Integrative Descriptions of Two New Tardigrade Species along with the New Record of Mesobiotus skorackii Kaczmarek et al., 2018 from Canada

: Two new tardigrade species from a moss sample collected in Canada, one representing Macrobiotus hufelandi complex and the second one belonging to the genus Bryodelphax , are described. Integrative analysis was undertaken based on morphological and morphometric data (using both light and scanning electron microscopy (SEM)) combined with multilocus molecular analysis (nuclear sequences, i.e., 18S rRNA, 28S rRNA and ITS-2 as well as mitochondrial COI barcode sequences). Based on COI sequences, Macrobiotus birendrai sp. nov. is most similar to Mac. canaricus (p-distance 17%), whereas Bryodelphax mareki sp. nov. is most similar to Bry. parvulus (p-distance 16%). Both species differ also from their congeners in some morphological and morphometric characters of adults and/or details of egg chorion. Additionally, a large population of Mesobiotus skorackii was found in the sample and this is the ﬁrst report of this species outside its terra typica in Kirghizia. The original description of this species was prepared based solely on the morphology and morphometry, therefore, here we provide updated data for this species enclosing morphometric and molecular data for the Canadian population.


Introduction
Canada is the second largest country in the world which extends its longitude from approximately 52 • to 141 • W to latitude approximately 42 • to 83 • N. It has such a distance that spans in six time zones and has a wide variety of climates. The highest peak in Canada which is Mount Logan reaches 5959 m asl and the country's landform structure can be considered a vast basin. Additionally, people living in two-thirds of the area experience very cold winters and short, cool summers. However, the interior plains of central southern area come with very cold winters, hot summers, and relatively sparse precipitation. Nonetheless, climate with hot, humid summers and cold, snowy winters also prevails in Southern Ontario and Quebec. Except for the west coast, all of Canada has a winter season with average temperatures below freezing and with continuous snow cover (https://www.britannica.com/place/Canada (accessed on 18 June 2021).
Tardigrada, also commonly known as water bears, inhabit in terrestrial and aquatic (freshwater and marine) environments. They can be found on aquatic plants and/or in lichens, leaf litter, mosses, soil, sediments [1][2][3]. To date, more than ca. 1300 species of tardigrades have been described throughout the world [4][5][6][7]. The genus Bryodelphax [8] is unique amongst Echiniscidae with some peculiar apomorphies like presence of 10 peribuccal papulae and plesiomorphies like ancestral type of the buccal apparatus, which makes Bryodelphax a good example of mosaic evolution in tardigrades [9,10]. Moreover, it is characterized by the presence of median plates 1 and 2 divided, median plate 3 not divided, and absence of notches on terminal plate. Up to now, 26 species were attributed to this genus [7]. The genus Macrobiotus [11] is one of the most species-rich and widespread genus in the phylum being also, the first formally described tardigrade genus. It is characterized by the presence of a rigid buccal tube with a straight ventral lamina lacking a ventral hook, 10 peribuccal lamellae, pharynx with two macroplacoids and microplacoid, symmetrical diploclaws and freely laid ornamented eggs [12]. Up to now, 118 species were attributed to this genus [7].
Tardigrade fauna of Canada is rather poorly known and up to now only 121 species have been reported from this region [13,14]. In this study, we applied integrative taxonomy for description of two new species from Canada belonging to the genus Bryodelphax and the Macrobiotus hufelandi complex. Moreover, we enriched this paper in additional molecular and morphometric data of the Canadian record of Mesobiotus skorackii Kaczmarek, Zawierucha, Buda, Stec, Gawlak, Michalczyk and Roszkowska [15], as the original description of this species was prepared based solely on the morphology and morphometry.

Sampling
A moss sample was collected in Banff National Park (AB, Canada) in March 2019. It 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 Stec et al. [16].

