in micrometres (µm).All structures were measured only if their orientation was appropriate.The pt index used is the ratio of the length of a structure to the length of the buccal tube, expressed as a percentage (Pilato 1981).Buccal apparatus type and claws were classified according to both Pilato and Binda (2010) and Vecchi et al. (2016b), while terminology used to describe the oral cavity armature (OCA) follows Pilato (1972) with modifications described in Michalczyk and Kaczmarek (2003).Body length was measured from the anterior extremity and the posterior end of the body, excluding the IV pair of legs.Measurements of the buccal apparatus, claws and eggs were based on recommendations by Kaczmarek and Michalczyk (2017).The An integrative description of Mesobiotus mandalori sp.nov.macroplacoid length sequence is presented according to Kaczmarek et al. (2014).The egg-shell morphology follows Kaczmarek et al. (2020a).Nomenclature of the cuticular bars on legs follow Kiosya et al. (2021).
All morphometric data were processed with the usage of the template "Parachela" ver.1.8, available from the Tardigrada Register (Michalczyk & Kaczmarek 2013) updated with the Thorpe's normalization of the data (as in Massa et al. 2021 according to Bartels et al. 2011) (SM.1).

Comparative genetic analysis
Before genomic DNA extraction, each specimen was identified in vivo using light microscopy.The DNA extraction was made from specimens using the Chelex®100 resin (Bio-Rad) extraction method (Casquet et al. 2012).Additionally, we applied modification in order to obtain tardigrade exoskeletons (Stec et al. 2020).The exoskeletons were removed from the obtained extract and were examined under stereomicroscope.Then, exoskeletons were sent back to the Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Poznań.
We sequenced four molecular markers, which differ in effective mutation rates: three nuclear fragments, i.e., 18S rRNA, 28S rRNA and ITS2 as well as one mitochondrial fragment, i.e., COI gene sequences.All applied primers have been listed in Table I.The 18S rRNA, 28S rRNA and COI gene fragments were amplified according to the protocols described in Kaczmarek et al. 2020b.In turn, the ITS2 sequences were amplified according to the protocol provided by Stec et al. 2018.Exonuclease I (20 U/μl, Thermo Scientific, Vilnius, Lithuania) and alkaline phosphatase FastAP (1 U/μl, Thermo Scientific, Vilnius, Lithuania) were applied to clean the PCR products.Sequencing in both directions was performed using the BigDyeTM terminator cycle sequencing method and ABI Prism 3130×l genetic analyser (Life Technologies).
All obtained sequences have been deposited in GenBank (for the accession numbers please see subsection "DNA sequences").
Obtained nuclear and mitochondrial DNA molecular markers were checked for quality and consensus sequences were created in BioEdit v. 7.2.5 (Hall 1999).Then, the Basic Local Alignment Search Tool (the BLAST; Altschul et al. (1990)) search at NCBI was used to verify the homology of the obtained nuclear sequences with molecular markers deposited in the GenBank database.The COI haplotypes were generated using DnaSP v5.10.01 program (Librado & Rozas 2009) and were translated into amino acid sequences using the EMBOSS-TRANSEQ application (Rice et al. 2000;Goujon et al. 2010) to check for indels, pseudogenes and internal stop codons.
Genetic comparisons between nuclear sequences obtained by us and molecular markers available in GenBank of the genus Mesobiotus were used to supplement phenotypic description of the new species.Single sequence of molecular markers representing each Mesobiotus species were downloaded from the database.All sequences obtained in our study, and sequences downloaded from GenBank database as originated from Mesobiotus, were aligned with ClustalW Multiple Alignment tool (Thompson et al. 1994), implemented in BioEdit v. 7.2.5, using default settings.Overall, aligned sequences were trimmed to 707 bp (20 species), 708 bp (14 species), 264 bp (14 species) and 568 bp (19 species) for 18S rRNA, 28S rRNA, ITS-2 and COI molecular markers, respectively.Downloaded nucleotide sequences which were too short after genetic comparisons or represented different fragments of DNA markers were not applied and as a result not all species belonging to the genus Mesobiotus were represented in all data sets.Mega X (Kumar et al. 2018) was applied to calculate the uncorrected genetic distances (p-distance) for each DNA fragment.Genetic distances were computed between species of the Mesobiotus species and the

Species identification
Identification of the new species was carried out according to the key to the Mesobiotus species and other original papers (Pilato et al. 2010;Tumanov 2018Tumanov , 2020;;Kaczmarek et al. 2020a;Stec 2021).

