New Records of Dactylobiotus parthenogeneticus Bertolani, 1982 Provide Insight into Its Genetic Variability and Geographic Distribution

Dactylobiotus 1982 provide insight into its genetic variability and distribution. 68 : 57-72. In sediment samples collected from three distinct European locations (United Kingdom, France, Poland), populations of Dactylobiotus parthenogeneticus were found. The original description of this species was based solely on the morphology observed with light microscopy and later supplemented by some additional SEM data of the buccal apparatus and DNA sequences of 18S rRNA and COI. Here we provide an updated description of the species by means of integrative taxonomy. The description comprises a comprehensive set of morphometric and morphological data from light and scanning microscopy as well as nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) fragments. Our analysis of haplotype diversity confirmed our morphological identification and showed that D. parthenogeneticus is widely distributed in Europe.

Tardigrades, known also as water bears, are a phylum of micro-invertebrates that inhabit freshwater and marine habitats but also limno-terrestrial environments with at least temporary moisture (NELSON et al. 2015). Tardigrade taxonomy started almost two centuries ago and currently over 1300 nominal taxa are recognized within the phylum DEGMA & GUIDETTI 2007;DEGMA et al. 2019).
The genus Dactylobiotus Schuster, 1980 was erected by SCHUSTER et al. (1980), and presently it comprises 18 species, which are known to be exclusively aquatic (KIHM et al. 2020). In this study, we focus on Dactylobiotus parthenogeneticus Bertolani, 1982 which has been recorded numerous times from several European countries such as Italy, Greece, Poland and Spain (BERTOLANI 1982a,b;BINDA & GUGLIELMINO 1982;BERTOLANI 1988;GUIL 2002;GUIDETTI et al. 2006b;POPRAWA et al. 2015) but also from Argentina, Bolivia and Mexico (MEYER 2013;KACZMAREK et al. 2015; MORENO-TALAMANTES et al. 2015). The genus Dactylobiotus was established 40 years ago, and so far none of its species were described using an inte-grative approach except recently discovered species Dactylobiotus ovimutans KIHM et al., 2020. Therefore, the morphological data for the majority of Dactylobiotus taxa were collected only with light microscopy and were not associated with genetic markers. This limitation and scarce genetic data are clearly visible in GenBank, where DNA sequences are provided for only three species: Dactylobiotus ambiguus (Murray, 1907) (28S rRNA and 18S rRNA), Dactylobiotus octavi Guidetti et al., 2006a (28S rRNA and 18S rRNA), and D. parthenogeneticus (18S rRNA and COI). In our work, we analysed three European populations of D. parthenogeneticus from Poland, France and Great Britain. We provide a new description of D. parthenogeneticus based on a detailed morphological examination with light and scanning electron microscopy as well as DNA sequences of the four standard molecular markers used in tardigrade taxonomy (18S rRNA, 28S rRNA, ITS-2 and COI). Based on our multifaceted approach and especially genetic comparisons, we confirmed that D. parthenogeneticus is distributed in aquatic habitats in at least three countries in Europe.
The samples were examined for tardigrades using the protocol by DASTYCH (1980) with modifications described in detail in STEC et al. (2015). For taxonomic analysis, animals and eggs isolated from the samples were separated 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 Table 1).

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, according to the protocol by MOREK et al. (2016). Slides were examined under an Olympus BX53 light microscope with phase contrast (PCM), connected with an Olympus DP74 digital camera. In order to obtain clean and extended specimens for scanning electron microscopy, tardigrades were processed according to the protocol by STEC et al. (2015). In short, specimens were first subjected to a 60°C water bath for 30 min to obtain fully extended animals, then to a water/ethanol and an ethanol/acetone series, then to CO 2 critical point drying and finally sputter coated with a thin layer of gold. Bucco-pharyngeal apparatuses were extracted according to the protocol of EIBYE-JACOBSEN (2001) as modified by G¥SIOREK et al. (2016). Specimens were examined under high vacuum in a Versa 3D Dual-Beam Scanning Electron Microscope at the ATOMIN facility of the Jagiellonian University, Kraków, Poland. All figures were assembled in Corel Photo-Paint X6, ver. 16.4.1.1281. For structures that could not be satisfactorily focused in a single light microscope photograph, a stack of 2-6 images were taken with an equidistance of ca. 0.2 ìm and assembled manually into a single deep-focus image in Corel Photo-Paint X6, ver. 16.4.1.1281.