Microscopy and Imaging
In total, 163 animals (74 Bryodelphax mareki sp. nov. + 45 Macrobiotus birendrai sp. nov. + 44 Mesobiotus skorackii) and 31 eggs (12 Macrobiotus birendrai sp. nov + 19 Mesobiotus skorackii) were mounted on microscope slides in the Hoyer's medium, and then examined under Olympus BX41 Phase Contrast light Microscope (PCM) associated with Olympus SC50 digital camera (Olympus Corporation, Shinjuku-ku, Japan). The 44 animals and 8 eggs were prepared for scanning electron microscopy (SEM) analysis according to the protocol in Roszkowska et al. [17] and examined under high vacuum in Hitachi S3000N SEM. Thirty-one specimens were prepared for genotyping.
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 2-10 images were taken every ca. 0.5 µm and then manually assembled into a single deep-focus image in Corel Photo-Paint 2017.

Morphometrics and Morphological Nomenclature
All measurements are given in micrometers (µm]). Structures were measured only if their orientation was suitable. Body length was measured from the anterior extremity to the end of the body, excluding hind legs. The sp index in Bryodelphax is the ratio of the length of a given structure to the length of the scapular plate expressed as a percentage (length of structure × 100⁄length scapular plate) [18] and later independently proposed as the psc index by Fontoura and Morais [19]. Ventral plates configuration in Bryodelphax is given according to Kaczmarek et al. [20]. The types of bucco-pharyngeal apparatuses and claws of Macrobiotidae were classified according to Pilato and Binda [21]. All measurements and terminology of adults and eggs of Macrobiotidae were prepared according to Kaczmarek and Michalczyk [22] and Kaczmarek et al. [23]. Terminology describing the oral cavity armature (OCA) in Macrobiotus and Mesobiotus follows Michalczyk and Kaczmarek [24] and OCA morphotypes are given according to Kaczmarek and Michalczyk [22]. The macroplacoid length sequence in Macrobiotus and Mesobiotus was indicated according to Kaczmarek et al. [25]. The pt ratio is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage [26]. The terminology of cuticular bars in macrobiotid legs follows Kiosya et al. [27]. The classification of type of egg process, sculpture on egg processes, egg processes bases and egg shell surface between processes are given according to Kaczmarek et al. [23]. Genus abbreviations follow Perry et al. [28].

Comparative Material
For identification and differentiation of the new species, the key by Gąsiorek et al. [30] for the genus Bryodelphax and the key by Kaczmarek and Michalczyk [22] for the genus Macrobiotus were used. We also compared our new species with the type material of Bry. aaseae Kristensen, Michalczyk and Kaczmarek [9], Bry. asiaticus Kaczmarek and Michalczyk [31]

Genotyping
Prior to DNA extraction, individual tardigrades from the three species were preliminarily identified in vivo using light microscopy (LM). Genomic DNA was extracted using a Chelex ®® 100 resin (Bio-Rad, Hercules, CA, USA) extraction method [42] with modification in order to obtain voucher specimens, i.e., tardigrade exoskeletons [43]. After DNA extraction we performed morphological analysis following the protocol of Kaczmarek et al. [43]. Then, all exoskeletons were deposited in the collection of the Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Poznań.
In total, four molecular markers were amplified: one mitochondrial gene, i.e., COIthe cytochrome oxidase subunit I; three nuclear markers, i.e., 18S rRNA-the small ribosome subunit and 28S rRNA-the large ribosome subunit as well as ITS-2-the internal transcribed spacer-2. The polymerase chain reaction (PCR) amplification was performed according to Kaczmarek et al. [44]. The sequences of primers applied to amplify molecular markers are listed in Table 1. All PCR reactions were conducted in a Biometra TProfessional thermocycler. Prior to the sequencing, the PCR products were treated with the FastAP Alkaline Phosphatase and thermosensitive Exonuclease I (Fermentas, Thermo Scientific, Waltham, MA, USA) according to the manufacturer's guidelines. Sanger DNA sequencing in both directions was performed by Macrogen (Amsterdam, The Netherlands).

