Integrative description of Macrobiotus kosmali sp. nov. (hufelandi group) from the Island of Madeira (Portugal)

Abstract Using integrative taxonomy, we describe Macrobiotus kosmali sp. nov. from Ribeira Brava, Madeira (Portugal). Based on morphological and morphometric data from both phase contrast light microscopy (PCM), as well as, from scanning electron microscopy (SEM), description of the new species is provided. Additionally, four DNA markers, three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) were used to calculate the p-distances between Macrobiotus kosmali sp. nov. and other species of the genus Macrobiotus. The uncorrected genetic distances indicated that the new species is the most similar to its four congeners, namely Mac. cf. recens and morphologically, new species is most similar to Mac. dariae, Mac. glebkai, Mac. noemiae, Mac. recens, Mac. rybaki and Mac. scoticus, but differs from them mainly in details of its egg chorion and egg processes. http://urn:lsid:zoobank.org:pub:B371F633-B35C-4578-8C75-9FA2B5E80588


Introduction
The archipelago of Madeira is located in the North Atlantic, approximately 630 km northwest from the West African coast (Casablanca, Morocco) and 900 km southwest from Europe (Lisbon, Portugal). It comprises the islands of Madeira, the largest island with an area of 741 km 2 , Porto Santo and the Desertas and together with the Selvagens, Canary Islands, Azores and Cape Verde archipelagos, makes up the biogeographical zone of Macaronesia. The Madeira climate is Mediterranean and deeply influenced by the northeast trade wind system, with weather conditions varying considerably between the south and north coasts and according to altitude (Gonçalves Silva & Ferreira 2019).
Species of the phylum Tardigrada, commonly called water bears, inhabit terrestrial and aquatic (freshwater and marine) environments. They are found in mosses, lichens, soil, leaf litter, sediments and on aquatic plants (Ramazzotti & Maucci 1983 with translation by Beasley 1995;Nelson et al. 2020a). Till date, more than ca. 1400 species of tardigrades have been described throughout the world (Guidetti & Bertolani 2005;Degma & Guidetti 2007Vicente & Bertolani 2013). Phylum Tardigrada is divided into two classes i.e., Heterotardigrada and Eutardigrada (Nelson et al. 2020b). The class Eutardigrada is later divided into two limnoterrestrial orders, Apochela and Parachela. The order Parachela, comprises the widespread, common and species rich superfamily Macrobiotoidea. Presence of a rigid buccal tube with a straight ventral bar lacking a ventral hook, pharynx with two macroplacoids and one microplacoid, 10 peribuccal lamellae, symmetrical diploclaws, pores in the cuticle, and freely laid ornamented eggs are main characteristics of nominal genus for the superfamily Macrobiotus C.A.S. Schultze 1834 (Stec et al. 2021a). The Macrobiotus genus is one of the most species-rich and widespread genera in entire the phylum Tardigrada, and it was also the first formally described tardigrade genus (Greven 2018). Till date, only four Macrobiotus species i.e., Macrobiotus echinogenitus Richters, 1903, Macrobiotus hufelandi C.A.S. Schultze, 1834, Macrobiotus occidentalis Murray, 1910 and Macrobiotus recens Cuénot, 1932 have been reported from the island of Madeira (Da Cunha & Do Nascimento Ribeiro 1962).
In this study, we applied integrative taxonomy in order to describe a new species of the genus Macrobiotus from the Island of Madeira.

Sampling
Moss sample was collected from riverbed of Ribeira Brava, Madeira (32°44ʹ29.8''N 17°01ʹ00.5''W), 517 metres asl in September 2019. 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 extraction and mounting techniques followed the protocol of Stec et al. (2015).

Microscopy and imaging
In total, 87 animals, 8 exuvium, 4 simplex and 31 eggs 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). Thirty animals and 30 eggs were prepared for Scanning Electron Microscope (SEM) analysis according to the protocol in Roszkowska et al. (2018), and examined under high vacuum in Hitachi S3000N SEM (Hitachi, Japan). The tardigrade exoskeleton was extracted from a pellet containing Chelex beads on the bottom of each tube and obtained exoskeletons were mounted on a microscope slide in Hoyer's medium for further morphological analysis Cesari et al. (2011).
All figures were assembled in Corel Photo-Paint 2017. For deep structures that could not be fully focused on a single photograph, a series of 2-10 images were taken every ca. 0.5 micrometres [μm] and then manually assembled into a single deepfocus image in Corel Photo-Paint 2017.

