Integrative Redescription of the Minibiotus intermedius (Plate, 1888)—The Type Species of the Genus Minibiotus R.O. Schuster, 1980

: In the present study, we used the integrative taxonomy approach to redescribe Minibiotus intermedius based on the newly found topotypic population in Marburg (Germany). As the original type material is not available, we designate a neotype to stabilize the taxonomy of the genus Minibiotus . Obtained mitochondrial COI barcode sequence and nuclear markers, i.e., 18S rRNA and 28S rRNA of M. intermedius from the neotype locality, were unique and distinct from those deposited in GenBank. In the ﬁrst redescription of M. intermedius , only four specimens and no eggs from the neotype locality were analyzed. Moreover, genetic analyses were not conducted and barcodes were not available. Therefore, the present study, by establishing the neotype and providing integrative data on the neotype population, helps to better deﬁne the Minibiotus taxonomy and prevents further misunderstandings in the future.


Introduction
Tardigrades inhabit aquatic (freshwater and marine) and terrestrial habitats, from the highest mountain peaks to deepest oceans, from the polar regions to the tropics. They are found in lichens, mosses, leaf litter, soil, sediments, and on aquatic plants [1]. Up to now, about 1400 species of hetero and eutardigrades have been described throughout the world [2][3][4][5].
The genus Minibiotus R.O. Schuster [6] was established more than forty years ago by Schuster et al. [6]. However, at first, the new genus was not easily accepted by other researchers who questioned its validity due to an insufficiently clear diagnosis [7,8]. Later, Claxton [9] published the most comprehensive revision of this genus based on animal and egg morphology, strengthening the generic diagnosis and, at the same time, its validity. In that time, 22 species were included into the genus Minibiotus; however, until now, many new species have been described. According to the current tardigrade checklist, 48 species are known in this genus [5]. The genus is mainly characterized by their antero-ventral mouth surrounded by 10 papulae, a narrow buccal tube with either a single or a double A single sample of mosses and lichens from a tree was collected in a mixed forest in Marburg (Germany) in September 2019 (sample code (SC) GR2, for more details see below). The sample was then packed in a paper envelope; dried at a temperature of ca. 20 • C; and delivered to the Department of Animal Taxonomy and Ecology at the Faculty of Biology, Adam Mickiewicz University in Poznań, Poland. The tardigrade collection, extraction, and mounting techniques followed the protocol of Dastych [34].

Microscopy and Imaging
A total of 38 specimens and eight eggs were mounted on microscope slides in Hoyer's medium, and then examined under an Olympus BX41 Phase Contrast light Microscope (PCM) associated with an Olympus SC50 digital camera (Olympus Corporation, Shinjuku-ku, Japan).
Three eggs were prepared for scanning electron microscope (SEM) analysis according to the protocol in Roszkowska et al. [35] and examined under a high vacuum in Hitachi S3000N SEM.
All figures were assembled in Corel Photo-Paint 2017. For deep structures that could not be fully focused in a single photograph, a series of 2-50 images was taken every ca. 0.5 µm depth and then manually assembled into a single deep-focus image in Corel Photo-Paint 2017.

Morphometrics and Morphological Nomenclature
All measurements are given in micrometres [µm]. The sample size was adjusted following recommendations by Stec et al. [36]. 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 type of bucco-pharyngeal apparatus and claws was classified according to Pilato and Binda [37]. The terminology used to describe oral cavity armature and egg shell morphology follows Michalczyk and Kaczmarek [38] and Kaczmarek and Michalczyk [39]. Macroplacoid length sequence is given according to Kaczmarek et al. [40]. The buccal tube length and the level of the stylet support insertion point were measured according to Pilato [41]. The pt index is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage [41]. Cuticular bars and muscle attachments under claws were classified according to Kiosya et al. [42]. All other measurements and nomenclature follow Kaczmarek and Michalczyk [39]. Morphometric data were handled using the "Parachela" ver. 1.8 template available from the Tardigrada Register [43]. Raw morphometric data for each analyzed species are provided as Supplementary Materials (SM. 1). Tardigrade taxonomy follows Bertolani et al. [16] and Stec et al. [18].

