Macrobiotus rebecchii sp. nov.: A New Limno-Terrestrial and Hermaphroditic Tardigrade from Kyrgyzstan

Simple Summary In a moss sample collected on a rock in Kyrgyzstan, I discovered a new hermaphroditic tardigrade belonging to the genus Macrobiotus C.A.S. Schultze, 1834. To document this new species (Macrobiotus rebecchii sp. nov.) I used detailed morphological data collected from animals and eggs under a contrast phase light microscope (PCM) and scanning electron microscope (SEM). I also obtained DNA sequences from specimens of the new species (18S rRNA, COI). The external appearance of Macrobiotus rebecchii sp. nov. is similar to Macrobiotus joannae Pilato & Binda, 1983, Macrobiotus punctillus Pilato, Binda & Azzaro, 1990 and Macrobiotus hannae Nowak & Stec, 2018, but it can be easily differentiated from them mainly by a different body granulation pattern. Abstract A new tardigrade species of the genus Macrobiotus C.A.S. Schultze, 1834 from Kyrgyzstan, is described and illustrated in this paper. Macrobiotus rebecchii sp. nov. is a hermaphroditic and limnoterrestrial species found in a moss growing on a rock in Toluk village. Specimens of the new species were examined for its morphological details using contrast phase light microscope (PCM) and scanning electron microscope (SEM). Genetic data in the form of DNA sequences of commonly used molecular markers were also obtained (18S rRNA, COI). Phenotypically the new species is most similar to Macrobiotus joannae Pilato & Binda, 1983, Macrobiotus punctillus Pilato, Binda & Azzaro, 1990, and Macrobiotus hannae Nowak & Stec, 2018, but can be easily differentiated from all of them by its body granulation pattern.


Introduction
The phylum Tardigrada includes aquatic micro-invertebrates represented by more than 1400 species [1][2][3]. Over a dozen new tardigrade species are described each year, expanding our knowledge, yet their biodiversity is poorly known. These animals have a global distribution and inhabit a wide variety of environments, from the greatest depths of the oceans to the highest mountain peaks, as well as extreme ephemeral habitats such as cryoconite holes and rock pools [4][5][6].
In this article, I describe a new species of the genus Macrobiotus, Macrobiotus rebecchii sp. nov., discovered in a moss sample collected from a rock in Toluk village in Kyrgyzstan. The description is based on morphological and morphometric analyses conducted with the use of phase contrast light microscopy (PCM) and scanning electron microscopy (SEM). Phenotypic data of the new species are supplemented by DNA sequences of the two molecular markers (18S rRNA and COI) commonly used in tardigrade taxonomy.

Sample Processing
A moss sample was collected from a rock in Toluk village in Kyrgyzstan in October 2018 by Barłomiej Surmacz and Witold Morek and was later examined for tardigrades using the protocol by Stec et al. [23]. To perform integrative taxonomic descriptions, the isolated animals and eggs extracted from the sample were split into three groups for specific analyses: morphological analysis with phase contrast light microscopy, morphological analysis with scanning electron microscopy, and DNA sequencing (for details please see section "Material examined" below).

Microscopy and Imaging
Specimens for light microscopy were mounted on microscope slides in a small drop of Hoyer's medium and secured with a cover slip. Slides were kept at room temperature until the medium was dry and solid. The dried slides were sealed with transparent nail polish and examined under a Leica DMLB light microscope with PCM and a digital camera. Immediately after mounting the specimens in the medium, the slides were checked under PCM for the presence of males and females in the studied population, as the spermatozoa in the testis or ovotestis are visible only for a few hours after mounting [12,24]. To obtain clean and extended specimens for SEM, tardigrades were processed according to the protocol by Stec et al. [23]. Specimens were examined under high vacuum in a Versa 3D DualBeam SEM at the ATOMIN facility of the Jagiellonian University, Kraków, Poland. All figures were assembled in Corel Photo-Paint X6. For structures that could not be satisfactorily focused in a single light microscope photograph, a stack of two to six images with an equidistance of ca. 0.2 µm were manually assembled into a single deep-focus image in Corel Photo-Paint X6.

