Observations on the “tenuis group” (Eutardigrada, Macrobiotidae) and description of a new Macrobiotus species

A new species of the tardigrade genus Macrobiotus is described. The species, designated M. ciprianoi n. sp., was isolated from a mixture of Provence broom leaf litter and mosses, and from rock mosses collected in the Sierra de Guadarrama, Madrid (Spain). Given that Macrobiotus ciprianoi n. sp. shares several characters to members of the “tenuis group”, we assessed the taxonomic homogeneity of the group. The new species differs from those of the “tenuis group” according to a unique set of characters related with claw shape, features of the buccal‐pharyngeal apparatus, and egg morphology. Our analysis of holotypes and/or paratypes of “tenuis group” species and other Macrobiotus species with similar characters (M. bondavallii and M. caelicola) reflects the heterogeneity of this group of species as currently described.


Introduction
identified a group of Macrobiotus species, which he subsequently formalized and called the ''tenuis group'', and indicated that ''it often proves difficult to discriminate between these species [tenuis group] in the absence of eggs, which are, on the contrary, always easy to identify''. The common characters of these species are: (1) a yellowish or light brown colour; (2) large size (over 600 mm long); (3) short ventral lamina (strengthening bar); (4) stylet support inserted a long distance from the posterior end of the buccal tube; (5) two macroplacoids, the first with a middle constriction that can be very deep; in such cases, the first macroplacoid can be interpreted as two; (6) slanting cuticular bars (between the apophyses and first macroplacoid) inside the pharynx; (7) long and thin double-claws, with secondary branches much shorter than primary branches, connected at roughly right angles; (8) hind claws always clearly larger than the others; and (9) large lunules, those of the fourth pairs larger than any other and with more or less clear dentations. Maucci (1987a) identified the following species within this group: Macrobiotus tenuis Binda and Pilato, 1972, Macrobiotus willardi Pilato, 1977, Macrobiotus mongolicus Maucci, 1987b, Macrobiotus ariekammensis Węglarska, 1965, Macrobiotus hyperonyx Maucci, 1982, Macrobiotus higginsi Maucci, 1987a, and Macrobiotus hystricogenitus Maucci, 1978. In 1999 to this group Macrobiotus kozharai Biserov, 1999. Tumanov (2005, 2007, besides defining the ''tenuis-type'' of claw excluding the group M. ariekammensis, added to this group Macrobiotus danilovi Tumanov, 2007, Macrobiotus tenuiformis Tumanov, 2007, and Macrobiotus voronkovi Tumanov, 2007 The polyphyletic status of the genus Macrobiotus (one of the largest tardigrade genera) (Guidetti and Bertolani 2001;Guidetti et al. 2005Guidetti et al. , 2007 and the finding of a new Macrobiotus species with several characters common to the ''tenuis group'' prompted us to examine known species of this group, to evaluate their homogeneity as a taxonomic group with a view to the future systematic re-organization of the genus. In the Iberian Peninsula, 23 Macrobiotus species have been identified (out of 150 species of the entire genus), but only 14 have been observed in the Madrid province (Guil 2002(Guil , 2004Guil and Guidetti 2005). This new Macrobiotus species found in a xerophilous shrub landscape of the granite mountain range of Sierra de Guadarrama, Madrid (Spain) thus adds to the number of species in this area.

New species
Seventeen specimens and five eggs (none with an embryo) of the new species were isolated from samples of a mixture of Provence broom (Cytisus purgans (L.)), leaf litter and mosses (Isothecium myosuroides Bridel, 1827; Hypnum cupressiforme Hedwig, 1801) and from samples of rock mosses (Tortella tortuosa (Hedwig) Limpricht, 1888), all collected from Sierra de Guadarrama (Madrid) in central Spain. Litter and moss samples were placed in water and left to soak overnight, after which samples were agitated, squeezed, and poured through two sieves (1.0 mm and 80.0 mm meshes). Sediment was collected into a Petri dish and then fixed with Carnoy liquid (three parts ethanol: one part acetic acid). Individual animals and eggs were mounted on microscope slides using Faure mounting medium (Ramazzotti and Maucci 1983).
Measurements were made at the highest magnification (6100) using a light microscope (LM) equipped for oil immersion differential interference contrast (DIC) and/or phase contrast (PhC) microscopy. The pt ratio reported in Tables I and II is the ratio of the length of a given structure to the length of the buccal tube (measured from the medio-dorsal ridge of the buccal armature to the end of the buccal tube), expressed as a percentage (Pilato 1981).