Morphometrics and morphological nomenclature
All measurements are given in micrometres (ìm). Sample size was adjusted based on recommendations by STEC et al. (2016). 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 the oral cavity armature and egg shell morphology is given in MICHALCZYK and KACZMAREK (2003) and KACZMAREK and MICHALCZYK (2017), respectively. Macroplacoid length sequence is given according to the method in KACZMAREK et al. (2014). Buccal tube length and the level of the stylet support insertion point were measured according to PILATO (1981). The pt index is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage (PILATO 1981). Buccal tube width was measured according to KACZMAREK and MICHALCZYK (2017) as the external and internal diameter at the level of the stylet support insertion point. Claws were measured according to BINDA and  PILATO (1999). Distance between egg processes was measured as the shortest line connecting base edges of the two randomly chosen closest processes (KACZMAREK & MICHALCZYK 2017). Morphometric data were handled using the "Parachela" ver.

Additional comparative material
For morphological comparison we used the original description as well as photomicrographs of the type series of D. parthenogeneticus deposited in the Roberto Bertolani collection taken by Piotr G¹siorek and Witold Morek (both of Jagiellonian University, Poland), thanks to the courtesy of Roberto Guidetti and Roberto Bertolani (University of Modena, Italy). Four additional SEM photomicrographs of the British population of D. parthenogeneticus were kindly provided by £ukasz Michalczyk and assembled within the figure plates (Figs 2 C-D and 8D, G).

Genotyping
The DNA was extracted from individual animals following a Chelex ® 100 resin (Bio-Rad) extraction method by CASQUET et al. (2012) with modifications described in detail in STEC et al. (2020a). We sequenced four DNA fragments: the small ribosome subunit (18S rRNA, nDNA), the large ribosome subunit (28S rRNA, nDNA), the internal transcribed spacer (ITS-2, nDNA), and the cytochrome oxidase subunit I (COI, mtDNA). All fragments were amplified and sequenced according to the protocols described in STEC et al. (2020a); primers and original references for specific PCR programs are listed in Table 2. Sequencing products were read with the ABI 3130xl sequencer at the Molecular Ecology Lab, Institute of Environmental Sciences of the Jagiellonian University, Kraków, Poland. Sequences were processed in BioEdit ver. 7.2.5 (HALL 1999) and submitted to GenBank.

Comparative molecular analysis
Since there are no published sequences of ITS-2, only the sequences of 18S rRNA, 28S rRNA and COI markers for species in the genus Dactylobiotus were downloaded from GenBank SANDS et al. 2008;CHEN et al. 2009, unpublished;JØRGENSEN et al. 2010;BERTOLANI et al. 2014;GUIL et al. 2019). However, nine 18S rRNA sequences (GQ925678-9, EF632436-42) and the only two 28S rRNA sequences (GQ849049 and MH079500) were not homologous with fragments sequenced in our study and thus excluded from further analysis. The sequences of each DNA marker were aligned separately using the AUTO (in the case of ITS-2 and COI) and the Q-INS-I strategy (in the case of ribosomal markers: 18S rRNA, 28S rRNA) of MAFFT version 7 (KATOH et al. 2002;KATOH & TOH 2008) and manually checked against non-conservative alignments in BioEdit. Then, the aligned sequences were trimmed to: 763 (18S rRNA), 769 (28S rRNA), 414 (ITS-2), 534 (COI) bp. All COI sequences were translated into protein sequences in MEGA7 version 7.0 (KUMAR et al. 2016) to check against pseudogenes. Uncorrected pairwise distances were calculated using MEGA7 and are provided as supplementary materials (Suppl. Mat. 2).

