New tardigrade records for the Baltic states with a description of Minibiotus formosus sp. n. (Eutardigrada, Macrobiotidae)

Abstract In sixteen moss, lichen and mixed (moss/lichen) samples, collected from Estonia, Latvia and Lithuania, 291 specimens, 48 simplexes, including one exuvium with 6 eggs, and 8 free-laid eggs of eutardigrades were found. In total, 17 species, together with one new to science, were identified (all are new records for the Baltic states): Astatumen bartosi, Diphascon (Adropion) prorsirostre, D. (Diphascon) bullatum, D. (D.) pingue pingue, D. (D.) recamieri, D. (D.) rugosum, Hypsibius convergens, H. dujardini, H. cf. scabropygus, Isohypsibius ronsisvallei, I. sattleri, Macrobiotus harmsworthi harmsworthi, M. hufelandi hufelandi, Milnesium asiaticum, Milnesium tardigradum tardigradum, Minibiotus formosus sp. n. and Paramacrobiotus richtersi. The new species is most similar to Minibiotus gumersindoi, but differs from it mainly by the presence of two types of cuticular pores, the absence of a triangular or pentagonal arrangement of pores above a single large pore on legs, the presence of granulation on all legs and a different macroplacoid length sequence. In this paper we also provide photographs and morphometrics of H. cf. scabropygus.


Introduction
The Baltic States, i.e. Estonia, Latvia and Lithuania, are located on the eastern coast of the Baltic Sea, and fall within the Palearctic ecozone (Holt et al. 2012). The topography of the three countries is dominated by lowlands with the highest peaks at ca. 300 m asl. The temperate climate is intermediate between maritime and continental. Even though the phylum Tardigrada is cosmopolitan and currently comprises ca. 1,200 species (Degma et al. 2013), so far only six tardigrade taxa have been reported from the Baltic States. Specifically, two from Estonia: Eremobiotus alicatai (Binda, 1969) and Isohypsibius cf. marcellinoi (Binda & Pilato, 1971), two from Latvia: Paramacrobiotus richtersi group and Macrobiotus hufelandi group and two from Lithuania: Macrobiotus sp. and Ramazzottius sp. (Šatkauskienė and Vosyliūtė 2010, Zawierucha and Kaźmierski 2012, Ziemelis et al. 2012.
In this study we report seventeen tardigrade species, which are all new records for the Baltic States. Moreover, one of these species is also new to science. The new species belongs to the genus Minibiotus R. O. Schuster, 1980O. Schuster, , that until 1988 contained only a single species, Minibiotus intermedius (Plate, 1888). In 1988 Pilato and Claxton (1988) described Minibiotus maculartus, and within the last decade fourteen new Minibiotus species have been described. Several species have also been transferred to Minibiotus from the genus Macrobiotus based on characters defined by Claxton (1998) and later supplemented by Guidetti et al. (2007) (Michalczyk and Kaczmarek 2003a, Guil and Guidetti 2005, Li et al. 2008, Fontoura et al. 2009a, b, Rossi et al. 2009, Meyer and Hinton 2009, Meyer and Domingue 2011Meyer 2012. Currently, the total number of Minibiotus species amounts to as many as forty seven.
In addition to the description of the new species, we also provide morphometric data and photographs of Hypsibius cf. scabropygus, a rare species that belongs to a large group of hypsibiids with at least partially sculptured dorsal cuticle and pharynx with two macroplacoids and without the microplacoid.