Genotyping
Three specimens of M. intermedius (isolates numbers: Min3GR, Min4GR, and Min6GR) were preliminarily identified in vivo using light microscopy (LM) prior to DNA extraction for genotyping analysis. Genomic DNA was extracted from individual animals using a Chelex ® 100 resin (Bio-Rad) method [44] with modifications described in detail in Kaczmarek et al. [45]. The tardigrade exoskeleton was extracted from a pellet containing Chelex beads on the bottom of each tube. Obtained exoskeletons were mounted on a microscope slide in Hoyer's medium for further morphological analysis and deposited in the collection of the Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Poznań (Poland).

Comparative Molecular Analysis
Obtained mitochondrial and nuclear sequences were checked for quality and were manually aligned in BioEdit v. 7.2.5 [50]. Basic local alignment search tool (BLAST) [51] searches were performed to verify the identity and homology of the obtained gene fragments with sequences deposited in the NCBI database. The COI sequence was translated into amino acid sequences with the invertebrate mitochondrial codon table and the 2th reading frame using the EMBOSS-TRANSEQ application [52,53].
For molecular comparisons, all sequences of species belonging to the genus Minibiotus were downloaded from the GenBank database and aligned using the ClustalW multiple alignment tool [54], implemented in BioEdit v. 7.2.5. Only the available GenBank sequences that represented a homologous fragment with nrDNA and mtDNA sequences obtained in our study were applied. Alignment sequences were trimmed to 472, 1138, and 678 bp for COI (13 sequences), 18S rRNA (6 sequences), and 28S rRNA (10 sequences) molecular markers, respectively. The uncorrected p-distances were calculated using pairwise deletion for the gap/missing data treatment option and the software MEGA X [55]. Detailed p-distance tables are presented as Supplementary Materials (SM.02).
Phylogenetic trees were computed using the software MEGA X by applying maximum likelihood (ML) analysis under the general settings of selected models with 1000 bootstraps. The best-fit substitution models were determined using jModelTest v. 2.1.4 [56] with the assumptions of both the Bayesian inference criterion (BIC) and the Akaike information criterion (AIC) [57]: the Hasegawa-Kishino-Yano with gamma (HKY + G) distribution (G parameter = 0.1899) [58] for COI sequences, the Kimura 2-parameter (K2) model [59] for 18S rRNA sequences, and the Kimura 2-parameter (K2 + G) model with gamma distribution (G parameter = 0.1709) for 28S rRNA sequences. The molecular markers of Macrobiotus porifini (GenBank accession numbers: COI-MT246659, 18S rRNA-MT241900, 28S rRNA-MT241897 [60]) were used as outgroups. Initial evolutionary trees for the heuristic search were generated by applying BioNJ and neighbor join algorithms to a matrix of pairwise distances (which was estimated using the maximum composite likelihood (MCL) approach). Finally, the topology of phylogenetic trees was selected with superior log likelihood value and visualized by FigTree v.1.4.3 and Inkscape 0.92.
Claws stout, of the hufelandi type ( Figure 3A,B). The primary branches had very large and distinct accessory points, a common tract, and an evident stalk ( Figure 3A,B). Under PCM, very poorly visible granulation was present on the IV pair of legs (visible mainly in larger specimens) ( Figure 3C, filled arrowhead). The lunulae was smooth on all legs. The cuticular bars under the claws were absent. Double muscle attachments were faintly marked under PCM ( Figure 3A, empty arrowhead).  Table 2) laid freely, white, spherical, or slightly ovoid ( Figure 4A-D). Processes were nail-shaped (shaped like the head of a screw) (Figures 4A-D and 5A-E). Each process was covered by a separate membrane ( Figure  5A,B,D). The heads of the processes were always wider than the process bases. In SEM, a central and deep depression was present at the top of the processes ( Figure 5B,C,E). This structure was also visible under PCM as a lighter circles at the processes tops (5D). Under PCM the egg surface between process smooth whereas under SEM usually striae extend from each process connecting it with the neighboring processes ( Figure 5B,C, arrows). These striae (which are in fact formed by membrane) formed four quadratic areas that surrounded each egg process resembling poorly marked areolae. Within each of these areas' wrinkles form 1-2 flat rose-like whorl structures ( Figure 5B,C, arrows).