Animals
Body transparent in juveniles and whitish in adults, but transparent after fixation in Hoyer's medium (Fig.1A). In live specimens, eyes are present but they dissolve in Hoyer's medium. Cuticle without pores but clearly a robust semilunar cuticular connection is present between external/posterior and internal/anterior claws ( Fig. 3A-B). Under SEM this connection is visible as discontinuous, being composed of extended lunulaelike thickenings under the claws on the lateral sides whereas its median portion is located within or under cuticle ( Fig. 3C-D). Claws on the first three pairs of legs similar in size but obviously longer on the hind legs. Mouth antero-ventral followed by ten short peribuccal lamellae, bucco-pharyngeal apparatus of the Macrobiotus type (Figs 4A-G, 5A-H and 6A-B). Under PCM, the oral cavity armature comprises only the second and the third band of teeth ( Fig. 4B-C). However, in SEM three bands of teeth are clearly visible with the first band being situated at the base of peribuccal lamellae and composed of 4-5 rows of scattered small conical teeth arranged around the oral cavity (Figs 5E and 6A-B). The second band of teeth is situated below the ring fold, and comprises 4-6 rows of small cone-shaped teeth which are larger than those of the first band and increase in size towards the third band of teeth (Figs 4B-C, 5E 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 buccal tube opening (Figs 4B-C, 5E and 6A-B). The third band of teeth is discontinuous and divided into dorsal and the ventral portions. Under PCM, the dorsal teeth are seen as three distinct transversal ridges whereas the ventral teeth appear as two separate lateral transverse ridges, between which a roundish median tooth is visible (Fig. 4B-C). In SEM, both dorsal and ventral teeth are also clearly distinct (Figs 5E and 6A-B). Under SEM, the dorsal teeth are sharpened at the end (Figs 5E and 6A-B), whereas the ventral portion of the third band of teeth comprises also several smaller additional teeth (Figs 5E and 6A-B). Under PCM in the lateral view of the buccal apparatus a strengthening bar (ventral lamina) with an incision determining a ventral  hook is clearly visible (Fig. 4D-E). The hook is visible as an invagination in the ventral lamina when observed from lateral view under SEM that starts with bifurcation visible clearly only in ventral view (Fig.  5A, C) and develops further below the common tract of the bifurcation. The ventral portion of the hook visible in PCM (Fig. 4D-E) is actually constituted by a common tract of the bifurcation and the branches of the bifurcation (Fig. 5A, C), while the opening of the hook under PCM (Fig. 4D-E) is the opening of the invagination visible under SEM (Fig. 5A, C) Measurements and statistics are given in Table 3.

Eggs
Laid freely, whitish, spherical (Figs 7A and 8A). Processes in the shape of short and wide cones with apexes divided into multiple (typically three to six) short, nodular, finger-like apices (Figs 7B-E and 8B-G, I). Under SEM, apices usually covered with microgranulation (Fig. 8I). Egg surface between the   processes seems to be smooth under PCM (Fig. 7D-E), whereas under SEM it is clearly wrinkled (Fig. 8B-F). Under PCM, the margins of processes bases seem to be serrated and surrounded by a crown of small thickenings/projections (Fig. 7D-E), which are internal strengthening structures stabilising the processes within the chorion, clearly visible under SEM when the chorion is broken (Fig. 8G-H) or vertical thickenings present on basal portions of processes walls ( Fig. 8B-C, E-F). Sometimes, micropores are present on the egg surface near the processes' base but they are visible only under SEM ( Fig. 8B-C, E). Eggs are sticky because they are covered by mucus which most likely enhances their adhesion to the substrate and maybe has also a protective function. This mucus is clearly visible under SEM as a web of flexible filaments that cover the egg surface (Fig. 8B-F) but is only faintly visible under PCM (Fig. 7D-E). Measurements and statistics are given in Table 4.