Material examined
Holotype. Holotype 535 mm long (see Table I Paratypes. Three specimens (see Table I) deposited in the non-insect invertebrate collection of the Natural History Museum of Madrid, Spain (Consejo Superior de Investigaciones Científicas, CSIC).

Diagnosis
Smooth cuticle lacking pearls (pores); colourless; dots present in the external base of all legs; two macroplacoids, the first with a deep middle constriction, and a microplacoid; claws with secondary branch inserted in main branch at a right angle; large lunules, those of the hind legs larger and with dentate or irregular margins; eggs with cone-shaped processes, the latter with a reticular network on their surface. tbl, total body length; btl, buccal tube length; vl, ventral lamina (strengthening bar); pt-vl, pt ratio of ventral lamina; ssi, stylet support insertion; pt-ssi, pt ratio of stylet support insertion; btd, inner buccal tube diameter; prl, macroplacoid row length; fpl, first macroplacoid length; spl, second macroplacoid length; m, microplacoid length; pb2-3, claw length of the second to third pair of legs; pt-pb2-3, pt ratio of claw length of the second to third pair of legs; sb2-3, length of the secondary branch of the second to third pair of legs; pb4, claw length of the fourth pair of legs; pt-pb4, pt ratio of claw length of the hind legs; sb4, length of the secondary branch of the hind legs; L-V, latero-ventral position of the specimen; -, missing measurements.

Description
Description of the holotype (see measurements in Table I): body colourless, length 535.0 mm. Cuticle smooth without pearls ( Figure 1), but with dots at the external base of each leg (more visible on hind legs). Ocular spots present. Buccal tube length 41.2 mm, internal diameter 3.9 mm (Figures 2A, 3A). Mouth terminal with peribuccal lamellae not well distinguishable on LM. Buccal armature comprises fine transverse ridge systems visible in the caudal region of the buccal cavity consisting of three dorsal and three ventral transverse ridges, very slender ( Figure 2B, C). Dorsal central crest arched with its convexity turned backwards ( Figure 2B). Stylet supports inserted in the buccal tube at a distance corresponding to 76.2% of its length (Table I). btl, buccal tube length; ptvl, pt ratio of the ventral lamina; ptssi, pt ratio for stylet support insertion; ptpb 1-3, pt ratio for claw length of one of the first three pairs of legs; ptpb 4, pt value for claw length of hind leg; ptpb4 -ptpb1-3, difference in pt ratios between claws of the first three pairs of legs and hind leg. a Mean values from Maucci (1987aMaucci ( , 1987b; b data from Kathman (1990); c data from Pilato (1977); d data from Biserov (1999); e data from Tumanov (2007); f type specimens plus moss specimens.
Claws with secondary branch inserted in main branch at a right angle (Figures 2D, E, 3C, 4A). Very short peduncle. Short common tract with basal septum delineating a very small basal portion ( Figure 2D, E). Fine accessory points on main claw branches. Large lunules at the base of claws of all legs. Lunules on legs I-III are smooth, those of the hind legs larger and dentate with small teeth (Figures 2D, E, 3D). Cuticular bars on legs absent.
In paratypes (lateral view), a long ventral lamina with a wide ventral crest is visible. Five eggs found were ascribed to Macrobiotus ciprianoi n. sp. on the basis that the other tardigrade species in the samples do not lay free, ornamented eggs (D. recamieri and E. canadensis) or show different egg ornamentation (M. richtersi).
Eggs laid free, spherical in shape and with processes on their surface ( Figure 3E-G). Processes are long conical or funnel-shaped with a network on their surface ( Figure 3F, G). Egg shell surface between processes is not visible on LM. Diameter of the eggs 99.8-103.7 mm. Process heights 11.9-19.8 mm (mean 15.6 mm) and diameters of process bases 6.9-9.9 mm (mean 8.2 mm).