Material and methods
Sixteen moss, lichen and mixed (moss/lichen) samples from trees, soil and stones were collected from 15 localities in Estonia, Latvia and Lithuania between the 29 April and the 5 May 2012 by the third author (more details below). Samples were collected and examined for tardigrades using standard methods (see Dastych 1980). After extraction, animals were mounted on microscope slides in Hoyer's medium. All specimens were examined measured and photographed using Phase Contrast Microscopy (PCM) or Scanning Electron Microscopy (SEM). In total 358 specimens (including 47 simplexes), one exuvium with 6 eggs, and 8 free-laid eggs were examined.
All measurements are given in micrometers [μm]. Structures were measured only if their orientation was appropriate. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. Buccal tube length and the level of the stylet support insertion point were measured according to Pilato (1981). Buccal tube width was measured as the external diameter at the level of the stylet support insertion point. Lengths of the claw branches were measured from the base of the claw to the top of the branch including accessory points for Minibiotus and according to Beasley et al. (2008) for Hypsibius. 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). Macroplacoid length sequence is given according to Kaczmarek et al. (2014b), i.e. macroplacoids are listed from the shortest to the longest and their relative sizes are denoted with appropriate inequality, approximation and/or equality signs (<, ≤, ≈, =). Morphometric data were handled using the 'Macrobiotoidea' ver. 1.1 template available from the Tardigrada Register (www.tardigrada.net/register, Michalczyk and Kaczmarek 2013).
For species identification and differentiation, keys in Claxton (1998), Fontoura and Pilato (2007), Kaczmarek et al. (2011), Michalczyk et al. 2012a, b and Ramazzotti and Maucci (1983, and original descriptions and redescriptions (Ehrenberg 1859, Ramazzotti 1959, 1962, Horning et al. 1978, Bertolani and Rebecchi 1993, Dastych 1988, 1990, Binda and Pilato 1992, Miller et al. 2005, Fontoura et al. 2009a, b, Meyer and Hinton 2009) as well as for insertion of the stylet muscles Pilato (2013) were used. Tardigrade taxonomy is presented according to Marley et al. (2011). Only specimens determined to species level are provided in the list of species (we omitted all specimens determined only to the species group level, e.g. the hufelandi group or the oberhaeuseri group). In the species list Roman numbers indicate sample codes (see sampling localities) and Arabic numbers indicate the number of specimens, exuvia/simplexes and eggs.
Raw data underlying the description of Minibiotus formosus sp. n. are deposited in the Tardigrada Register (Michalczyk and Kaczmarek 2013)   Milnesium asiaticum Tumanov, 2006 Localities and specimen numbers. XV: 1 specimen. Remarks. Our specimen corresponds perfectly to the original description. Milnesium asiaticum was originally described from Kirghizstan and subsequently found in the Svalbard archipelago (Tumanov 2006, Kaczmarek et al. 2012.
Remarks. Specimens correspond perfectly with the redescription by Michalczyk et al. (2012a, b). This species was reported from many localities throughout the World, however records prior to Michalczyk et al. (2012a, b) need to be verified. So far, all confirmed localities are exclusively European (Michalczyk et al. 2012a, b Pilato, 1987 Diphascon (Diphascon) bullatum Murray, 1905 Localities and specimen numbers. XIII: 1 specimen.
Remarks. Specimens correspond well with the limited original description (Murray 1905) and also with later descriptions (Argue 1974and Dastych 1980, 1988. This species is very similar to D. (D.) patanei (Binda & Pilato, 1971) and, as suggested by Dastych (1988), these two species could be synonymous. Thus, to clarify the taxonomic status of D. (D.) patanei, a re-description of D. (D.) bullatum is necessary based on material from locus typicus in Scotland (the type material probably does not exist) (Dastych 1988). 2014a), H. convergens used to be considered cosmopolitan, but it is most likely a complex of very similar (possibly also cryptic) species found throughout the world. The original H. convergens description no longer conforms to modern standards and therefore a redescription is required. Nevertheless, the examined specimens correspond perfectly with the original description and the H. convergens characteristics reviewed by Miller et al. (2005).
Remarks. H. dujardini belongs to the cosmopolitan convergens-dujardini complex of species (McInnes 1994, Miller et al. 2005, Kaczmarek and Michalczyk 2009a, Kaczmarek et al. 2014a, and used to be considered cosmopolitan, but it is most likely a complex of similar (possibly cryptic) species found throughout the world. Due to the limited original description, H. dujardini needs a modern redescription. Nevertheless, the examined specimens correspond perfectly with the original description and the H. dujardini characteristics reviewed by Miller et al. (2005).
Description (measurements in Table 1). Adults. Body transparent/white (after preparation), eyes present in 6 of 15 (40%) specimens mounted in Hoyer's medium ( Fig. 1). Dorsal cuticle sculptured: from head to legs II without tubercles but clearly thickened, from legs II to the caudal end of the body (including legs IV) with irregular tubercles and platelets. Tubercles increasing in size from the anterior to the posterior part of the body, reaching maximum dimensions between legs III and IV, where tubercles sometimes merge and form irregular platelets (Figs 2-5). Irregular tubercles 1.0-6.0 μm in diameter. Ventral cuticle smooth (i.e. without sculpturing). Gibbosities and cuticular pores absent.
Bucco-pharyngeal apparatus of the Hypsibius type, without the ventral lamina, and with forked apophyses for stylet muscles (Fig. 6). Peribuccal lamellae absent. Teeth in the oral cavity armature absent or not visible under PCM. Pharyngeal bulb with apophyses and with two granular macroplacoids (both, without constrictions). Macroplacoid length sequence 2<1. Microplacoid and septulum absent.
Claws of the Hypsibius type, internal claws much smaller and of a different shape than the external claws (Fig. 7). All main branches with large accessory points. Smooth, indistinct areoles under claws usually visible only on posterior claws IV. Cuticular bars under claws I-III absent but a small bar is present near the posterior claw IV (Fig. 7, arrow).