Remarks
In comparison with the original description the following morphological characters are newly reported for the species: the presence of the first band of teeth in the oral cavity armature visible only under SEM and the presence of constrictions in the first and second macroplacoids. Furthermore the updated description provides much more detailed morphological characterisation of the claws, oral cavity armature as well as egg ornamentation. Finally we did not notice any obvious variation in the observed morphological characters between specimens from the three distinct population examined in our study.

Genetic comparisons
Genetic distances showed small differences between the three Dactylobiotus populations examined in this work. All populations share the same 18S rRNA haplotype, whereas each population exhibits distinct 28S rRNA and ITS-2 haplotypes (Fig. 9A-B). The genetic distances are: 0.13-0.26% for 28S rRNA and 0.24-0.97% for ITS-2. The comparison with other 18S rRNA sequences from GenBank also shows very low genetic differences that range from 0.00% to 0.26%. Similarly, for COI all populations examined in this study exhibited at least one distinct, population-specific haplotype, however one of the two haplotypes in the Polish population is identical with the haplotype present in the French population (Fig. 9C). Moreover, comparisons with other COI sequences from GenBank confirmed that the three newly found populations represent D. parthenogeneticus as genetic distances between the haplotypes and the COI sequence of AY598771 are very small (2) A B C and range from 0.37% to 0.75%. The COI sequence of AY598771 comes from a pond located ca. 6 km from the pond where the type population of D. parthenogeneticus was found, thus it can be considered as a barcode reliably representing this species. The peat bog where the species was originally discovered (type locality) has been destroyed (Roberto Bertolani, pers. com.). Moreover, the comparison also showed that other COI sequences labelled as Dactylobiotus sp. (EF632523-9; The South Shetland Islands, Antarctica) belong to a different species as they all differ from all haplotypes of D. parthenogeneticus in more than 17% (please see Suppl. Mat. 2 for detailed matrices with genetic distances calculated between all analysed sequences).