Morphometrics and Morphological Nomenclature
All measurements are given in micrometres (µm). The sample size was adjusted following the recommendations of Stec et al. [25]. Structures were measured only if their orientation was suitable. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. The terminology used to describe oral cavity armature and eggshell morphology follows Michalczyk & Kaczmarek [26] and Kaczmarek & Michalczyk [27]. The macroplacoid length sequence is given according to Kaczmarek et al. [28], while the morphological states of the cuticular bars on the legs follow Kiosya et al. [29]. The length of the buccal tube and the level of the insertion point of the stylet support were measured according to Pilato [30]. The pt index is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage [30]. All other measurements and nomenclature follow Kaczmarek & Michalczyk [27]. Specifically, the width of the buccal tube was measured as the external and internal diameter at the level of the stylet support insertion point. The heights of the claw branches were measured Animals 2022, 12, 2906 3 of 16 from the base of the claw (i.e., excluding the lunula) to the top of the branch, including accessory points. The distance between the egg processes was measured as the shortest line connecting the base edges of the two randomly chosen closest processes. Morphometric data were handled using the 'Parachela' ver. 1.8 template available from the Tardigrada Register [31] and are given in Supplementary Materials (Spreadsheets S1). The tardigrade taxonomy follows Bertolani et al. [32] and Stec et al. [18].

Genotyping
Before DNA isolation, two animals were temporarily mounted in a drop of water on microscope slide, secured with cover slip, checked under the light microscope, and immediately removed from the slide to avoid damage. DNA was extracted from the individual animals following a Chelex ® 100 resin (Bio-Rad) extraction method by Casquet et al. [33] with modifications described in detail by Stec et al. [34]. Two DNA fragments were sequenced: the small ribosome subunit (18S rRNA, nDNA), and the cytochrome oxidase subunit I (COI, mtDNA). All fragments were amplified and sequenced according to the protocols described by Stec et al. [34]. Primers and original references for specific PCR programs are listed in Table 1. The sequencing products were read with the ABI 3130xl sequencer at the Institute of Systematics and Evolution of Animals of the Polish Academy of Sciences, Kraków, Poland. Sequences were processed in BioEdit ver. 7.2.5 [35] and submitted to GenBank.  [39]. For genetic comparison, all published 18S rRNA and COI sequences of these taxa [32,39] were downloaded from GenBank. The sequences were aligned using the default settings (in the case of COI) and the Q-INS-I method (in the case of 18S rRNA) of MAFFT version 7 [40,41] and manually checked against non-conservative alignments in BioEdit. The aligned sequences were then trimmed to 758 (18S rRNA), and 657 (COI), bp. All COI sequences were translated into protein sequences in MEGA11 [42] to check against pseudogenes. The uncorrected pairwise distances were calculated using MEGA11 and are provided in the Supplementary Materials (Spreadsheets S2).   . Panel D also shows minute granulation that is not visible in PCM (the same as in Figure 3F) but here it can hardly be discriminated from dirt, which is also present on this fragment of the dorsal cuticle. Scale bars in μm. . Panel D also shows minute granulation that is not visible in PCM (the same as in Figure 3F) but here it can hardly be discriminated from dirt, which is also present on this fragment of the dorsal cuticle. Scale bars in µm. . Panel D also shows minute granulation that is not visible in PCM (the same as in Figure 3F) but here it can hardly be discriminated from dirt, which is also present on this fragment of the dorsal cuticle. Scale bars in μm.  granulation seen in the caudal region of the last body segment (paratype); (C) a pulvinus-shaped cuticular bulge on the internal surface of leg II and an additional cuticular fold positioned distally (holotype); (D) granulation on the hind leg and a band of sparse granulation seen in the caudal region of the last body segment (paratype). The filled flat arrowhead indicates a single continuous cuticular bar above the claws, the filled indented arrowhead indicates an additional cuticular fold on the internal leg surface, and the empty indented arrowheads indicate body granulation faintly visible in PCM in the proximity of the well-visible caudal band of granulation, which is marked by a yellow dashed line. Scale bars in μm.  there is an evident dentation in the lunulae in legs IV ( Figure 4B,D). A single continuous cuticular bar with central constriction is present above claws I-III ( Figure 4A,C), with shadowed extensions narrowing toward double muscle attachments ( Figure 4A; visible only in PCM). Sometimes, additional shadowed areas are present just above the lunulae ( Figure 4A; visible only in PCM). A horseshoe-shaped structure connects the anterior and posterior lunules in leg IV ( Figure 4B).

