Integrative description of Mesobiotus anastasiae sp. nov. (Eutardigrada, Macrobiotoidea) and first record of Lobohalacarus (Chelicerata, Trombidiformes) from the Republic of South Africa

A new species of the genus Mesobiotus is described from the Republic of South Africa using a traditional morphological approach (light and scanning electron microscopy) combined with molecular analysis (18S rRNA, 28S rRNA, ITS-2 and COI markers). Mesobiotus anastasiae sp. nov. differs from all known Mesobiotus species by having a unique combination of characters of the adult animals and the eggs. Adults of the new species have an oral cavity armature without elongate teeth in the second band, while the processes of the egg chorion have a basal collar and distinct rows of large pores. An updated key to the species of the genus Mesobiotus, including 66 of 70 currently described species, is given. An aquatic mite species from the Lobohalacarus weberi complex (freshwater Halacaridae) co-occurs with M. anastasiae sp. nov., suggesting that the newly described tardigrade inhabits constantly wet moss cushion habitats.


Introduction
The Tardigrada Doyère, 1840 is a phylum of microscopic multicellular animals widely distributed in nature. Currently there are more than 1300 species described (Degma et al. 2009(Degma et al. -2020, but this is very likely an underrepresentation of the actual number of taxa, as the global diversity of tardigrades is poorly investigated (Bartels et al. 2016).
The tardigrade fauna of the Republic of South Africa is the most investigated on the African continent (Middleton 2003;McIness et al. 2017). However, the current state of knowledge is far from complete. Among 46 tardigrade species reported from the Republic of South Africa (McIness et al. 2017;Meyer et al. 2018;Stec et al. 2020), 22 records (48%) belong to poorly described species, or members of (semi-)cryptic species complexes, whose attribution needs verification. To date, only one species of South African tardigrade (Minibiotus ioculator Stec, Kristensen & Michalczyk, 2020) was described using an integrative approach, including light and scanning electron microscopy investigation, as well as sequencing of molecular markers (Stec et al. 2020).
In 2008 I received a few moss samples collected in the Republic of South Africa. One of these samples contained a new species of the genus Mesobiotus Vecchi, Cesari, Bertolani, Jönsson & Guidetti, 2016. Currently this genus includes 69 species (Kaczmarek et al. 2020; see also Tumanov 2018b andTumanov &Pilato 2019), being the second-largest genus in the family Macrobiotidae Thulin, 1928. A recent integrative redescription of M. harmsworthi (Murray, 1907), the type species of the genus , gave a powerful impetus for further research on the real diversity of Mesobiotus. To date, however, only a small fraction of its species have been genetically characterized, so phylogenetic relationships within the genus remains poorly resolved (Kaczmarek et al. 2020).
In this paper, I describe a new Mesobiotus species using integrative taxonomy. The detailed morphological description is supplemented by DNA sequences of four standard genes used in tardigrade taxonomy and phylogenetics (the nuclear 18S rRNA, 28S rRNA, ITS-2, and the mitochondrial COI).
An aquatic mite species of the genus Lobohalacarus Viets, 1939(freshwater Halacaridae Murray, 1877, belonging to the Lobohalacarus weberi complex was also found in the sample, being a new record for this genus in the south African region.

Sampling
The moss sample was collected at the top of Table Mountain (approx. 33°57′43.9″ S, 18°24′38.0″ E, ≈ 1000 m a.s.l.), in the Republic of South Africa, by Irina Nikolaeva, on January 10, 2008. Material was stored within paper envelopes at room temperature. Tardigrade specimens were extracted from rehydrated samples using the standard technique of washing them through two sieves (first with ≈ 1 mm mesh size and second with 35 μm mesh size; Tumanov 2018a). The contents of the finer sieve were examined under a Leica M205C stereo microscope.