Discussion
Our study provides detailed morphological and genetic data on an aquatic tardigrade species, Dactylobiotus parthenogeneticus, collected from three distinct localities and analysed with integrative taxonomy approach. These results will enhance future species identifications but also will contribute to studies on tardigrade phylogeny with the set of four molecular markers. The DNA sequences and haplotype analysis confirmed our initial morphological identification and affirmed that this species is most probably very common in Europe.
To date, there are only a few studies that have investigated the distribution of a single tardigrade species using genetic data in Europe (e.g. CESARI et al. 2009;JØRGENSEN et al. 2007JØRGENSEN et al. , 2013G¥SIOREK et al. , 2019bMOREK et al. 2019a) as well as on other continents (e.g. ZAWIERUCHA et al. 2018;G¥SIOREK et al. 2019c;JACKSON & MEYER 2019;KACZMAREK et al. 2020;SUGIURA et al. 2020). However, none of these studies was conducted on an exclusively aquatic, freshwater-dwelling tardigrade species. The most similar of all of these to our research in terms of tardigrade habitat were studies conducted by CESARI et al. ZAWIERUCHA et al. (2018). The first one focused on the distribution of Acutuncus antarcticus (RICHTERS, 1904), the most abundant and common tardigrade species in Antarctica, which lives in freshwater ecosystems and terrestrial microhabitats in soil, grass, algae, moss and lichen in non-glacial areas (MURRAY 1910;DASTYCH 1991). The second one analysed the geographic distribution pattern of Cryoconicus kaczmareki ZAWIERUCHA et al., 2018, a dark-pigmented tardigrade inhabiting cryoconite holes in mountain glaciers in China and Kyrgyzstan. Thus, our work can be considered as the first small-scale phylogeographic study on an exclusively aquatic tardigrade, which could have different dispersal modes compared to terrestrial species due to weak or absent anhydrobiotic abilities and -at the same time -the encystation capability of aquatic tar-digrades (e.g. GUIDETTI et al. 2006b;JANELT & POPRAWA 2020). For example, epizoochory, which was suggested for some terrestrial tardigrades (MOGLE et al. 2018;ROBERTSON et al. in press), may play a vital role in species transmission between water bodies by aquatic birds and mammals both on the intra-and inter-continental scale. This seems to be relevant as D. parthenogeneticus has already been reported from Argentina, Bolivia and Mexico (see the Introduction section). Since some recent works have demonstrated or suggested the existence of cryptic/pseudocryptic taxa in tardigrades (e.g. FAURBY et al. 2008;FONTOURA & MORAIS 2011;GUIDETTI et al. 2016;STEC et al. 2018;GUIDETTI et al.2019;MOREK et al. 2019b;SURMACZ et al. 2019;STEC et al. 2020aSTEC et al. , 2020b, these reports of D. parthenogeneticus from outside Europe and based only on morphological observations must be regarded with a dose of scepticism until genetically confirmed. Conversely, since the comparison of morphometric data obtained in our study with data presented by MORENO-TALAMANTES et al. (2015) showed no differences between Polish and Mexican populations, this suggests an extremely wide distribution range. This would not be very surprising especially since D. parthenogeneticus is a parthenogenetic species, and recent works have already demonstrated such an extensive distribution for asexual tardigrades in distinct genera, e.g. Paramacrobiotus Guidetti et al., 2009, Richtersius Pilato and Binda, 1989and Echiniscus Schultze, 1840(see G¥SIOREK et al. 2019dGUIDETTI et al. 2019;KACZMAREK et al. 2020;STEC et al. 2020a;STEC et al. 2020b).
As mentioned in the introduction, almost all Dactylobiotus species were originally described using only traditional morphological techniques with light microscopy and often with small sample sizes of animals and eggs. Previously KACZMAREK et al. (2008) and MORENO-TALAMANTES et al. (2015) listed three species with uncertain taxonomic positions, but we also noted a fourth species. The first of these is Dactylobiotus macronyx (Dujardin, 1851), whose validity was questioned by many taxonomists due to the very inadequate original description and the lack of a modern redescription (CUÉNOT 1932;MARCUS 1936;RAMAZZOTTI & MAUCCI 1983;BINDA & PILATO 1999;GUIDETTI et al. 2006a;KACZMAREK et al. 2008). The description states that the species lays smooth unornamented eggs within exuviae, which is atypical not only for the genus, but also for the entire order Macrobiotoidea. Thus, following also previous recommendations by BINDA and PILATO (1999) and GUIDETTI et al. (2006b) Fig. 2D in this work clearly shows that the claws of the exuviae belong to the recently established isohypsibioid aquatic genus Grevenius G¹siorek et al., 2019a. Thus, considering that the description is based on animals and eggs that represent different tardigrade orders, and that the species identification without eggs is almost impossible in the genus Dactylobiotus, here we also designate this species as nomen dubium: Dactylobiotus kansae Beasley et al., 2009 nom. dub. Two other Dactylobiotus species with highly insufficient descriptions are Dactylobiotus aquatilis Yang, 1999 and Dactylobiotus henanensis Yang, 2002. These descriptions do not contain any information on egg morphology, which is crucial for species identification within the genus; they lack detailed descriptions and/or measurements of other taxonomically important characteristics, such as cuticle morphology, claws and buccal apparatus; and they do not contain a proper differential diagnosis with other similar taxa. Since a correct identification of these species is impossible, we also propose to designate them as nomina dubia: Dactylobiotus aquatilis Yang, 1999 nom. dub. and Dactylobiotus henanensis Yang, 2002 nom. dub.