Integrative description of two new species of the genus Mesobiotus (Eutardigrada, Macrobiotoidea) from Russia, with an updated phylogeny of the genus

. In this study, we describe two new species of Mesobiotus based on morphological data collected through light and scanning electron microscopy. Descriptions include DNA sequences of four commonly used molecular markers (18S rDNA, 28S rDNA, ITS-2, and COI). Mesobiotus efa sp. nov. was discovered in North-West Russia and belongs to the group of species with smooth cuticle, harmsworthi -type OCA, typical Mesobiotus claws IV with unindented lunules, and egg chorion with reticulated processes in form of ‘sharp wide cones’ or ‘cones with long slender endings’, egg process bases with well-developed crone of dark thickenings without ﬁ nger-like projections, and egg shell surface between the processes with ridges without reticulation, areolation or semi-areolation. It can be distinguished from all know species of this group by a unique combination of morphological and morphometric characters. Mesobiotus vulpinus sp. nov. was found in the Russian Far East, and is similar to Mesobiotus mauccii by having an egg chorion with polygonal relief. The new species can be distinguished from M. mauccii by having a narrower buccal tube, by details of oral cavity armature, and by longer egg chorion processes. Furthermore, we provide results of the phylogenetic analyses of the genus Mesobiotus conducted in this study.


Introduction
Tardigrades are a group of microscopic segmented animals widely distributed in the nature (Nelson 2018).Despite its strictly aquatic lifestyle this group successfully invaded terrestrial ecotopes being associated with habitats that periodically contains liquid water -moss cushions, lichens, soil, and leaf debris (Nelson 2018).Semiterrestrial tardigrades (tardigrades that live in terrestrial habitats subjected to periodic desiccation) comprise most of the species diversity of this group.
The genus Mesobiotus with 76 currently described species (Degma & Guidetti 2023;Vecchi et al. 2023) is a second large genus within Macrobiotidae.Modern integrative redescription of its type species Mesobiotus harmsworthi (Murray, 1907) given by Kaczmarek et al. (2018) together with the revisions of the genus morphology and phylogeny (Kaczmarek et al. 2020;Stec 2022) provided a strong base for the description of new species of Mesobiotus.
Fauna of semiterrestrial tardigrades of Russia is poorly investigated (see Tumanov et al. 2022).All records of species of Mesobiotus for this territory originates from the publications that precede the modern revision of the Macrobiotidae taxonomy and should be considered dubious except for Mesobiotus montanus (Murray, 1910) noted for several regions of northern Russia (Biserov 1991(Biserov , 1996)), and Mesobiotus altitudinalis (Biserov, 1997(Biserov, -1998) ) described from North Ossetia.Biserov (1991) noted Mesobiotus furciger (Murray, 1907, as Macrobiotus) for Udmurtia.In our opinion, this record should be attributed as belonging to the unknown species of the polyphyletic Mesobiotus furciger morphogroup (according to Stec 2022).Numerous records of M. harmsworthi, M. harmsworthi coronatus, and M. harmsworthi obscurus are not valid due to changes in Mesobiotus taxonomy that have taken place since their publication (Pilato et al. 2000;Kaczmarek et al. 2018Kaczmarek et al. , 2020;;Stec 2022).
In this paper, we describe two new species of Mesobiotus which have been found during the investigation of the tardigrade fauna of Russia.The detailed morphological description is supplemented by DNA sequences of four standard genes used in tardigrade taxonomy and phylogenetics (the nuclear 18S rRNA, 28S rRNA, ITS-2, and the mitochondrial COI).We also performed a multigene phylogenetic analysis in order to determine the position of new species on the Mesobiotus phylogenetic tree and to reconstruct an updated phylogeny of the genus.

Sampling
The moss samples were collected in the vicinity of the cities of St Petersburg and Vladivostok.Material was stored within paper envelopes at room temperature.Tardigrade specimens were extracted from rehydrated samples using the standard technique of washing them through two sieves (fi rst with ≈ 1 mm mesh size and second with 29 μm mesh size; Tumanov 2018a ).The contents of the fi ner sieve were examined under a Leica M205C stereo microscope.