Variability
Ranges and means of the morphometric characters are reported in Table I. Only two paratypes have ocular spots (in the other types, ocular spots are not visible probably due to interference by the mounting medium). In smaller paratypes, probably juveniles, the constriction in the first macroplacoid is shallower.
In M. ciprianoi n. sp. specimens collected from rock mosses, ocular spots visible in eight of the 13 fixed specimens. In smaller specimens, most likely juveniles: first macroplacoid has a shallower constriction, and first macroplacoid is larger than the second (Table I). The second macroplacoid has a widening in its posterior zone, which is not visible in smaller specimens.

Etymology
The new species was named after the late Dr Cipriano Ló pez Almeida, grandfather of the first author (N. Guil).

Taxonomic remarks
Macrobiotus ciprianoi n. sp. can be easily distinguished from the other Macrobiotus species according to a set of morphological characters: claw shape, bucco-pharyngeal apparatus characteristics, and egg morphology. The new species shares several characters with species of the ''tenuis group'' (first macroplacoid with a deep constriction, secondary branch inserted in main branch at a right angle, and large lunules on all legs), yet differs with respect to its claw shape, presence of more rounded macroplacoids, and eggs with coneshaped processes, whose surfaces are reticulate. Macrobiotus tenuis ( Figure 4H) and M. kozharai are the most similar species to Macrobiotus ciprianoi n. sp. Nevertheless, M. ciprianoi n. sp. differs from M. tenuis in that it has larger lunules on the claws of the hind leg, a shorter common claw tract, and in the shape of the egg processes (the eggs of M. tenuis bear truncated cone-shaped processes), and from M. kozharai in that its claws and common claw tract are shorter, and it lacks a posterior band of teeth. Other tenuis species with two macroplacoids (first with a deep constriction) similar to M. ciprianoi n. sp. are M. danilovi, M. tenuiformis, and M. voronkovi (Tumanov 2007). These three species have a different buccal armature, slender and larger claws of the tenuis-type, and their macroplacoids are larger and thinner compared to those of M. ciprianoi n. sp. Macrobiotus danilovi has similar shaped egg processes to the new species, although the conical processes of M. ciprianoi n. sp. are longer and more slender. Macrobiotus tenuiformis and M. voronkovi have completely different shaped egg processes to M. ciprianoi n. sp. such that their distinction is unmistakable.
If the first, deeply constricted macroplacoid is interpreted as two macroplacoids (i.e. three macroplacoids in total), then M. ciprianoi n. sp. could be mistaken for M. wilardi ( Figure 4B). This tenuis species has three macroplacoids and eggs bearing cone-shaped processes with elongated tips. Nevertheless, Macrobiotus willardi and Macrobiotus ciprianoi n. sp. can be clearly differentiated since the new species has a fine buccal armature with no visible band of teeth (both anteriorly and posteriorly), shorter macroplacoids, a smaller microplacoid closer to the third macroplacoid, and smaller accessory points. Moreover, the secondary branch of the claws of M. ciprianoi n. sp. is almost straight and forms a right angle with the main branch ( Figures 2D, E, 3C), whereas in M. willardi, the secondary branch is curved and the angle made with the main branch is more acute ( Figure 4B).

Morphological remarks regarding Macrobiotus species of the ''tenuis group'' or similar species
In this section, we list some of the characters observed in species of the ''tenuis group'' (or related species) that have not been previously described or were erroneously indicated in their original descriptions.

Macrobiotus ariekammensis
( Figures 4C, 5B, D) Characters previously not cited in descriptions of M. ariekammensis and/or M. adelges (Węglarska 1965;Dastych 1977Dastych , 1985 are: cuticular pearls over the entire surface of the animal, larger pearls in anterior (mainly around the mouth opening) and caudal dorsal regions; buccal tube with a clear bend in an anterior position (immediately behind the mouth opening). At this level, the animal has a large dorsal tooth on the internal surface of the buccal tube ( Figure 5B, D); in lateral view, the ventral portion of the buccal tube is shorter and ends at the level of the stylet sheaths; ventral lamina has a wide ventral crest characterized by two lobes in lateral view ( Figure 5D). Contrary to that reported by Dastych (1985), the buccal armature contains a posterior band of small teeth (transverse crests absent).