Discussion
The new species belongs to Macrobiotus hufelandi morphogroup sensu Stec et al. [18]. By having (i) three bands of teeth in the oral cavity armature that are well visible under light microscope, (ii) entire body cuticle covered by granulation (sometimes visible only in SEM), (iii) eggs with inverted goblet shaped processes and (iv) evident reticulation on the egg surface between processes, the new species is the most similar to three other Macrobiotus taxa, namely M. joannae reported from its type locality in Australia [38], and several uncertain localities in central, eastern, and south-eastern Russia [49] and from Italy [32], M. hannae known only from its type locality in Poland [39], and Macrobiotus punctillus

Material Examined
Thirty-six animals, 65 eggs mounted on microscope slides in Hoyer's medium, 10 animals and 11 eggs examined in SEM, and two animals processed for DNA sequencing.

Etymology
The species is named after Lorena Rebecchi, a world-renowned tardigrade specialist from the University of Modena and Reggio Emilia (Modena, Italy). She intensively studied reproduction in Tardigrada, including the ovotestis maturation pattern in the hermaphroditic species Macrobiotus joannae which is similar to the new species described in this study.

Animals
Body transparent in juveniles and white in adults, after fixation in Hoyer's medium brownish ( Figure 1A). Eyes present. Round and oval pores (0.4-0.7 µm in diameter), scattered randomly on the entire cuticle (on the ventral side of the body distributed more sparsely) ( Figure 1B-D), including the external and internal surface of all legs. Evident granulation on the external surface of all legs I-III is visible under PCM and SEM (Figures 2A and 3A). Granulation is also present on the lateral and dorsal surfaces of legs IV (Figures 2B,D and 3D,E). A pulvinus-shaped cuticular bulge is present centrally on the internal surface of all legs I-III and an additional cuticular fold positioned distally (Figures 2C and 3C). This structure is visible only if the legs are fully extended and well oriented on the slide or SEM stubs. In addition to the typical patches of leg granulation, a band of granulation is present on the dorso and latero-caudal surface of the last body segment (Figures 2B,D, 3B,D and 10C). This band of sparse dorsal granula-tion extends posteriorly and connects symmetrically with the granulation on both legs IV ( Figures 2B,D, 3B,D and 10C). Leg granulation as well as caudal band of granulation are always clearly visible in PCM. However, except for this distinct granulation, the entire animal body is covered by evenly distributed, minute granulation, which is visible only under SEM ( Figure 3F). The size of these microgranules, with diameters ranging from 0.05 to 0.07 µm, is below the resolution of the light microscope. This granulation can be slightly bigger only occasionally in the proximity of the mentioned well-visible caudal band, and on such occasions is very faintly visible in PCM ( Figure 2B,D) but always well visible in SEM ( Figure 3B,D).
Claws small and slender, of the hufelandi type ( Figure 4A-D) with primary branches with distinct accessory points, a long common tract and an evident stalk connecting the claw to the lunula ( Figure 4A-D). The lunulae in legs I-III are smooth ( Figure 4A,C), while there is an evident dentation in the lunulae in legs IV ( Figure 4B,D). A single continuous cuticular bar with central constriction is present above claws I-III ( Figure 4A,C), with shadowed extensions narrowing toward double muscle attachments ( Figure 4A; visible only in PCM). Sometimes, additional shadowed areas are present just above the lunulae ( Figure 4A; visible only in PCM). A horseshoe-shaped structure connects the anterior and posterior lunules in leg IV ( Figure 4B).
Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type, with the ventral lamina and ten small peribuccal lamellae followed by six buccal sensory lobes ( Figures 5A and 6A,B). Under PCM, the oral cavity armature is of the hufelandi type-three bands of teeth are always visible ( Figure 5B,C). The first band of teeth is composed of numerous extremely small cones arranged in four to six rows located anteriorly in the oral cavity, just behind the bases of the peribuccal lamellae ( Figures 5B,C and 6A,B). The second band of teeth is located between the ring fold and the third band of teeth and comprises 4-5 rows of small cones, slightly larger than those of the first band ( Figures 5B,C and 6A,B). The teeth of the third band are located within the posterior portion of the oral cavity between the second band of teeth and the opening of the buccal tube ( Figures 5B,C and 6A,B). The third band of teeth is discontinuous and divided into the dorsal and ventral portions. Under PCM, the dorsal teeth are seen as three distinct transverse ridges, whereas the ventral teeth appear as two separate lateral transverse ridges between which a roundish median tooth is visible ( Figure 5B,C). In SEM, both dorsal and ventral teeth are also clearly distinct ( Figure 6A,B). Under SEM, the margins of the dorsal teeth are serrated ( Figure 6A), whereas the margins of the ventral teeth are evidently less serrated ( Figure 6B). Pharyngeal bulb spherical, with triangular apophyses, three anterior cuticular spikes (typically only two are visible in any given plane), two rod-shaped macroplacoids and a large triangular microplacoid ( Figure 5A). The macroplacoid length sequence being 2 < 1. The first and the second macroplacoid are constricted centrally and subterminally, respectively ( Figure 5D,E). Measurements and statistics are given in Table 2.

Eggs
Laid freely, white, spherical and ornamented ( Figures 7A-E and 8A-D). The surface between processes of the hufelandi type, i.e., chorion surface covered by evident reticulum (Figures 7A,B and 8A-C). The reticulation is uniform across the entire surface. There are several rows of pores between processes, and the mesh nodes and bars are often wider than the pore diameter (the second character is more evident in SEM than in PCM; Figures 7A,B and 8A-C). The pores in the reticulum are circular or slightly oval (0.25-0.60 µm in diameter), and under SEM almost all pores are seen to contain one or more small round or elongated granules ( Figure 8A-C). The processes are in the shape of inverted goblets with slightly concave conical trunks and well-defined terminal discs ( Figures 7A-E and 8A-D). Faint annulations are visible under SEM on the process trunk ( Figure 8A-D). A crown of gently marked thickenings is visible around the bases of the processes as darker dots in PCM ( Figure 7A,B) and as wrinkled bases in SEM ( Figure 7A-C). The terminal discs are cog-shaped, with a concave central area and 10-18 distinct teeth ( Figures 7A-E and 8A-D). The terminal discs, and especially their teeth, are covered by small granules (visible only under SEM) that probably serve to improve the adhesive properties of the egg processes ( Figure 8A-D). Measurements and statistics are given in Table 3.

Reproduction
The type population of M. rebecchii sp. nov. is hermaphroditic. In each of the analysed adult gravid individuals two types of gametes were observed. The observation of individuals freshly mounted in Hoyer's medium revealed the ovotestis filled with spermatozoa ( Figure 9) and developing oocytes.