Morphometrics and morphological nomenclature
All measurements are given in 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 types of bucco-pharyngeal apparatuses and claws were classified according to Pilato and Binda (2010). All measurements and terminology of adults and eggs of Macrobiotus were prepared according to Kaczmarek and Michalczyk (2017). Terminology describing the oral cavity armature (OCA) follows Michalczyk and Kaczmarek (2003) and OCA morphotypes are given according to Kaczmarek and Michalczyk (2017). The macroplacoid length sequence was indicated according to Kaczmarek et al. (2014a). The pt ratio is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage (Pilato 1981). The terminology of cuticular bars in macrobiotid legs follows Kiosya et al. (2021). Genus abbreviations follow Perry et al. (2019).
Morphometric data were handled using the "Parachela" ver. 1.8 template available from the Tardigrada Register (Michalczyk & Kaczmarek 2013). Raw morphometric data for the new species are given in Supplementry Materials (SM.05).

Comparative material
For identification of the new species, we used the key by Kaczmarek and Michalczyk (2017) for genus Macrobiotus. All the new species description after Kaczmarek and Michalczyk (2017) were also checked.

Genotyping
Before molecular analysis, each tardigrade specimen was examined in vivo under a light microscope (LM). In order to obtain voucher specimens, genomic DNA was extracted from individual animals following a Chelex 100 resin (BioRad) extraction method provided by Casquet et al. (2012) with modifications described in Stec et al. (2020a).
Molecular data analysis. The BLAST (Basic Local Alignment Search Tool; Altschul et al. (1990)) search at NCBI was used to verify the identity and homology of the amplified molecular markers with sequences deposited in the GenBank database. Obtained nuclear and mitochonrial DNA sequences were checked for quality and trimmed to the same length in BioEdit v. 7.2.5 (Hall 1999). For each individual was then created consensus sequence. The sequences of nuclear molecular markers were aligned using the ClustalW Multiple Alignment tool (Thompson et al. 1994), implemented in BioEdit v. 7.2.5 using default settings. In turn, the sequences of mitochondrial gene fragment were translated into amino acid sequences using the EMBOSS-TRANSEQ application (Rice et al. 2000;Goujon et al. 2010) to test against pseudogenes and check for indels, as well as internal stop codons and unambiguously aligned without inserting gaps. The COI haplotypes were retrieved using DNASP v.5.10.01 software (Librado & Rozas 2009).
Genetic comparisons between obtained and available in the GenBank molecular markers of the genus Macrobiotus were performed to supplement phenotypic description of the new species. Single sequence of molecular markers representing each Macrobiotus species were applied. Sequences which were too short after genetic comparisons or represented different fragments of DNA markers due to the application of various primers for the amplification were not used and as a result not all species were represented in all data sets. Overall, aligned sequences were trimmed to 554 (36 species), 651 (32 species), 258 bp (34 species) and 433 bp (39 species) for 18S rRNA, 28S rRNA, ITS-2 and COI molecular markers, respectively. Mega X (Kumar et al. 2018) was applied to calculate the uncorrected genetic distances (p-distance) for each DNA fragment and to perform the nucleotide sequence composition of the analysed molecular markers. Genetic distances computed between species of the Macrobiotus species and the GenBank accession numbers of applied sequences provided in the Supplementary Materials (SM.1-SM.4).
Material examined. The 131 animals, i.e., holotype and 130 paratypes (animals: 86, exuvium: 8, simplex: 4 and eggs: 31) mounted on microscope slides in Hoyer's medium, 30 animals and 30 eggs prepared for SEM and ten animals prepared for molecular analyses. However, DNA sequences were obtained from two adult specimens (exoskeleton: 1/S, 2/S) which were later mounted on microscope slide in Hoyer's medium and included into type series.
Type depositories. Holotype (M8/7) and 120 paratypes (animals: 81, exuvium: 6, simplex: 4 and eggs: 29) (slides: M8/*, where the asterisk can be substituted by any of the following numbers: 1, 4-16, 18-26, 1/ S, 2/S) were deposited at the Department of Animal Taxonomy  Etymology. The authors would like to dedicate this species to Roman Kosmala, one of the most recognizable Polish sculptors. Author of many monuments, obelisks and commemorative plaques. Table I). Body transparent after fixation in Hoyer's medium, eyes present in all fixed specimens (Figure 1(a), (b)). Entire cuticle covered with elliptical pores with (1.4-2.9 µm in length and 0.9-1.8 µm in width) distributed throughout the body and clearly visible in both PCM and SEM (Figure 1(c), (d)). The edges of cuticular pores are evidently thicker compared with surrounding cuticle in the SEM. Tiny granules inside pores absent. Bucco-pharyngeal apparatus of the Macrobiotus type, with ventral lamina and ten peribuccal lamellae (Figure 2(b)). Mouth antero-ventral. Oral cavity armature of the hufelandi type under PCM with all three bands of teeth visible. First band of teeth made of numerous small cones below peribuccal lamellae. The second band composed of numerous minute teeth and consists of 3-4 rows of teeth visible in PCM. The third band of teeth comprises with a system of three dorsal (Figure 2(a), (f)) and three ventral transverse ridges (Figure 2(b)). Pharyngeal bulb spherical with triangular apophyses, two rod-shaped macroplacoids and a triangular microplacoid. Macroplacoid length sequence 2 < 1 (Figure 3(a)). The first macroplacoid with central constriction (Figure 2 Table II). Eggs spherical, white, ornamented and laid freely ( Figure 5(a), (c)). Egg chorion surface between processes porous and reticulated ( Figure 5(c-f), (i), (j)). Pores of egg surface mesh small i.e., 0.3-1.1 μm in diameter with roundish and/or irregular shapes. In PCM, pores visible in the form of reticulation around the egg processes base whereas in SEM, clearly visible pores around the base and entire chorion surface ( Figure 5(c-f), (i), (j)). The bases of egg processes are surrounded by a crown of evident thickenings visible as dark extensions/short striae radiated from the processes bases in PCM, but 130 P. Kayastha et al. they are also distinct as wider bars of the "reticulum" extending from the processes bases in SEM ( Figure 5(c-f)). Processes in the shape of long and thin cones, sometimes with small terminal discs ( Figure 5(g), (i), (j)) and sometimes bifurcated ( Figure 5(h)). Egg processes, from the middle to the top, covered with tiny granules which are clearly visible in SEM only ( Figure 5