Microscopy and imaging
Tardigrades found were fi xed with acetic acid or relaxed by incubating live individuals at 60°C for 30 min (Morek et al. 2016) and mounted on slides in Hoyer's medium.Permanent slides were examined under a Leica DM2500 microscope equipped with phase contrast (PhC) and differential interference contrast (DIC).Photographs were taken using a Nikon DS-Fi3 digital camera with NIS software.

Morphometrics and terminology
The sample size for morphometrics was chosen following the recommendations of Stec et al. (2016).Structures were measured only if their orientations were suitable.Body length was measured from the anterior end of the body to the posterior end, excluding the hind legs.The buccal tube was measured from the dorsal crests of the oral cavity armature (OCA) to the caudal end of the buccal tube, not including the buccal apophyses.Terminology for the structures within the bucco-pharyngeal apparatus and for the claws follows those of Michalczyk & Kaczmarek (2003) and Pilato & Binda (2010).Elements of the buccal apparatus, claws and eggs were measured according to Kaczmarek & Michalczyk (2017).The macroplacoid length sequence is given according to Kaczmarek et al. (2014).Cuticular structures under claws on legs I-III are described according to Kiosya et al. (2021).All measurements are given in micrometres (μm).The pt index used is the percentage ratio between the length of a structure and the length of the buccal tube (Pilato 1981), and is presented here in italics.Morphometric data were handled using ver.1.6 of the "Parachela" template, which is available from the Tardigrada Register (Michalczyk & Kaczmarek 2013).

Genotyping
DNA was extracted from individual specimens using QuickExtract ™ DNA Extraction Solution (Lucigen Corporation, USA; see description of complete protocol in Tumanov 2020b).Preserved exoskeletons were recovered, mounted on a microscope slide in Hoyer's medium and retained as the hologenophore (Pleijel et al. 2008).
Four genes were sequenced: a small ribosome subunit (18S rRNA) gene, a large ribosome subunit (28S rRNA) gene, internal transcribed spacer (ITS-2), and the cytochrome oxidase subunit I (COI) gene.PCR reactions included 5 μl template DNA, 1 μl of each primer, 1 μl DNTP, 5 μl Taq Buffer (10×) (−Mg), 4 μl 25 mM MgCl 2 and 0.2 μl Taq DNA Polymerase (Thermo Scientifi c ™ ) in a fi nal volume of 50 μl.The primers and PCR programs used are listed in electronic supplementary material (see Supp. fi le 1).The PCR products were visualised in 1.5% agarose gel stained with ethidium bromide.All amplicons were sequenced directly using the ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the help of an ABI Prism 310 Genetic Analyzer in the Core Facilities Center "Centre for Molecular and Cell Technologies" of St Petersburg State University.Sequences were edited and assembled using ChromasPro software (Technelysium, USA).The COI sequences were translated to amino acids using the invertebrate mitochondrial code, MEGA11 (Tamura et al. 2021), in order to check for the presence of stop codons and therefore of pseudogenes.Uncorrected pairwise distances were calculated using MEGA11 with gaps/missing data treatment set to "pairwise deletion".All obtained sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/-accession numbers available in the species descriptions).

Phylogenetic analyses
Sequences of 18S, 28S, ITS-2, and COI markers representing all species of Mesobiotus for which at least two of the abovementioned markers were available in GenBank at the time of the analysis were downloaded.Sequences of appropriate length that were homologous to the sequences obtained and originated from publications with a reliable attribution of the investigated taxa were selected, with addition of the newly obtained sequences (Table 1).Richtersius coronifer (Richters, 1903) (Macrobiotoidea, Richtersiusidae) was used as an outgroup.
Sequences were automatically aligned with the MAFFT algorithm (Katoh et al. 2002) with the software AliView ver.1.27 (Larsson 2014); the alignments were cropped to a length of 983 bp for 18S, 770 bp for 28S, 566 bp for ITS-2, and 657 bp for COI.Sequences of all genes were concatenated using SeaView ver.4.0 (Gouy et al. 2010) (fi nal alignment presented in Supp.fi le 2).Maximum-likelihood (ML) topologies were constructed using IQ-TREE software multicore ver.1.6.12(Kalyaanamoorthy et al. 2017;Minh et al. 2020).The best substitution model and partitioning scheme for posterior phylogenetic analysis was automatically chosen by IQ-TREE software for each of 6 partitions (18S/28S/ITS-2/COI 1-2-3 codon positions) (see Supp. fi le 3).Bayesian analysis of the same datasets was performed using MrBayes ver.3.2.6,GTR model with gamma correction for intersite rate variation (8 categories) and the covariation model (Ronquist & Huelsenbeck 2003).Analyses were run as two separate chains (default heating parameters) for 20 million generations, by which time they had ceased converging (fi nal average standard deviation of the split frequencies was less than 0.01).The quality of chains was estimated using built-in MrBayes tools.MrBayes program was run at the CIPRES ver.3.3 website (Miller et al. 2010).Bayesian analysis quality was verifi ed using the program Tracer ver.1.7.1 (Rambaut et al. 2018).