Mesobiotus mandalori sp. nov. (Figures 3-7, Tables II-III)
Only a small number of the new species character lengths were found to be non-isometric with regard to the buccal tube length following Thorpe's normalization (SM.1).The range of pt indexes (min-max) for these characters did not differ significantly between the ranges estimated on the non-normalized data and the Thorpe's normalized data (SM.1).Furthermore, results for simple linear regression analysis performed for measurements and pt value using GraphPad Prism 8.0 are presented in Tables II and III  For measured value all of the characters showed significance with p value < 0.0001, however, only SSIPpt showed significance with p value < 0.05.
Etymology.The name mandalori alludes to the fictional planet of Mandalore (home world of Mandalorians), from the Star Wars Universe.The first ever visual depiction of Mandalore showed this world as being covered by dense forests, and its inhabitants living in villages hidden in the tree canopies.This reflects the type locality of the new species, i.e. moss on trees in deep forest.
Material examined.In total 72 specimens (animals and eggs) were examined.The 39 animals and 20 eggs were mounted on microscope slides in Hoyer's medium, one animal and two eggs were fixed on SEM stubs and 10 animals were processed for DNA sequencing.Description of the new species.Animals (all measurements and statistics in Table IV).Live specimens white, but transparent after fixation in Hoyer's medium (Figure 3a).Eyes present in all examined specimens    An integrative description of Mesobiotus mandalori sp.nov.
(Figure 3a).Body cuticle smooth and without pores (Figure 3b).Granulation on legs I-IV present and clearly visible in PCM (Figure 4c).Claws of the Mesobiotus type with minute stalk, distinct distal part of the basal portion and short common tract (Figures 4a-c).Primary and secondary branches diverge at a point near half the claw height.All primary branches with accessory points (Figures 4a-b and d).The claws of the fourth pair of legs slightly longer than others (Figures 4c-d Bucco-pharyngeal apparatus of Mesobiotus type (Figures 5a-b) with ventral lamina and 10 peribuccal lamellae (Figure 6a).Oral cavity armature (OCA) of the harmsworthi type, with three bands of teeth visible in PCM (Figures 6a-b).First (anterior) band of small teeth, in form of granules, clearly visible in PCM (Figures 6a-b, filled unindented arrowheads), a second (posterior) band of teeth in the form of a row composed of comma-shaped ridges, parallel to the main axis of the buccal tube (Figures 6a-b, empty unindented arrowheads); and finally the third band in the form of a system of three dorsal and three ventral transverse  6a).Lateral ventral ridges are curved, longer and thicker than the middle one, but thin toward the external endings, while the middle ventral ridge is shorter and roundish, and has the form of a singular tooth (Figure 6b).Pharyngeal bulb with apophyses, three rod-shaped macroplacoids and a large microplacoid located very closely to the last macroplacoid (Figures 3a, 5 a-b and 6c-d).Macroplacoid length sequence 1 > 3 > 2. First and second macroplacoid without constrictions and protrusions.Third macroplacoid with shallow, distinct subterminal constriction, but without terminal protrusion (Figures 6c-d, filled arrow).
Eggs (all measurements and statistics in Table V).Spherical, white and laid freely.Egg chorion with processes in the shape of wide sharp cones (Figures 7a-b, e-j) with thick, but flexible, shorter or longer endings (often broken) (Figures 7e-j).In some processes, its apical parts bifurcated or divided into even more short filaments (Figures 7h-j).In apical parts of some egg processes, small bubble-like internal structures are clearly visible in PCM (Figures 7g and i).Processes with two-layer walls (Figures 7g-j) with a net of trabecular structures between both layers, forming reticular design, clearly visible in PCM (Figures 7c and h).In SEM, the surface of processes smooth or rarely with small pores on its bases (Figures 7c-e).Additionally, reticular design is sometimes visible under the smooth walls (Figure 7e).Egg processes bases surrounded by a crown of thickening (Figures 7a-f).Egg shell covered with small granules (in PCM), which in SEM are visible as small pores and wrinkles (Figures 7d-f).