Short Diagnosis
The body cuticle was smooth without pores and granulation, but very poorly visible granulation on the IV pair of legs was present. Large eye spots were also present. The buccal tube with an anterior and a posterior bend and slightly thicker below the stylet insertion point. The pharyngeal bulb had apophyses, three granular macroplacoids, and a small microplacoid (length sequence 2 ≤ 3 < 1). Eggs with processes (28-32 around the circumference) in the shape of a screw's head were covered by a separate membrane.

Description of the Neotypic Population
Animals (measurements and statistics in Table 1). Body white in live specimens and transparent after fixation in Hoyer's medium (Figure 1). Eyes were present in all specimens after mounting in the Hoyer's medium (Figure 1). The body cuticle was smooth without pores and granulation (Figure 1).
Mouth antero-ventral surrounded by ten peribuccal papulae or shortened lamellae. The bucco-pharyngeal apparatus of the Minibiotus type (Figure 2A) had an anterior and a posterior bend (clearly visible in lateral view). The buccal tube was slightly thicker below the stylet insertion point (Figure 2A, arrow). Under PCM, only a third band of teeth was faintly visible (Figure 2A).
Claws stout, of the hufelandi type ( Figure 3A,B). The primary branches had very large and distinct accessory points, a common tract, and an evident stalk ( Figure 3A,B). Under PCM, very poorly visible granulation was present on the IV pair of legs (visible mainly in larger specimens) ( Figure 3C, filled arrowhead). The lunulae was smooth on all legs. The cuticular bars under the claws were absent. Double muscle attachments were faintly marked under PCM ( Figure 3A, empty arrowhead).
Eggs (measurements and statistics in Table 2) laid freely, white, spherical, or slightly ovoid ( Figure 4A-D). Processes were nail-shaped (shaped like the head of a screw) ( Figures 4A-D and 5A-E). Each process was covered by a separate membrane ( Figure 5A,B,D). The heads of the processes were always wider than the process bases. In SEM, a central and deep depression was present at the top of the processes ( Figure 5B,C,E). This structure was also visible under PCM as a lighter circles at the processes tops (5D). Under PCM the egg surface between process smooth whereas under SEM usually striae extend from each process connecting it with the neighboring processes ( Figure 5B,C, arrows). These striae (which are in fact formed by membrane) formed four quadratic areas Diversity 2022, 14, 356 9 of 15 that surrounded each egg process resembling poorly marked areolae. Within each of these areas' wrinkles form 1-2 flat rose-like whorl structures ( Figure 5B,C, arrows).

Comparisons with Other Genetic Sequences of Minibiotus Taxa
All the obtained sequences of M. intermedius were unique and distinct from those deposited in GenBank. The COI molecular marker exhibited a single sequence. In the conservative 18S rRNA gene fragment, we observed no differences between two sequences. In 28S rRNA, two haplotypes were found (three sequences), with a p-distance of 0.6%.
The ranges of uncorrected genetic p-distances between M. intermedius and other species/taxa belonging to the genus Minibiotus are as follows (please see SM2):

Morphological Differential Diagnosis
Minibiotus intermedius, by the morphology of adults (smooth dorsal and ventral cuticle and absence of pores) and eggs (processes in shape of a screw's head and covered by a separate membrane), is most similar to: M. continuus (Pilato and Lisi [67]), M. floriparus (Claxton [10]), and M. taiti (Claxton [10]). However it differs from:

Establishing of the New Neotype and Neoparatypes of M. intermedius
Taking into consideration that M. intermedius was described in 1888 by Plate, based on specimens from Chile and Germany, we can probably assume that the type material of M. intermedius no longer exists. What is more accurate diagnose of the species were poorly described in the past so that it was necessary to establish a neotype series of this species. Claxton [9] redescribed M. intermedius and established neotype altogether with three syntypes for four specimens collected in Marburg on 27 August 1994. Importantly, Claxton did not report any eggs from this locality, and she unjustifiably assigned eggs from other localities (Africa, Australia, Europe, New Zealand, and North and South America) for this redescription. This action should be criticized as it is commonly known that cryptic species or complexes of extremely similar species are often reported for tardigrades. In such a case, one should be extremely careful while assigning morphotype of eggs to the animal morphotype, especially in groups such as macrobiotids, in which egg ornaments hold a number of characters which are important in species identification. Moreover, the exact location (with geographic coordinates) in Marburg was also not reported by Claxton [9]. In addition, so far, DNA barcodes are unknown for M. intermedius, as confident species identification is currently impossible.
For this reason, considering all issues regarding the Claxton's neotype, we decided that existing neotype and syntypes are invalid due to the lack of eggs from the locality, which makes the correct identification of the species impossible (see ICZN, article 75.3.4 and 75.3.5). In such a situation, we designate a new neotype (specimen) altogether with 45 neoparatypes (37 specimens and 8 eggs) of M. intermedius collected from the type locality in Marburg (Germany), which is in agreement with ICZN article 75.3.5. The detailed characterization of the neotype population by integrated analysis can stabilize the taxonomy of the genus and allow for more detailed exploration of its diversity. Specimens of the neotype series were deposited in the Department of Animal Taxonomy and Ecology of Adam Mickiewicz University in Poznań, Poland, as well as in the Institute of Systematics and Evolution of Animals of the Polish Academy of Sciences, Kraków, Poland. All the above-mentioned statements are in accordance with the International Commission on Zoological Nomenclature (ICZN) acts dedicated to establishing a neotype series.

Discussion
In our study, we used integrative taxonomy to describe M. intermedius specimens from one of reported terra typica-Marburg (Germany). Our analysis has shown that our specimens and their eggs are morphologically very similar to specimens and eggs used for the first redescription of this species by Claxton [9]. However, in the population studied by us, eggs were a little smaller than those described by Claxton [9], i.e. egg bare diameter: 40.0-45.0 µm (in [9]) vs. 46.3-54.5 µm (in studied population), egg full diameter: 45.0-52.0 µm vs. 52.1-61.4 µm. Moreover, a ring of tiny pores on the top of egg processes was reported by Claxton [9], though these pores were absent in the population in our study. However, as noted previously, eggs described by Claxton [9] were not collected in type locality in Marburg, but in many different localities. Therefore, in this situation, they cannot be confidently assigned to the true M. intermedius and it is very likely that they may belong to a completely different Minibiotus species. The detailed redescription presented by us in this study with detailed phenotypic and genetic data for the neotype population will effectively prevent similar misunderstandings in the future. This will directly contribute to the knowledge of the true distribution range of this species, which are most probably overestimated at present due to the mentioned identification problem [68][69][70][71][72][73][74].
Molecular markers (i.e., COI, 18S rRNA, and 28S rRNA sequences) were only available for four Minibiotus species and seven taxa belonging to an undefined species of the genus Minibiotus. The ML phylogenetic reconstructions based on the limited molecular data sets yielded a congruent topology ( Figure 6) with two main clades: the first clade comprised M. intermedius (without pores) and M. gumersindoi (with pores) and the second clade contained M. furcatus (with pores). For M. pentannulatus (with pores) and M. ioculator (without pores), only COI and 28S rRNA sequences were available which were clustered together with the second clade. It is obvious that the taxonomic position of some sequences deposited in GenBank, described as M. furcatus, still needs further verification. We are not convinced which of these sequences have been correctly flagged, and all of them need revision. However, we assumed that the two COI sequences, i.e., deposited in GenBank as JX683828 and JX683829, seem to be questionable because they were clustered with the first clade. Diversity 2022, 14, x FOR PEER REVIEW 11 of 15  In summary, we propose a new population of the M. intermedius from Marburg as a type population of this species. Re-establishing the type species of the genus Minibiotus with provided integrative description should facilitate the description of new taxa within this genus.