Institutional acronyms
The specimens examined are kept at the following institutions and collections (the curator is given in parentheses):

Adult animals
Body elongated (Fig. 1) (morphometrics in Table 2, raw morphometric data are provided in the Supp.fi le 4).Fresh specimens uncolored or whitish with slightly greenish gut content, transparent after fi xation in Hoyer's medium.Black eyes present (Figs 1A, 3A, black arrowheads), often dissolving after slide mounting.Cuticle smooth in LM, with fi ne uniform sculpture consisting of minute conical granules with pointed apices visible under SEM only (Fig. 2A).All legs with granulated areas consisted of small granules, poorly discernible or, sometimes, completely invisible in LM.Legs I-III with small granulated areas on the external surfaces, near the claw bases (Fig. 2B-C), the internal leg surfaces without granulation, with distinct pulvinus.Legs IV with better-developed granulation mainly dorsally to the claws (Fig. 2D) and around the claw bases (Fig. 4C-D, white arrowhead).
Buccal-pharyngeal apparatus of Macrobiotus type (Fig. 3A) with the ventral lamina and ten peribuccal lamellae.Oral cavity armature (OCA) of harmsworthi type (according to Kaczmarek et al. 2020) with three bands of teeth visible in LM.Evident fi rst (anterior) band consists of a wide band of numerous minute teeth visible as dots in LM (Fig. 3D, F-G).Second band consists of a row of longitudinally elongated triangular teeth (Fig. 3D-G).Third band comprises three dorsal and three ventral transverse ridges (Fig. 3D-G).Medio-ventral ridge usually never divided into separate parts, only single specimen was found with detached lateral part of the medio-ventral ridge.Ventrally OCA with numerous additional teeth between the second and the third teeth bands (Fig. 3F-G).Pharyngeal bulb with apophyses, three macroplacoids and a large microplacoid (Fig. 3B-C).Macroplacoid length sequence is 2 < 3 ≤ 1.First macroplacoid is anteriorly narrowed, third macroplacoid with distinct subterminal constriction (Fig. 3B-C).
Claws of Mesobiotus type with minute stalk, distinct distal part of the basal portion, short common tract and developed internal septum, defi ning a distal part (Fig. 4A-C, E).Primary and secondary branches diverge below the half of the claw height, main branches with well-developed accessory points (Fig. 4A, C-D).Claws of fourth pair of legs slightly longer than claws of fi rst three pairs of legs (Fig. 4C).All claws with smooth lunules (Fig. 4).Anterior (internal) and posterior (external) claws of the legs IV are similar in shape (Fig. 4D).Lunules on posterior claws distinctly larger than on anterior claws (Fig. 4C-D).Single continuous cuticular bars are present below claw bases of the fi rst three pairs of legs (Fig. 4A-B, black arrowhead) with poorly developed muscle attachment points below (Fig. 4B).Claws of the legs IV are connected with a wide but poorly sclerifi ed horseshoe-like structure, visible in PhC only (Fig. 4E, black arrowhead).