Comparative Genetic Analysis
Obtained mtDNA and nrDNA sequences were quality checked and consensus sequences were created for individual tardigrades in BioEdit v. 7.2.5 [49]. All COI sequences were translated into amino acid sequences to check against pseudogenes using the EMBOSS-TRANSEQ application [50,51]. The translation was performed with the invertebrate mitochondrial codon table. To verify the homology of the amplified DNA region, Basic Local Alignment Search Tool [52] searches at the National Centre for Biotechnology Information NCBI were applied.
All obtained sequences were deposited in GenBank and the accession numbers are listed in Table 2. For molecular comparisons, all sequences of the mtDNA and nrDNA fragments of the genera Bryodelphax, Macrobiotus and Mesobiotus were downloaded from the GenBank and trimmed to the same length in BioEdit v. 7.2.5. The COI sequences could be unambiguously aligned without inserting gaps. In turn, the sequences of nrDNA were aligned using ClustalW Multiple Alignment tool [53] implemented in BioEdit v. 7.2.5. with default settings. Uncorrected pairwise distances were calculated using MEGA X [54].

Taxonomic Account
Phylum: Tardigrada Doyère, 1840 [55] Class         Material examined: The 74 animals, i.e., holotype + 73 paratypes (females: 37; undefined sex: 34 and 2 exuviae) mounted on microscope slides in Hoyer's medium, 40 animals prepared for SEM and 20 animals prepared for molecular analyses (not included in the type series). However, DNA sequences were obtained from only five specimens (exoskeletons) which was later mounted on microscope slide in Hoyer's medium and included into type series.  Material examined: The 74 animals, i.e., holotype + 73 paratypes (females: 37; undefined sex: 34 and 2 exuviae) mounted on microscope slides in Hoyer's medium, 40 animals prepared for SEM and 20 animals prepared for molecular analyses (not included in the type series). However, DNA sequences were obtained from only five specimens (exoskeletons) which was later mounted on microscope slide in Hoyer's medium and included into type series.

Description of the new species
Adult females (measurements and statistics in Table 3). Body light yellow in live specimens (transparent after mounting in Hoyer's medium) ( Figure 1A-C), eyes absent or not visible after mounting on microscope slides. Small and conical primary and secondary clavae present. Cirri internus and externus with poorly developed cirrophores. Cirri internus always shorter than cirri externus. Cirri A of a typical length for Bryodelphax, i.e., up to 25% of the total body length. Only lateral appendages cirri A present apart from head appendages.
Dorsal sculpture, visible in PCM, composed of intra-cuticular pillars (visible as dark dots/granules) and pores (visible as white dots) ( Median plates 1 and 2 divided by smooth transverse stripe, median plate 3 undivided. Median plate 2 largest among all median plates. Paired plates 1 and 2 also divided transversely into two parts by smooth stripes. Ventral side with three rows of greyish plates (formula: III:2-2-1). First row with two plates just below the head ( Figure 3A,D, filled arrowheads). Three genital plates surrounding the gonopore (two lateral, in line with the gonopore) and the third one situated posteriorly to the gonopore ( Figure 3B-D, empty arrowheads)).
Papilla-like structure on leg I hardly visible under PCM but visible in SEM ( Figures 1C and 4A,B, arrow). Papillae on leg IV present ( Figures 1C and 4D, arrowhead). Dentate collar absent on leg IV ( Figure 1A-C). All claws slender, claws IV always slightly longer than claws I-III. External claws smooth, internal ones with a small spur pointing downward and placed very close to the claw bases ( Figure 4A-D). The female gonopore with the typical six-petal rosette. Males