Remarks.
Hypsibius scabropygus has been recorded from many localities, mostly in the Holarctic (McInnes 1994). In general, our specimens correspond to the original description by Cuénot (1929Cuénot ( , 1932 and later descriptions by Marcus (1930) (=Hypsibius callimerus spec. nov.), and by Ramazzotti and Maucci (1983). However, importantly, none of the above mentioned descriptions reported a bar between anterior and posterior claws IV, which is present in all our specimens. Given the bar is small, it is possible that it was overlooked by Cuénot and later authors. If, however, H. scabropygus does not have the bar, then our specimens should probably be classified as a new species. Thus, until H. scabropygus is redescribed, our Latvian and Estonian records should be regarded as H. cf. scabropygus. As there is a possibility of our specimens belonging to a new species, we provide standard morphometrics (Table 1) and photographs (Figs 1-7).
Remarks. Our specimen corresponds perfectly with characters of A. bartosi proposed by Dastych (1988) with the main difference between A. bartosi and A. trinacriae being the absence/presence of cuticular bars on legs II-III. Due to the notorious difficulties in differentiating the two species, the actual distribution of A. bartosi cannot currently be described with confidence. McInnes (1994) cited this species from several localities in Europe and from single African, Asian and South American sites. Isohypsibius ronsisvallei Binda & Pilato, 1969 Localities and specimen numbers. III: 1 specimen.
Remarks. The species has previously been reported from several, mostly European, localities in the Holarctic (McInnes 1994).

Superfamily
Remarks. The species belongs to the harmsworthi group which is widely distributed across a broad range of ecosystems throughout the world (McInnes 1994, Kaczmarek et al. 2014a). In the last decade many new species within this group were described from a variety of localities (Michalczyk and Kaczmarek 2003b, Pilato et al. 2004, Tumanov 2005a, b, Pilato et al. 2006a, Kaczmarek et al. 2007, Kaczmarek and Michalczyk 2009b, Rossi et al. 2009, see also Kaczmarek et al. 2011 for the diagnostic key to the group). Due to many uncertain reports of M. harmsworthi harmsworthi, especially in older literature, the distribution of the species is currently unknown. Specimens found in the present study correspond well to the characters presented in Pilato et al. (2000) and were successfully identified with the key by Kaczmarek et al. (2011).
Remarks. The species belongs to the hufelandi group which is widely distributed across a broad range of ecosystems throughout the world (McInnes 1994, Kaczmarek et al. 2014a). In the last decade new species belonging to this group have been described from various localities , Dastych 2002, Fontoura et al. 2008, Bartels et al. 2009, Kaczmarek and Michalczyk 2009b, Bertolani et al. 2011, Biserov et al. 2011, Pilato et al. 2012, see also Bertolani and Rebecchi 1993 for the diagnostic key to the group). Due to many uncertain reports of M. hufelandi hufelandi, especially in older literature, the distribution of the species is currently unknown. Specimens found in the present study correspond well with the redescription by Bertolani and Rebecchi (1993).