Eggs
One adult female with mature oocites was islolated and cultivated for three days until the eggs were layed.After that the female was taken for the DNA extraction and gene sequencing (voucher slide SPbU 275(211)) while the layed eggs were taken for the morphological analysis using LM.
Eggs spherical, white, ornamented and laid freely (Figs 5A-C, 6A; morphometrics in Table 3).Chorion with conical processes that can be attributed to the "cones with long slender endings and fi laments" and "reticular design with "bubbles" morphotypes" (according to Kaczmarek et al. 2020).Egg processes with wide bases and thinned and fl exible apices usually well differentiated (Figs 5, 6A-B, D).Processes (with the exception of the thinned apical parts) with bilayered walls, with a net of trabecular structures between the internal and external layers, forming irregular rounded meshes of different size, so the processes seem to be reticulated in LM (Fig. 5).Apical parts of the processes with bubble-like internal   structure (Fig. 5), rarely bifurcating (Fig. 5A, F-G, black arrowheads), usually with a tuft of very short (0.5-2,75 μm) apical and subapical fi laments (Figs 5E, G-H, 6D, white arrowheads).Large pores (ca 1 μm in diameter), mostly indiscernible in LM and well-visible in SEM, are present on the basal part of all processes, forming a single row (Figs 5I, black arrowhead, 6B-D, black arrowheads).Process bases with well-developed crone of dark thickenings, visible in LM (Fig. 5A-D, H).Egg surface between the processes without areolation or pores but with a system of irregularly distributed wrinkles poorly discernible in LM as irregularly distributed granules and well-visible in SEM (Figs 5D, 6A-C).

Reproduction
No males were found.

Adult animals
Body elongated (Fig. 7) (morphometrics in Table 4, raw morphometric data are provided in the Supp.fi le 5).Fresh specimens uncolored or whitish with slightly greenish gut content, transparent after fi xation in Hoyer's medium.Black eyes present, often dissolving after slide mounting.Cuticle smooth in LM, with fi ne uniform sculpture consisting of minute conical granules with pointed apices visible under SEM only (Fig. 8A).All legs with granulated areas consisted of small granules, usually well visible in LM.Legs I-III with small granulated areas on the external surfaces, near the claw bases (Fig. 8B-C, black arrowhead), the internal leg surfaces without granulation, with indistinctly demarcated large pulvinus, visible in SEM only (Fig. 10A, white arrowhead).Legs IV with better-developed granulation mainly dorsally to the claws (Fig. 8D-E, white arrowhead) and around the claw bases (Fig. 10E, H, black arrowheads).
Buccal-pharyngeal apparatus of Macrobiotus type (Fig. 9A) with the ventral lamina and ten peribuccal lamellae (Fig. 8F).Oral cavity armature (OCA) of harmsworthi type (according to Kaczmarek et al. 2020) with three bands of teeth visible in LM.Evident fi rst (anterior) band consists of a wide band of numerous minute teeth visible as dots in LM (Figs 8F, white arrow, 9E, G).Second band consists of a row of longitudinally elongated triangular teeth (Fig. 9D-H).Third band comprises three dorsal and three ventral transverse ridges (Figs 8F white arrowhead, 9D-H).Medio-ventral ridge often divided in two or three separate teeth (Fig. 9H).Latero-ventral ridges often with strong indentations (Fig. 9G), sometimes almost fragmented to separate teeth.Rare additional teeth are present ventrally, between the second and the third teeth bands (Fig. 9F-H).Pharyngeal bulb with apophyses, three macroplacoids and a large microplacoid (Fig. 9B-C).Macroplacoid length sequence is 2 < 3 ≤ 1.First macroplacoid is anteriorly narrowed, third macroplacoid with poorly developed subterminal constriction (Fig. 9B-C).
Claws of Mesobiotus type with minute stalk, distinct distal part of the basal portion, short common tract and developed internal septum, defi ning a distal part (Fig. 10B, D-E).Primary and secondary branches diverge below the half of the claw height, main branches with well-developed accessory points (Fig. 10B-F).Claws of fourth pair of legs slightly longer than claws of fi rst three pairs of legs (Fig. 10E).All claws with smooth lunules (Fig. 10B-C, E, G).Anterior (internal) and posterior (external) claws of the legs IV are similar in shape (Fig. 10E).Single continuous cuticular bars of characteristic shape (two wide short bars connected by thin angular strip) are present below claw bases of the fi rst three pairs of legs (Fig. 10B, D, black arrowhead) with poorly developed muscle attachment points below (Fig. 10B,  D).Claws of the legs IV are connected with a poorly sclerifi ed horseshoe-like structure, visible in PhC only (Fig. 10H, white arrowhead).