Discussion
The new species belongs to Macrobiotus hufelandi morphogroup sensu Stec et al. [18]. By having (i) three bands of teeth in the oral cavity armature that are well visible under light microscope, (ii) entire body cuticle covered by granulation (sometimes visible only in SEM), (iii) eggs with inverted goblet shaped processes and (iv) evident reticulation on the egg surface between processes, the new species is the most similar to three other Macrobiotus taxa, namely M. joannae reported from its type locality in Australia [38], and several uncertain localities in central, eastern, and south-eastern Russia [49] and from Italy [32], M. hannae known only from its type locality in Poland [39], and Macrobiotus punctillus Pilato, Binda & Azzaro, 1990 [50] known only from its type locality in Chile [50]. However, it can be easily distinguished from all of them by a different pattern of body granulation visible under a light microscope (LM; Figure 10). Specifically, M. joannae and M. punctillus have clearly visible granulation evenly covering the entire body surface ( Figure 10A; [38,39,50]); M. hannae has minute body granulation evenly covering the entire body surface, but it is visible only in SEM and not in LM ( Figure 10B; [39]), while M. rebecchii sp. nov. also has body granulation covering the entire body surface but comprises two types of granules: bigger granules that are visible under LM and form a specific band on the dorso-and latero-caudal surface of the last body segment, and smaller granulation which is not visible under LM ( Figure 10C). The new species has also better developed and evident dentation in the lunulae of the hind legs compared to M. hannae, and very similar to those of M. joannae [39], whereas the lunulae of the hind legs are smooth in M. punctillus [50]. The new species also exhibits shadowed extensions of the cuticular bar above the claws in legs I-III narrowing toward double muscle attachments, which are absent in M. hannae and M. joannae. Although original descriptions of M. joannae and M. hannae did not report a crown of thickenings around the processes bases, they are visible in figures presented in Nowak & Stec [39]. Similarly, an additional cuticular fold positioned distally on the internal surface of legs I-III was not mentioned directly for M. hannae but is clearly visible in the figures provided in the original description [39]. In addition, there are no obvious differences in the morphometric characters between the new species and the M. joannae and M. hannae, as all its ranges and values do overlap. There are however small morphometric differences between M. rebecchii sp. nov. and M. punctillus, namely: larger body size µm for egg processes height and width in M. punctillus), and wider terminal discs on egg processes (4.8-8.2 µm in the new species vs. 3.7-3.9 µm in M. punctillus).
Genetic comparison and lack of differences between 18S rRNA sequences of the three compared species (M. rebecchii sp. nov., M hannae, M. joannae) confirmed their close relationship. This is also in agreement with their morphological similarity. Importantly, a high divergence in the mitochondrial marker between the new species and M. hannae demonstrates their distinctiveness and additionally supports the hypothesis of the new species. Such cases of extreme morphological similarity, lack of differences in nuclear markers, and evident interspecific divergence in COI, have been recently reported by several studies which described new macrobiotid taxa (e.g., [19,[51][52][53][54][55][56]). Finally, the results presented herein, and the new species description from Kyrgyzstan, which is very similar to M. joannae and M. hannae, support the conclusions of Nowak & Stec [39] who questioned the validity of previous European records of M. joannae. Therefore, the confirmed geographic distribution of the three nominal hermaphroditic Macrobiotus species discussed in this study (M. rebecchii sp. nov., M hannae, M. joannae) seems to be limited to their type localities, unless other records are positively verified with the tools of integrative taxonomy.

Conclusions
The results of my study demonstrate morphological as well as genetic evidence for the distinctiveness of the newly found species from its congeners, supporting at the same time the erection of the new tardigrade species, Macrobiotus rebecchii sp. nov. As a result of this discovery, the number of tardigrade species recorded in Kyrgyzstan has now increased to 17. The new species is also the third Macrobiotus to be formally described in this country.

Funding:
The study was supported by the grant from the European Commission's programme "Transnational Access to Major Research Infrastructures" to SYNTHESYS (grant no. DK-TAF-2693 to DS) and by the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The author confirms that the data supporting the findings of this study are available within the article and its supplementary materials. The DNA sequences generated in this study are available in GenBank.