Differential diagnosis
Mesobiotus mandalori sp.nov.belongs to the harmsworthi group (according to Kaczmarek et al. 2011Kaczmarek et al. , 2018) ) with eggs with conical and reticulated processes with nonbranching apical parts and processes bases surrounded by the crown of dot-like structures or radial ridges.The new species is similar in those features to 19 taxa, however the new species differs from: 1. Mesobiotus binieki (Kaczmarek et al. 2011) (known only from the type locality in Bulgaria) An integrative description of Mesobiotus mandalori sp.nov.(Binda & Pilato 1987) (known from Italy, Seychelles Tunisia (type locality) and New Zealand (Binda & Pilato 1995;Pilato & Binda 1996)) by the presence of eyes, absence of long flexible filaments on the top of egg processes, absence of the system of radial ridges on egg shell and the smaller number of egg processes on egg circumference (10-12 in the new species vs 15-23 in Meb.diffuses).4. Mesobiotus dimentmani (Pilato et al. 2010) (known only from the type locality in Israel) by lower pt of the buccal tube external width (11.8-14.5 (Pilato et al. 2000) (known from the Aeolian Islands and several other islands in the Tyrrhenian Sea, Italy (Pilato et al. 2000)) by the presence of eyes, presence of indented lunules on IV pairs of legs, lower pt of the buccal tube external width (11.8-14.5 in the new species vs 17.9-21.1 in Meb.patiens).The comparison is based on morphometric data provided by Pilato et al. (2000) and Pilato and Lisi (2009).11.Mesobiotus perfidus (Pilato & Lisi 2009) (known only from the type locality on Seychelles Islands) by the presence of the first band of teeth in the OCA, presence of granulation on legs I-III and indented lunules on IV pairs of legs and having shorter apical parts of the processes i.e. much shorter than conical basal parts (long spiniform apical parts, longer than conical basal parts in Meb.perfidus).12. Mesobiotus philippinicus Mapalo et al. 2016 (known (Pilato et al. 2000) (known from Sardinia (type locality), a few other localities in Italy, and from Israel (Pilato et al. 2000(Pilato et al. , 2010) ) by having a lower pt of external width of the buccal tube (11.8-14.5 in the new species vs 17.5-29.1 in Meb.simulans), and having apical parts of the egg processes bifurcated or divided into even more short filaments.The comparison is based on the Meb.simulans description by Pilato et al. (2000), and morphometric data provided by Pilato and Lisi (2009).19.Mesobiotus wuzhishanensis (Yin et al. 2011)
The genus Mesobiotus is a relatively new taxon that was excluded from the genus Macrobiotus in 2016 based on molecular and morphological data (Vecchi et al. 2016b) and initially consisted only of 55 species previously assigned to the genus Macrobiotus (Vecchi et al. 2016b;Degma & Guidetti 2009-2024).Four other species of this genus were described already in 2016 (including one described by Vecchi et al. (2016b); Degma & Guidetti 2009-2024), followed by more.Up to end of 2022 17 new species of genus Mesobiotus were discovered (Degma & Guidetti 2009-2024;Kaczmarek et al. 2018;Tumanov 2018Tumanov , 2020;;Tumanov & Pilato 2019;Stec 2019Stec , 2021Stec , 2022)).But studies on this genus did not end on description of new species.Recently the type species of the genus of Meb.harmsworthi (Murray 1907) was redescribed (Kaczmarek et al. 2018).Moreover, as was mentioned in the introduction, the genus Mesobiotus is morphologically divided into two main species groups Meb.harmsworthi group, and the Meb.furciger (see e.g.Kaczmarek et al. 2020a).It was recently shown that those two groups are not monophyletic and that their existence has no support in molecular phylogenetic data (Stec 2021;Stec et al. 2021;Short et al. 2022).As a result, they should be referred to as "morphogroups" only (Short et al. 2022) which are not taxonomic ranks.However, some tardigradologists began to wonder whether using this terminology is justified.Nevertheless, this term is often used in systematics papers, mostly because of its utility in describing newly found species (see e.g.Stec 2021;Short et al. 2022) and in this context it is used in this work.
As stated previously, to date 75 species have been described in Mesobiotus genus (Degma & Guidetti 2009-2024) (11 species in Meb.furciger group and 28 species in Meb.harmsworthi group), which makes around 5.1% of all known tardigrade species (Degma & Guidetti 2009-2024).Among all these species only Meb. harmsworthi (besides the newly described one) was reported from territory of Poland (Dastych 1980(Dastych , 1988) ) (also found in Wielkopolska Province (Kaczmarek & Michalczyk 2003)).However, these records were published before the formal redescription of Meb.harmsworthi and the presence of this species in Poland is doubtful.
As mentioned above, the current state of knowledge of both the tardigrade fauna of Poland and the diversity of the Mesobiotus genus is far from complete, and faunistic research in these areas could still provide new discoveries.
). Anterior (internal) and posterior (external) claws of legs IV similar in shape, however posterior claws slightly longer than anterior ones.Lunules under claws I-III smooth and slightly dentate on under claws IV (Figures 4a-b, filled unindented arrowheads; 4D, filled unindented arrowheads).Thin singular cuticular bars (Figures 4a-b, empty unindented arrowheads) and double muscle attachments under claws I-III present (Figures 4a-b, filled arrows).In addition, horse-shoe-shaped structures connecting anterior and posterior claws of IV pair of legs also present (Figure 4c, filled indented arrowhead).