Eggs
No eggs with developed embryos were found, but taking into account that M. vulpinus sp.nov.was the only tardigrade species present in the sample we believe that the adult specimens and the eggs belong to the same species.
Eggs spherical, white, ornamented and laid freely (Figs 11A, 12A, C; morphometrics in Table 5).Chorion with conical processes that can be attributed to the "sharp narrow cones" and "reticular design with "bubbles" morphotypes" (according to Kaczmarek et al. 2020).Egg processes in form of elongated cones with poorly differentiated basal and apical parts (Figs 11B, E-F, 12-13).Processes (with the  exception of the elongated apical parts) with bilayered walls, with a net of trabecular structures between the internal and external layers, forming irregular rounded meshes of different size, so the processes seem to be reticulated in LM (Fig. 11).Apical parts of the processes with bubble-like internal structure (Fig. 11F), rarely bifurcating (Figs 11E, 13A).Processes surface bears annulations, visible in SEM only (Figs 12B,D,13).Rare large pores (1.4-2.3 μm in diameter), poorly discernible in LM and well-visible in SEM, are present on the basal part of all processes, below the half of the process height, forming a single row (Figs 11H, white arrowheads, 12, 13, white arrowheads), Second row of distinctly smaller and more numerous pores is located in the most basal part of each process (Fig. 13, black arrowheads).Process bases with poorly developed, sometimes almost invisible crone of dark thickenings (Fig. 11C,  E-F).Egg surface between the processes with distinct polygonal relief consisted of ridges forming hexagonal (rarely pentagonal) cells around each process 12).Points of ridges intersection bears small bulbous processes (Figs 11B, 12B, D, 13B).Both ridges and bulbous processes with internal trabecular structures, similar to the main processes walls.Egg surface between the processes bases and the ridges of polygonal relief with a system of smaller radial ridges and pores discernible both in LM and SEM 12B,D,13).

Reproduction
No males were found.

Discussion
Phenotypic differential diagnosis of Mesobiotus efa sp.nov.
Within the genus, Mesobiotus efa sp.nov.belongs to the group of species with smooth cuticle, harmsworthi-type OCA, typical Mesobiotus claws IV with unindented lunules, and egg chorion with reticulated processes in form of "sharp wide cones" or "cones with long slender endings" (these two types of processes are often poorly distinguishable), egg process bases with well-developed crone of dark thickenings without fi nger-like projections, and egg shell surface between the processes with ridges without reticulation, areolation or semi-areolation.
Within this species complex Mesobiotus efa sp.nov.differs from: Mesobiotus altitudinalis (known only from the type locality in Russia; Biserov 1997Biserov -1998) )  Mesobiotus binieki (Kaczmarek, Gołdyn, Prokop & Michalczyk, 2011) (known only from the type locality in Bulgaria; Kaczmarek et al. 2011) by having medio-ventral ridges of OCA always unbroken, and by having egg processes with less differentiated basal and apical parts (in M. binieki the basal parts are in shape of very short and wide cones while the apical parts are long thin spines without developed internal bubbles.Mesobiotus patiens (Pilato, Binda, Napolitano & Moncada, 2000) (known from the Aeolian Islands (type locality) and several islands in the Tyrrhenian Sea, Italy; Pilato et al. 2000) by having numerous additional teeth in ventral OCA (no such teeth in M. patiens), and by having smaller eggs (egg diameter without processes is 67.5-69.1 μm in M. efa sp.nov.and 75-87 μm in M. patiens) with distal part of the processes better developed with well-visible internal bubbles (in M. patiens distal part of the processes reduced, thin and short, often broken, without internal bubbles).

Mesobiotus coronatus (de
Mesobiotus rigidus (Pilato & Lisi, 2006) (known only from the type locality in New Zealand; Pilato & Lisi 2006) by having eggs with system of radial ridges on the egg shell surface between processes poorly visible in LM (well-visible in M. rigidus) and presence of bifurcated processes and tuft of short fi laments on the processes' top (egg processes never subdivided in M. rigidus).(Pilato 1974: 67).Rarely in some eggs of M. vulpinus these processes are small, similar to those in M. mauccii (Fig. 12A-B).

Genetic comparison of
Mesobiotus mauccii was also noted from several Asian locations: North China (Beasley & Miller 2007) Full matrices with p-distances are provided in the Supp.fi le 6.