Genetic variability
Aligned sequences (obtained in present study and downloaded from GenBank) were trimmed to 591, 498 and 700 bp for COI (four sequences; two species), 18S rRNA (eight sequences; four species and two sequences of Bryodelphax sp.) and 28S rRNA (14 sequences; seven species and two sequences of Bryodelphax sp.) molecular markers, respectively. Only sequences of Bryodelphax downloaded from GenBank that coincided with our four aforementioned molecular markers were selected.
In the case of the COI molecular marker, only three sequences of Bry. parvulus [8] were available in GenBank. Analysis of the p-distances between our sequences (GenBank accession numbers: MW655785-87) and three sequences of Bry. parvulus was from 16% (GenBank accession numbers: JX683827, JX683826, unpublished) to 18% (GenBank accession number: HM193405 [61]). In the case of the 18S rRNA molecular marker (GenBank accession numbers of our sequences: MW680639-40), no genetic differences were observed when compared with Bry. parvulus (GenBank accession numbers: HM193371 [61]; JX676189, [62]) and p-distance between other sequences, i.e., Bryodelphax sp. (GenBank accession numbers: EU266963 and EF632433 [63]) and Bry. tatrensis [64] (GenBank accession numbers: JX676188 and JX676190 [62]) was 0.01%. The ranges of uncorrected genetic p-distances between our 28S rRNA sequences (GenBank accession numbers:    Table 4. Measurements (in µm) and pt values of selected morphological structures of individuals of Macrobiotus birendrai sp. nov. mounted in Hoyer's medium (N-number of specimens/structures measured; RANGE refers to the smallest and the largest structure among all measured specimens; SD-standard deviation, pt-ratio of the length of a given structure to the length of the buccal tube expressed as a percentage).       Material examined: The 57 specimens, i.e., holotype (slide: CN8.43) + 56 paratypes (adults: 44 and eggs: 12) were mounted on microscope slides in Hoyer's medium, four eggs prepared for SEM and five animals prepared for molecular analyses (not included in type series). However, DNA sequences were obtained from one female specimen (exoskeleton) which was later mounted on microscope slide in Hoyer's medium and included into type series. Etymology: The first author would like to dedicate this species to her father-Birendra Prasad Lal Karna.
Description of the new species. Adults (measurements and statistics in Table 4). Body transparent after fixation in Hoyer's medium, eyes present in all fixed specimens ( Figure 5A). Entire cuticle covered with conspicuous round and lenticular pores (0.6-1.8 µm in diameter) distributed randomly ( Figure 5B). However, larger pores present, on dorsal side, at the anterior and posterior part of the body. Bucco-pharyngeal apparatus of the Macrobiotus type, with ventral lamina and 10 peribuccal lamellae ( Figure 6A). Mouth antero-ventral. Oral cavity armature of the hufelandi type, with first and the second band composed of numerous minute teeth (visible as granules in PCM) and third composed of three dorsal and three ventral transverse ridges ( Figure 6B-D). Pharyngeal bulb spherical with triangular apophyses, two rod-shaped macroplacoids and a triangular microplacoid. Macroplacoid length sequence 2 < 1 ( Figure 6A,E). The first macroplacoid with central constriction ( Figure 6E, arrowhead), second with sub-terminal constriction. Claws of the hufelandi type ( Figure 7A Eggs (measurements and statistics in Table 5). Eggs spherical, ornamented and laid freely with egg chorion of the hufelandi type ( Figure 8A,B). Pores of egg surface mesh circular, similar in size and rather small, i.e., 0.2-0.8 µm in diameter ( Figure 8C,E,F). Processes in the shape of inverted concave cups with terminal discs (Figure 8D-H). Terminal discs concave with serrated margins or with small irregular teeth ( Figure 8D-H).
Short diagnosis: Adults (measurements and statistics in Table 6). Body white in living animals and transparent after fixation in Hoyer's medium, eyes present, cuticle smooth. Buccopharyngeal apparatus of the Macrobiotus type, with ventral lamina and ten peribuccal lamellae. Mouth antero-ventral. Oral cavity armature of the harmsworthi type. Pharyngeal bulb spherical with triangular apophyses, three rod-shaped macroplacoids and a triangular microplacoid. Macroplacoid length sequence 2 < 3 < 1. The first macroplacoid narrower anteriorly, the second without constrictions and the third with a small, subterminal constriction. Claws of the Mesobiotus type. Lunules under claws I-III smooth and slightly dentated under claws IV. Thin cuticular bars under claws I-III present. Granulation hardly visible on legs I-III, whereas on legs IV always clearly marked.
Eggs (measurements and statistics in Table 7). Eggs laid freely, white and spherical. Egg processes in the shape of short and wide sharpened cones. Egg processes reticulated and surrounded by six areolae delimited by thin brims which are often discontinuous, thus areolae are not always fully formed (semi-areolation). Surface inside the areolae with clearly visible wrinkles.