Microscopy and imaging
The tardigrades found in moss samples were fixed with acetic acid (a few drops of the concentrated acetic acid were added to the glass staining block containing tardigrade specimens in ddH 2 O) and mounted on slides in Hoyer's medium. The mite specimen was directly mounted on a slide in Hoyer's medium without fixation. Light microscopy (LM): resulting permanent slides were examined under a Leica DM2500 microscope equipped with phase contrast (PhC) and differential interference contrast (DIC). Photographs were made using a Nikon DS-Fi3 digital camera with NIS software.
For scanning electron microscopy (SEM), specimens were dehydrated in an ascending ethyl alcohol series (10%, 20%, 30%, 50%, 70%, 96%) and acetone, critical-point dried in CO 2 , mounted on stubs and coated with gold. A Tescan MIRA3 LMU scanning electron microscope was used for observations (Centre for Molecular and Cell Technologies, St. Petersburg State University).
was measured from the dorsal crests of the oral cavity armature (OCA) to the caudal end of the buccal tube, not including the buccal apophyses. Terminology for the structures within the bucco-pharyngeal apparatus and for the claws follows those of  and . Elements of the buccal apparatus, claws and eggs were measured according to Kaczmarek & Michalczyk (2017). The macroplacoid length sequence is given according to Kaczmarek et al. (2014). All measurements are given in micrometres (μm). The pt index used is the percentage ratio between the length of a structure and the length of the buccal tube (Pilato 1981), and is presented here in italics. Morphometric data were handled using ver. 1.6 of the "Parachela" template, which is available from the Tardigrada Register (Michalczyk & Kaczmarek 2013).

Genotyping
DNA was extracted from two individual animals using QuickExtract ™ DNA Extraction Solution (Lucigen Corporation, USA) using the protocol kindly provided by Torbjørn Ekrem, Norwegian University of Science and Technology (see Supplementary file 1). Preserved exoskeletons were recovered, mounted on a microscope slide in Hoyer's medium and retained as the hologenophore (Pleijel et al. 2008).
Four genes were sequenced from one specimen: a small ribosome subunit (18S rRNA) gene, a large ribosome subunit (28S rRNA) gene, internal transcribed spacer (ITS-2), and the cytochrome oxidase subunit I (COI) gene. The primers and PCR programs used are provided in Table 1.
The PCR products were visualized in 1.5% agarose gel stained with ethidium bromide. All amplicons were sequenced directly using the ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) using an ABI Prism 310 Genetic Analyzer in the Core Facilities Center 'Centre for Molecular and Cell Technologies' of St. Petersburg State University. Sequences were edited and assembled using ChromasPro software (Technelysium, USA). The COI sequences were translated to amino acids using the invertebrate mitochondrial code, implemented in MEGA7 (Kumar et al. 2016), in order to check for the presence of stop codons and therefore of pseudogenes.
All sequences of the genus Mesobiotus available in GenBank at the time of the analysis were downloaded, and those originating from published works with reliable attribution of the investigated taxa were selected (see Appendix 1). Sequences of the 28S gene published by Vecchi et al. (2016) (KT226079-KT226087) and Guil et al. (2019) (MH079488, MH079489) were excluded from comparison as they do not overlap with the sequences obtained in this study. Sequences were aligned using the Muscle algorithm (Edgar 2004) with default settings, as implemented in SeaView ver. 4.0 (Gouy et al. 2010). Uncorrected pairwise distances were calculated using MEGA7 (Kumar et al. 2016) with gaps/ missing data treatment set to "complete deletion". All obtained sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/).

Mite identification
A review paper (Bartsch 1989) was used for the preliminary identification of the mite specimen found. Other publications devoted to the descriptions of Lobohalacarus species and subspecies, and zoogeographical records for this genus (Harvey 1988;Bartsch 1995aBartsch , 1995bBartsch , 2008Bartsch , 2018, were used for the final identification.