Description (measurements in
Mouth antero-ventral. Ten peribuccal papulae present. Bucco-pharyngeal apparatus of the Minibiotus type (Figs 10-11). Oral cavity armature absent or not visible under PCM. Buccal tube with a poorly visible ventral lamina and with an anterior and a posterior bend (both visible in lateral view only, Fig. 11). Buccal tube walls thickened just below the stylet supports insertion point. Pharyngeal apophyses triangular, very near to the first macroplacoid. Three granular macroplacoids and a minute microplacoid present in the pharyngeal bulb. All macroplacoids of similar but not identical sizes, the macroplacoid length sequence: 2<1<3. Septulum absent.
Claws of the Macrobiotus type (Figs 12-13). Primary branches of claws with thin, but obvious accessory points detaching at the apogee of the primary branch curve.
Smooth lunules present on all legs, distinctly larger under external and posterior claws. Bars and other cuticular thickenings on legs absent. Table 3). White/transparent, laid freely (Fig. 14). Spherical, without areolation. Processes in the shape of short, smooth, slightly flexible cones (Fig. 15). Processes are distributed on the surface of the egg close one to another but never in contact. Surface between processes smooth under PCM (Fig. 15).

Eggs (measurements in
Remarks. Since ventral lamina is very poorly visible, the measurements of this structure are not included in Table 2. Three unembryonated eggs have been found alongside the described specimens. Given that M. formosus sp. n. was the only Minibiotus species in the Figures 8-11. Minibiotus formosus sp. n.: 8 habitus (holotype, ventral view) 9 dorsal cuticle with pores (holotype) 10-11 bucco-pharyngeal apparatus (10 dorso-ventral projection, paratype 11 lateral view, paratype). All PCM. sample and because no Ramazzottius Binda & Pilato, 1986 was found in the samples, we assumed that these eggs belong to the new species.
Type depositories. Holotype 23 paratypes and 3 eggs are deposited in the Department of Animal Taxonomy and Ecology at the Adam Mickiewicz University (Poznań, Poland).

Differential diagnosis
The new species is most similar to M. gumersindoi Guil & Guidetti, 2005, but it differs from it by: the presence of two types of cuticular pores (small and large) in the new species vs pores of a uniform size in M. gumersindoi, the absence of a triangular or a pentagonal arrangement of pores placed above a single large pore on legs, the presence of granulation on legs, a different macroplacoid length sequence (2<1<3 in the new species vs 1=2=3 in M. gumersindoi), and by slightly larger macroplacoids (I: 1.3-1.9 μm; II: 1.2-1.7 μm III: 1.4-2.2 μm in the new species vs 1.0 μm in M. gumersindoi).
Other species to which M. formosus sp. n. is similar by some characteristics of adult and/or egg morphology (e.g. pores in transverse bands, eggs with conical processes), include species listed below. The new species differs specifically from: • M. bisoctus (Horning et al. 1978) by: the absence of trilobed and star-shaped pores (although their presence was not mentioned in the original description, they are clearly visible in Fig. 114 in Horning et al. (1978), and by stylet supports inserted in a more anterior position (pt=49.5-56.2 in the new species vs pt≈60.3 in M. bisoctus (according to Claxton 1998)). • M. eichhorni    Table 2 below and Table 1 in Michalczyk and Kaczmarek 2004 for exact differences in dimensions of placoids and claws). • M. furcatus (Ehrenberg, 1859) (according to Binda and Pilato 1992) Table 2 below and Table 2 in ). • M. keppelensis Claxton, 1998 (Ramazzotti, 1959) (Ramazzotti, 1962) by the presence of cuticular pores, the presence of granulation on all legs, and by fully developed lunules (only small open lunules present in M. subintermedius). • M. vinciguerrae Binda & Pilato, 1992 by: pores arranged in bands, the absence of tri-and quadrilobed pores, the presence of two types of pores (small and large)

Genus: Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar & Wolf, 2009
Paramacrobiotus richtersi (Murray, 1911) Localities and specimen numbers. XI: 2 specimens (including 1 simplex) and 1 egg. Remarks. Paramacrobiotus species (until recently a collection of species within Macrobiotus) can be divided into three groups: areolatus, huziori and richtersi, with respect to the combination of two traits: the presence/absence of the microplacoid in the pharynx and the type of egg areolation. Paramacrobiotus richtersi, considered cosmopolitan, is recognised as the nominal species for a group of very similar taxa that require careful taxonomic examination of adults and egg morphology for correct identification. In the last decade many new species of this group have been described from various localities (e.g. Pilato et al. 2004, Tumanov 2005b, Michalczyk and Kaczmarek 2006a, b, Michalczyk et al. 2006, b, Degma et al. 2008, Bartels et al. 2009, Pilato et al. 2012, with more recent additions including molecular data (Guidetti et al. 2009, Schill et al. 2010.