Phylogenetic analysis
General topology of the obtained consensus phylogenetic tree (Fig. 14) conforms to the results of the most recent analyses performed by Stec (2022) and Vecchi et al. (2023).The monophyletic genus Mesobiotus comprises a complex of basal Antarctic clades paraphyletic in both Bayesian, and ML analyses consisted of two clearly separated subclades: the fi rst incorporates M. hilariae Vecchi, Cesari, Bertolani, Jönsson, Rebecchi & Guidetti, 2016 and undescribed species of M. harmsworthi morphogroup (Short et al. 2022) and the second incorporates at least four well-supported subclades of undescribed species of M. furciger morphogroup (Short et al. 2022;morphogroups according Stec 2022).
The second main subclade, which incorporates all non-Antarctic taxa, revealed monophyletic in our analysis, but with weak support (0.85 in Bayes and 76 in ML).This is in contrast with the results of Stec (2022), where the high support for the monophyly of this clade was obtained.This clade consists of two monophyletic clades: the fi rst comprising two South Asian species (M.dilimanensis from the Philippines and M. marmoreus from Vietnam) and the second including all other species of Mesobiotus.This second subclade incorporates a larger subclade consisting of two distinct species complexes: the fi rst including Holarctic species and the second including mostly tropical or subtropical species.Sister group to this "Holarctic +Tropical" subclade is a small subclade, consisting of only three species (M.cf.barabanovi from Kyrgyzstan, M. gr.furciger from Norway, and Mesobiotus sp. from Finland).The Holarctic subclade includes M. huecoensis from USA, M. peterseni from Greenland, M. harmsworthi from Svalbard, M. gr.harmsworthi from Russia and the monophyletic clade comprises M. occultatus from Svalbard and M. efa sp.nov.from North-West Russia as sister groups.The position of M. huecoensis is instable -in the Bayesian analysis it is a sister group to all other (North Holarctic) species of the subclade, while in the ML analysis it is a sister group to M. peterseni (with weak support, 67%).Such ambiguity can be the result of the data incompleteness for this species: only 18S rRNA and COI sequences are available.
The most notable differences from the results of previous studies relate to the structure of a relatively large 'tropical' species complex.Within this clade, we obtained three moderately-to well-supported subclades with poorly resolved relationships between them.For the fi rst time, we state the presence of a moderately-supported monophyletic clade comprising all known South African species (M.anastasiae Tumanov, 2020, M. maklowiczi Stec, 2022, andM. diegoi Stec, 2022) It is interesting to note presence of three independent clades consisting of species from Vietnam and the Philippines: M. dilimanensis + M. marmoreus; M. imperialis + M. philippinicus, and M. datanlanicus + M. insanis.Such a zoogegraphic pattern can be evidence for the strong ancient connections between tardigrade faunas of these regions.A close relationship of the newly described species from the Russian Far East (Primorsky Krai) to one of the South Asian clades is not surprising.The presence of tropical elements in the invertebrate fauna of Primorsky Krai is a well-known phenomenon (Likharev 1953;Korovchinsky 2006;Markova et al. 2015;Ganin 2018;Garibian 2020).This region is usually considered as a refugium of the Neogene tropical fauna escaping the infl uence of the last glaciation (Likharev 1953).The morphological similarity of M. vulpinus sp.nov. to M. maucchii, known from China and, possibly, the Andaman Islands supports its close affi nity to the tropical species complex.

Fig. 8 .
Fig. 8. Mesobiotus vulpinus sp.nov., cuticular sculpture and oral cavity armature (OCA).A-B, E-F.Paratype (SPbU Tar_33).C-D.Holotype (SPbU 320(10)).A. High magnifi cation of the sculpture of the dorsal body surface, SEM.B. Dot-like sculpture on the external surface of leg III, SEM. C. Dotlike sculpture on the external surface of leg III, PhC, black arrowhead inticates the zone of sculpture.D. Dot-like sculpture on the dorsal side of hind leg, PhC.E. Dot-like sculpture on the dorsal side of hind leg, SEM, white arrowhead indicates the zone of sculpture.F. Mouth opening with dorsal OCA visible, SEM, white arrow indicates the fi rst band of teeth, white arrowhead indicates the dorsal crests of the third band of teeth.Scale bars A-B, F = 2 μm; C-E = 5 μm.