Morphological description
Body elongated (Fig. 1A-B) (morphometrics in Table 2, raw morphometric data are provided in Supplementary file 2). Fresh specimens uncolored or whitish with slightly greenish gut content, transparent after fixation in Hoyer's medium. Four specimens with eyes ( Fig. 1A), usually welldiscernible after slide mounting, three specimens without eyes; in two specimens processed for DNA extraction the presence or absence of eyes was not registered. Dorsal cuticle in LM with poorly visible network-like pattern ( Fig. 2A). SEM investigation revealed presence of flat tubercles all over the body surface ( Fig. 2B-C). Additionally, the body surface is covered with regularly distributed small granules (invisible in LM; Fig. 2B). In the dorso-lateral position, above the bases of the hind legs, zones of concentrated granules are present (Fig. 2C). All legs with granulated areas. Legs I-III with small granulated areas on the external surfaces, near the claw bases (Fig. 4A), invisible or extremely poorly visible in LM (Fig. 4D, black arrowhead), the internal leg surfaces without granulation, with indistinct pulvinus (Fig. 4B, white asterisk). Legs IV with better-developed granulation dorsally (Fig. 2D) and around the claw bases (Fig. 4G).
Buccal-pharyngeal apparatus of Macrobiotus type ( Fig. 3A) with ventral lamina and ten peribuccal lamellae. Oral cavity armature (OCA) of modified krynauwi type (according to Kaczmarek et al. 2020) with three bands of teeth visible in LM. Evident first (anterior) band consists of a single uneven line of relatively large and slightly longitudinally elongated teeth ( (rarely) posterior-most rows of the second band are slightly larger than others, sometimes teeth of the anterior-most row are very slightly longitudinally elongated. Third band comprises three dorsal and three ventral transverse ridges (Fig. 3H, J, M, O). Medio-ventral ridge is usually more or less clearly divided into two separate parts (Fig. 3J, O). Pharyngeal bulb with apophyses, three macroplacoids and a large microplacoid (Fig. 3A). Macroplacoid length sequence is 2 < 3 = 1. First macroplacoid is anteriorly narrowed, third macroplacoid without distinct subterminal constriction, but with strong terminal protrusion, directed towards pharynx lumen ( Fig. 3B-E, black arrowheads).
Claws of Mesobiotus type with minute stalk, distinct distal part of the basal portion, short common tract and developed internal septum, defining a distal part. Primary and secondary branches diverge at a point near half the claw height, main branches with long accessory points, which at a large distance from the main claw ( Fig. 4C, E-F, H). Claws of fourth pair of legs slightly longer than claws of first three pairs of legs (Fig. 4H). All claws with smooth lunules (Fig. 4C, E-F, H). Anterior (internal) and posterior (external) claws of legs IV are similar in shape, with equally sized lunules. Poorly developed bar-like cuticular thickenings are present below claw bases of the first three pairs of legs (Fig. 4C, black arrowhead). Claws of legs IV are connected with a wide horseshoe-like structure (Fig. 4F, black arrowhead).      Table 3). Chorion with conical processes that can be attributed to the "sharp wide cones with collars" and "reticular design with "bubbles"" morphotypes (according to Kaczmarek et al. 2020). Egg processes with wide bases and thinned and flexible apices (Figs 5C-I, 6D-F). Basal parts of the processes with bilayered walls, with a net of trabecular structures between the internal and external layers, forming irregular rounded meshes of different size, so the processes seem to be reticulated in LM ( (Pilato, 1974). Paratype egg (UNICT 2132). A-B. Egg processes, white arrowhead indicates polygonate mesh-like pattern, black arrowheads indicate the collar, black arrows indicate pores, DIC. Scale bars: 10 µm.
irregularly distributed ridges and small pores between them ( Fig. 6D-F). In some eggs underdeveloped small processes are present among normal processes (Fig. 6B, E).

Reproduction
The new species is dioecious. Adult males were identified by having testis filled with spermatozoa, visible under PhC on mounted slides. Males of M. anastasiae sp. nov. exhibit no secondary sexual dimorphism.

DNA sequences
Sequences of good quality for the four aforementioned molecular markers were obtained from one specimen (voucher slide SpbU 256 (11) During sample processing, a single mite specimen from the family Halacaridae (Chelicerata, Trombidiformes) was found (Fig. 8A). In having an undivided ventral shield (Fig. 8B) and developed frontal spine (Fig. 8C) this specimen undoubtedly belongs to the Lobohalacarus weberi complex of species, and is the first record of this genus from southern Africa (Bartsch 2018). Apart from its nominative variable species Lobohalacarus weberi (Romijn & Viets, 1924), this complex includes several similar species and subspecies with unclear taxonomic status (Bartsch 1995a(Bartsch , 2018. The studied specimen is similar to Lobohalacarus weberi tristanensis Bartsch, 1995 (known from Tristan da Cunha Islands only) by having genu I with two ventral spines (Fig. 8A, black arrows) and genital sclerites with two pairs of genital acetabula (Fig. 8D, white arrowheads), but differs from this form by having five pairs of perigenital setae (Fig. 8B, white arrowheads).

Discussion
Phenotypic differential diagnosis of Mesobiotus anastasiae sp. nov.
Adult animals Within the genus Mesobiotus, only two species have OCA of the krynauwi type -without the row of the longitudinally elongated teeth in the second band and with a developed first row of teeth: M. krynauwi (Dastych & Harris, 1995) and M. tehuelchensis (Rossi, Claps & Ardohain, 2009).