Figure 4 .
Figure 4. Mesobiotus mandalori sp.nov.-PCM images of claws I, III and IV: (a) -Claw I with smooth lunules (paratype); (b) -Claw III with smooth lunules (paratype); (c) -Claw IV with dented lunules (holotype).(d) -Lunules under claws IV with dented lunules (paratype).Filled flat arrowhead indicates lunules; filled indented arrowheads indicate the granulation; empty indented arrowheads indicate the indicates horseshoe structure connecting the anterior and the posterior claws; empty arrow indicates a single continuous cuticular bar under the claws; filled arrow indicates paired muscle attachments Scale bars in μm.

Figure 6 .
Figure 6.Mesobiotus mandalori sp.nov.-PCM images of oral cavity armature (OCA) and placoid morphology: (a) -dorsal view of OCA (paratype); (b) -ventral view of OCA (paratype); (c) -dorsal view of placoid morphology; (d) -ventral view of placoid morphology.Filled flat arrowheads indicate peribuccal lamellae; empty flat arrowheads indicate the first band of teeth; filled indented arrowheads indicate the second band of teeth; filled indented arrowheads indicate the third band of teeth, filled arrows indicate the middle ridge dorsal and ventral respectively; empty arrows indicate subterminal constrictions in the third macroplacoid.Scale bars in μm.

Figure 7 .
Figure 7. Mesobiotus mandalori sp.nov.-PCM and SEM images of the eggs, egg surface, egg processes surface and midsections: (a) PCM image of the eggs; (b) SEM image of the egg; (c) PCM image of the egg surface; (d) SEM image of the egg surface; (e-f) SEM images of the egg processes surface; (g-j) PCM images of the egg processes midsections.Filled flat arrowhead indicates reticular design; filled indented arrowheads indicate crowns of strong thickenings around the process base; empty indented arrowheads indicate pores;filled arrow indicates bubble-like internal structures, Filled flat arrowheads indicate reticular design; filled arrows crowns of strong thickenings around the process base; empty arrows indicate pores.Scale bars in μm.

Table I .
Primers with their original references used for sequencing of three molecular markers of Mesobiotus mandalori sp.nov.
as well as Figures 1 and 2 (SSIP represents Stylet support insertion point, BTEW represents Buccal Tube External width, BTIW represents Buccal Tube Internal Width, VLL represents Ventral Lamina Length, M1 represents Macroplacoid 1 length, M2 represents Macroplacoid 2 length, M3 represents Macroplacoid 3 length, Mi represents Microplacoid length, MR represents Macroplacoid Row length, PR represents Placoid Row length m represents measured value in µm and pt represents pt value).

Table IV .
Measurements [in µm]and pt values of selected morphological structures of Mesobiotus.mandalori sp.nov.Table contains measurements [in µm] and pt values of selected morphological structures of the newly described species."N" stands for the number of specimens/structures measured; "RANGE" refers to measurements taken for the smallest and the largest structure among all measured specimens; SD stands for standard deviation.
ridges (Figures6a-b, indented arrowheads).Dorsal ridges similar in length and equally thick, however the lateral ones are thinning toward the external endings (Figure

Table V .
Measurements [in µm] of selected morphological structures of eggs of Mesobiotus mandalori sp.nov.Table contains measurements [in µm] structures of eggs of newly described species.Where "N" stands for the number of specimens/structures measured; "RANGE" refers to measurements taken for the smallest and the largest structure among all measured specimens; SD stands for standard deviation.
by generally shorter claws (see Table II in the present paper and Table III in Yin et al.