Fig. 12 .
Fig. 12. Mesobiotus vulpinus sp.nov., egg.A-D.Paratypes (SPbU Tar_65).A, C. Total view of the eggs, SEM.B, D. Details of the egg surface, SEM.Note the difference in the degree of development of small tubercles and numerous small pores on the egg surface.Scale bars: A-B = 20 μm; C-D = 5 μm.
by having typical Mesobiotus claws while M. altitudinalis has thin elongated claws, especially on legs IV, by having numerous additional teeth in OCA ventrally, by having eggs with smaller egg processes (processes height 11.1-21.6μm in M. efa sp.nov.vs 22.0-35.0μm in M. altitudinalis), and by having egg surface between processes without pores.Mesobiotus baltatus (McInnes, 1991) (known only from the type locality in Spain; McInnes 1991) by having no pigmented bands (present in M. baltatus) and by having well-developed crone of dark thickenings around the egg processes (absent in M. baltatus).

Fig. 14 .
Fig. 14.Phylogeny of Mesobiotus Vecchi, Cesari, Bertolani, Jönsson, Rebecchi & Guidetti, 2016 based on concatenated 18S + 28S + ITS-2 + COI sequences.Numbers at nodes indicate Bayesian posterior probability values (BI, fi rst values) and bootstrap values (ML, second values).Black dots indicate the nodes supported by values of 1.0/100% with both methods.Low support values (below 0.9 in BI and below 70% in ML) not shown.Scale bar and branch lengths refer to the Bayesian analysis.

Table 1
(continued on next page).Complete list of sequences used in the phylogenetic analysis.Sequences produced in this study are marked in bold.

Table 1 (
continued).Complete list of sequences used in the phylogenetic analysis.Sequences produced in this study are marked in bold.

Table 2 .
Summary of morphometric data for Mesobiotus efa sp.nov.Measurements are given in μm, pt values in % (the pt index is the percentage ratio between the length of a structure and the length of the buccal tube).

Table 3 .
Measurements (in μm) of selected morphological structures of eggs of Mesobiotus efa sp.nov.Abbreviations: N = number of eggs/structures measured, range refers to the smallest and the largest structure among all measured specimens; SD = standard deviation).

Table 4 .
Summary of morphometric data for Mesobiotus vulpinus sp.nov.Measurements are given in μm, pt values in % (the pt index is the percentage ratio between the length of a structure and the length of the buccal tube).

Table 5 .
Measurements (in μm) of selected morphological structures of eggs of Mesobiotus vulpinus sp.nov.Abbreviations: N = number of eggs/structures measured, range refers to the smallest and the largest structure among all measured specimens; SD = standard deviation).
Massa et al. 2021)esari, Rebecchi & Jönsson, 2021rica only;Pilato et al. 2000;Kaczmarek et al. 2015)by Kaczmarek et al. 2018(pt for anterior/posterior claws of legsin M. coronatus), and by having larger eggs (egg diameter without processes is 67.5-69.1 μm in M. efa and 42-55 μm in M. coronatus) with larger processes (process height is 11.1-21.6μm in M. efa and up to 9.2 μm in M. coronatus).Mesobiotus emiliaeMassa, Guidetti, Cesari, Rebecchi & Jönsson, 2021(known only from the type locality in Sweden;Massa et al. 2021), by having slightly larger eggs (egg diameter without processes is 67.5-69.1 μm in M. efa sp.nov.and46.9-64.6 μm in M. emiliae) with higher egg processes (process height is 11.1-21.6μm in M. efa and 7.9-10.6μm in M. emiliae), by having egg processes with relatively longer apical parts, and by having larger inter-process distances (2.2-6.6 μm in M. efa and 0.5-1.6 μm in M. emiliae).Mesobiotus helenae Tumanov & Pilato, 2019 (known only from the type locality in New Zealand; Tumanov & Pilato 2019) by having medio-ventral ridges of OCA always unbroken (divided in M. helenae), having shorter claws (pt for posterior claws of legs IV are 22.4-29.0 in M. efa sp.nov.and30.7-31.4 in M. helenae), and by having smaller eggs (egg diameter without processes is 67.5-69.1 μm in M. efa and 71.0 μm in M. helenae) with less numerous processes (number of processes on the egg circumference is 12-14 in M. efa and 22 in M. helenae), and processes walls with well-developed internal reticulation (poorly visible in M. helenae).Mesobiotus imperialis Stec, 2021 (known only from the type locality in Vietnam; Stec 2021) by having medio-ventral ridges of OCA always unbroken (divided in M. imperialis), by having lunules on legs IV always smooth (slight indentation visible in about 50% of observed specimens of M. imperialis), by having egg surface between processes without pores (in M. imperialis pores are present and visible in LM as light dots), and by having a single row of large pores around the smooth egg processes (in M. imperialis egg processes with numerous depressions and pores not organised in rows) -the last character detectable in SEM only.Mesobiotus nikolaevae Tumanov, 2018 (known only from the type locality in Croatia; Tumanov 2018b) by having more numerous additional teeth in ventral OCA, by having egg surface between processes without pores (in M. nikolaevae pores are present and visible in LM as light dots), by having ridges between egg processes poorly visible in LM (well-developed, forming a reticulate-like pattern in M. nikolaevae), and by having a single row of large pores around the egg processes (in M. nikolaevae egg processes with irregularly distributed small pores) -the last character detectable in SEM only.Mesobiotus occultatusKaczmarek, Zawierucha, Buda, Stec, Gawlak, Michalczyk & Roszkowska,  2018 (known only from Spitsbergen;Kaczmarek et al. 2018) by having medio-ventral ridges of OCA always unbroken (often divided in M. occultatus, the character not mentioned in the original description(Kaczmarek pers.com. 2 Nov. 2019)), by having eggs with less tightly distributed processes (interprocess distance is 2.2-6.6 μm (mean 4.4 μm) in M. efa sp.nov.and 1.4-4.2μm (mean 2.6 μm) in M. occultatus), and by having a single row of large pores around the egg processes (smaller pores, less regularly distributed over the processes in M. occultatus).
Full matrices with p-distances are provided in the Supp.fi le 6.