DNA sequences
We obtained good quality sequences for the applied molecular markers: COI: single sequence; 631 bp long; 18S rRNA: two sequences; 667-715 bp long; 28S rRNA: single sequence; 735 bp long.

Genetic variability
Aligned sequences (obtained in our study and downloaded from GenBank) were trimmed to 565, 474 and 713 bp for COI (14 sequences, selected from GenBank-one sequence per species; (see Supplementary Materials-SM6)), 18S rRNA (two sequences selected from GenBank-one sequence per species; see below) and 28S rRNA (13 sequences selected from GenBank-one sequence per species) molecular markers, respectively.

Discussion
Out of 10 provinces and three territories of Canada, limno-terrestrial tardigrades have been reported in eight provinces and two territories. Up to now, no tardigrades have been reported from Northwest Territories, Nova Scotia nor Prince Edward Island. The highest number of tardigrade species were recorded from Nunavut (70) and the lowest Manitoba (only one). Moreover, 18 species were recorded from Alberta, 55 from British Columbia, 33 from New Brunswick, 29 from Newfoundland and Labrador, 12 from Ontario, 13 from Quebec, 3 from Saskatchewan and 5 from Yukon [13,14]. Only one species of the genus Bryodelphax, i.e., Bry. parvulus has been recorded from British Columbia and Nunavut. In case of the genus Macrobiotus, four species i.e., Mac. echinogenitus Richters [81], Mac. hufelandi, Mac. occidentalis Murray [82] and Mac. virgatus Murray [82] were recorded from Alberta, British Columbia, New Brunswick, Newfoundland and Labrador, Nunavut, Ontario and Quebec. Among these, only Mac. hufelandi belongs to the hufelandi group. Three species of the genus Mesobiotus, i.e., Meb. harmsworthi (Murray [83]), Meb. montanus (Murray [82]) and Meb. pilatoi (Binda and Rebecchi [84]) were recorded from Alberta, British Columbia, New Brunswick, Newfoundland and Labrador, Nunavut, Ontario and Quebec [14]. What is more intriguing, some species reported from Canada in the past are now considered as group of species or species with problematic taxonomical status [14].
Summarizing, from Canada only 121 tardigrade species and subspecies are known. Taking into consideration the area of the country (ca. 10 million km 2 ) and its diversity such as habitats and ecosystem, it is obvious that this number is highly underestimated. For comparison, USA, with the similar country area, has more than 220 species reported [14]. This contrast is even more spectacular while comparing Canadian tardigrade fauna with the number of tardigrade species from much smaller areas like e.g., Costa Rica (ca. 51,000 km 2 and 63 species known), Finland (ca. 340,000 km 2 and 68 species known), Italy (ca. 300,000 km 2 and 234 species known) or Poland (ca. 312,000 km 2 and 111 species known) [68,[85][86][87][88][89]. The number of tardigrade species from Canada is expected to be much higher than reported up to date, especially that in the present study, in one analyzed sample, we found two species new for science and one new record for the country.

Conflicts of Interest:
The authors declare no conflict of interest.