DNA sequences
We obtained good quality sequences for the applied molecular markers: Differential diagnosis. By having porous/reticulated egg surface between processes, reduced/absent Figure 2. Macrobiotus kosmali sp. nov.: a -oral cavity armature (paratype, PCM) from dorsal side; b -oral cavity armature (paratype, PCM) from ventral side; c -mouth with ten peribuccal lamella (paratype, SEM); d -f-oral cavity armature (paratype, SEM). Filled unindented arrowhead represents first band of teeth, empty unindented arrowhead represents second band of teeth, filled indented arrowhead represents third band of teeth from dorsal side and empty indented arrowhead represents third band of teeth from ventral side. Scale bars in µm.   ); b -claws I (paratype, PCM) with pulvunis like structure; cclaws IV (paratype, PCM); d -claws III (paratype, SEM); e -claws IV (paratype, SEM). Filled indented arrowhead represents smooth lunulas, empty indented arrowhead represents divided cuticular bars, filled unindented arrowhead represents pulvinus-like structure, empty arrow represents doubled muscle attachments and filled arrow represents granulations present on legs. Scale bars in µm.

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P. Kayastha et al. ; c -f -the surface between egg processes (paratype, PCM and SEM respectively); g -egg processes with small terminal discs (paratype, PCM); h -egg processes with bifurcation at top (paratype, PCM); i -j -egg processes with cap like disc and trunks covered with tiny granules (paratype, SEM). Filled indented arrowhead represents bubbles inside egg processes trunk, empty indented arrowhead represents pores on egg chorion surface, filled unindented arrowhead represents bifurcated egg processes, empty unindented arrowhead represents tiny granules on egg processes trunk. Scale bars in µm.  (Pilato & Bertolani 2004), by: a different oral cavity armature type (hufelandi type in the new species vs patagonicus type in the Mac. dariae), presence of smooth lunules on IV pair of legs, longer buccal tube ( Mac. glebkai known from type locality in Ulyanovsk (Biserov 1990) Table I in this paper for new species and  Table II
With the discovery of Mac. kosmali sp. nov. the Macrobiotus species identified in the Island of Madeira increased to 5. However, Mac. kosmali sp. nov. is very similar to Mac. recens reported from Madeira by Da Cunha and Do Nascimento Ribeiro (1962) in the past. Based on this we should probably consider that specimens reported in the past also belong to Mac. kosmali sp nov. instead of Mac. recens. We should also treat with extreme caution all past records of Mac. recens which are outside the original type locality. Moreover, other Macrobiotus species reported from Madeira i.e., Mac. echinogenitus, Mac. hufelandi and Mac. occidentalis are burdened with major taxonomic problems and should be considered as doubtful reports which need a confirmation in further studies.