Eggs
Based on the morphology of the processes, eggs of M. anastasiae sp. nov. should be attributed to the "sharp wide cones with collar" morphotype (Kaczmarek et al. 2020). Within the genus Mesobiotus only two species have this type of egg processes: M. joenssoni Guidetti et al., 2019 and M. mauccii (Pilato, 1974) (the former species' eggs were erroneously attributed to the "sharp narrow cones" morphotype in Kaczmarek et al. (2020)). In all three species, the conical processes have a wide base with a developed collar (or "circular thickness" (Guidetti et al. 2019)), situated on the process wall above the egg surface. Additionally, the egg processes of all three species bear large pores on their side walls. , by having a thinner buccal tube (external buccal tube width is 4.1-6.0 µm vs ca 11 µm in M. mauccii type material), by having egg processes with numerous pores arranged in lines above and below the collar (rare single pores are distributed all over the process surface in M. mauccii), and in lacking ridges on the egg surface between the processes, forming a mesh-like pattern with polygonate cells circling each process (Fig. 7A-B).

Genotypic differential diagnosis of Mesobiotus anastasiae sp. nov.
The ranges of uncorrected genetic p-distances between the studied population of Mesobiotus anastasiae sp. nov. and other species of the genus Mesobiotus, for which sequences are available from GenBank (see Appendix 1), are as follows: COI: 19.15%-26.60% (mean 21.66%), with the most similar being M. gr. furciger from Norway (MH195153, , and the least similar being M. furciger (Murray, 1907) from Antarctica (JX865308, Czechowski et al. 2012). Full matrices with p-distances are provided in Supplementary file 3.

Ecological notes
The location where the investigated moss sample was taken was characterized by the collector as a wet depression with a small temporary pond. But the genus Lobohalacarus, which was also found in the sample, is known to inhabit constant water bodies, primarily subterranean waters and areas where hypogean waters meet the surface (Bartsch 1995a), so the presence of this mite in the sample likely indicates that the type location for Mesobiotus anastasiae sp. nov. can be a permanently wet moss cushion. The presence of the single mite specimen can also be the result of an accidental introduction, but this seems to be less probable, because of the absence of permanent bodies of water in close proximity to the sampling site (I. Nikolaeva pers. com.) and the limited abilities of Halacaridae to tolerate desiccation. A key to the Mesobiotus species was published recently (Kaczmarek et al. 2020). Unfortunately, two species (M. nikolaevae Tumanov, 2018 andM. helenae Tumanov &Pilato, 2019) were not included in this work, and three additional species (M. datanlanicus Stec, 2019, M. dilimanensis Itang, Stec, Mapalo, Mirano-Bascos & Michalczyk, 2020and M. joenssoni Guidetti, Gneuß, Cesari, Altiero & Schill, 2019 are not present because the key predates their descriptions. Further, some minor inaccuracies in the key were revealed. Here I present an updated key to the species of the genus Mesobiotus, including all abovementioned omitted species, as well as the new species described here. The key is based on the version published by Kaczmarek et al. (2020). Mesobiotus meridionalis (Richters, 1909) nom. inq., M. polaris (Murray, 1910) nom. inq., M. stellaris (du Bois-Reymond Marcus, 1944) nom. inq. and M. armatus (Pilato & Binda, 1996) nom. inq. were excluded from the key, following Kaczmarek et al. (2020).  (Pilato & Lisi, 2009) 4. Cuticle with pores without granulation on the body surface or on legs, oral cavity armature without longitudinally elongated teeth in the second band, egg processes in shape of cones with long slender endings, egg processes with "bubbles", egg processes  (Pilato & Sperlinga, 1975) 7. Cuticular sculpture appears in PhC as thin reticulate pattern, oral cavity armature without longitudinally elongated teeth in the second band, with one row of elongated teeth in the first band, egg processes with collar and large pores above it ..  (Binda, Pilato & Lisi, 2005 (Pilato & Lisi, 2006) -Egg shell surface smooth, egg processes with reticular design, number of egg processes on the circumference 21 or less, width of egg processes bases 8.9 µm or more .  (Pilato, Binda & Lisi, 2004) -Lunules under claws IV with indented margin, eyes present, areolation on the egg surface absent, egg process bases with crown of thickenings ...... M. pseudocoronatus (Pilato, Binda & Lisi, 2006) 11. The egg processes in shape  (Binda & Pilato, 1994) 13. The egg processes "mammillate-  (Pilato & Patanè, 1998 (Pilato & Binda, 1996) 26. Egg processes bases elongated into long stripes, which form the semi-areolation (the branches at least in some cases not connected to each other) ..  (Dastych, 1984) 30. Granulation on legs I-III absent, egg processes in shape of cones with long slender endings, number of egg processes on the circumference ca 20 .  (Pilato & Lisi, 2006