Phenotypic differential diagnosis of Mesobiotus vulpinus sp. nov.
(Pilato, 1974)us Mesobiotus only M. mauccii(Pilato, 1974)(described from South China; Pilato 1974) has egg chorion with polygonal relief.Mesobiotus vulpinus sp.nov.differs from M. mauccii by having eyes, by having narrower buccal tube (pt for the buccal tube external width is 13.0-18.2 in M. vulpinus and 23.12 in M. mauccii (buccal tube measurements was taken from the type specimen photo), by having stylet supports inserted in more anterior position (pt for the stylet support insertion point is 74.7-77.6 in M. vulpinus and 79.49 in M. mauccii, by having no additional teeth in dorsal OCA and only few additional teeth in ventral OCA (M.mauccii has additional teeth both in dorsal and ventral OCA, ventral additional teeth are numerous and organized in several rows), by having longer egg processes (29.8-36.1 μm in M. vulpinus and 15-19 μm in M. mauccii) with less differentiated basal and apical parts, by lack of collar around the process base, and by usually evidently developed small bulbous processes in the intersection points of polygonal relief ridges (Fig.12C-D) (intersection points with poorly developed thickenings in M. mauccii: "the vertices of these polygons are particularly prominent and almost form a tubercle"

comparison of Mesobiotus vulpinus sp. nov.
& Takeda 2000)te Pasa (1980)00;Abe & Takeda 2005)aman Island(Maucci & Durante Pasa 1980), and Japan(Utsugi 1988;Abe & Takeda 2000;Abe & Takeda 2005).AllChina records mostly conform to the original description of M. mauccii, while in Abe &Takeda's (2005)photographs egg processes are longer and evidently different in shape, being more similar to the M. vulpinus sp.nov.eggprocesses.Also the buccal tube seems to be narrower in Japanese specimens than in type material of M. mauccii (see Abe & Takeda 2005: fi g. 3) and eyes are present, like in M. vulpinus.In our opinion, it is very likely that the Japanese records of M. mauccii are in fact M. vulpinus or a similar species.The Andaman record is the most questionable as the photograph of an adult specimen attributed to M. mauccii inMaucci & Durante Pasa (1980)in fact belongs to an unknown species of Paramacrobiotus (see Abe& Takeda 2000)and the only evidence of the presence of this species on the Andaman Islands is the photo of a damaged egg.The ranges of uncorrected genetic p-distances between the studied population of Mesobiotus vulpinus sp.nov.andotherspecies of the genus Mesobiotus, for which sequences are available from GenBank, are as follows: COI: 24.60%-33.03%(mean29.61%),with the most similar being M. diegoi Stec, 2022 from South Africa (OP143857, OP143858, Stec 2022), and the least similar being M. dilimanensis from the Philippines (MN257047,Itang et al. 2020).