Macrobiotus hyperonyx
( Figures 4D, 5C) Buccal tube has a clear bend in its anterior portion (just after the mouth opening). At this level, the animal has a large dorsal tooth on the inner surface of the buccal tube ( Figure 5A, C), which was not cited in the original description (Maucci 1982). In lateral view, the ventral portion of the buccal tube is shorter and ends at the stylet sheaths. Unlike the original description, the buccal armature comprises a posterior band of small teeth (transverse crests absent).

Macrobiotus hystricogenitus
( Figure 4E) Contrary to the description by Maucci (1978), the buccal armature comprises a posterior band of small teeth (due to the position of the buccal tube, it was not possible to detect the presence of transverse crests).

Macrobiotus caelicola
( Figures 4F, 5E, F) Buccal armature consists of a posterior row (two rows in a large specimen) of quite large, rounded teeth. At the position of the lateral transverse crests, two rounded teeth occur at both dorsal and ventral locations; central transverse crest is absent. Internal surface of the anterior portion of the buccal tube has a large dorsal tooth (not cited in the original description; Kathman 1990) ( Figure 4E, F). Slanting cuticular bars (between apophyses and first macroplacoid) can be observed in the pharynx. Table II compares the measurements and/or pt ratios of some of the sclerified structures of the ''tenuis group'' species and M. hufelandi (as type species of the genus) with those reported by Maucci (1987a). Our data are generally similar (or within the standard error ranges) to those reported by this author (Maucci, 1987b). The only large discrepancies were the pt ratios for claw length (Table II).

Discussion
According to data reported in the taxonomic remarks (Table II) and those provided in the original descriptions of the species, the common characters identified by Maucci (1987aMaucci ( , 1987b for the ''tenuis group'' should be reconsidered as follows: N Yellowish or light brown colour: this character is not present in most of the ''tenuis group of species (M. willardi, M. hystricogenitus, M. kosharai, M. tenuis, M. danilovi, M. tenuiformis, and M. voronkovi). Ramazzotti and Maucci (1983) reported that specimens from a M. tenuis population were yellowish in colour but in the original description by Binda and Pilato (1972), the type specimens are colourless. N Large size: specimens of the ''tenuis group'' can attain a large size (over 600 mm in length; Maucci 1987b) yet this is also true of other Macrobiotus species.
N Short ventral lamina: given the subjective and relative nature of the terms ''long'' or ''short'' for ventral lamina length and stylet support insertion point on the buccal tube, we used the figures reported by Claxton (1998) for Minibiotus species as reference measurements. Claxton (1998) reported pt ratios for ventral lamina length and distance to stylet support insertion point of (62 and (73, respectively, for Minibiotus species (generally considered to have a short ventral lamina and stylet support inserted a long distance from the posterior end of the buccal tube). Most species of the ''tenuis group'' have ventral lamina pt ratios that are lower or similar to those of Minibiotus species (Table II). Higher pt ratios were recorded, however, for specimens of M. ariekammensis and M. hyperonyx (Table II). N Stylet support inserted a long distance from the posterior end of the buccal tube: only M.
tenuis has a pt ratio for the stylet support insertion point lower than that of the Minibiotus species, but all species of the ''tenuis group'' show low pt values, which are clearly lower than for Macrobiotus hufelandi (type species of the genus) (Table II).
N Slanting cuticular bars (between the apophyses and first macroplacoid) inside the pharynx: these cuticular bars appear in all species of the ''tenuis group'', but this character is often neglected in the description of the species, e.g. see taxonomic remarks and descriptions of Macrobiotus bondavallii (Figure 1 in Manicardi 1989), Macrobiotus kurasi Dastych, 1980(Figure 2 in Dastych 1980, and Macrobiotus marlenae Kaczmarek andMichalczyk, 2004 (Figure 3 in Kaczmarek andMichalczyk 2004). This could indicate the observation of this character in species not belonging to the ''tenuis group''. Moreover, its presence is also recorded in species of different genera (Tumanov 2005).
N Hind claws always clearly larger than others: all species of the ''tenuis group'' have longer primary claw branches on the hind leg than on the other pairs of claws (this character is not clearly specified for M. kosharai ;Biserov 1999). Macrobiotus hystricogenitus, M. higginsi, M. willardi, M. tenuis, M. danilovi, and M. mongolicus (see Table II) differ amongst themselves in terms of their pt ratios for hind claw length compared to the length of other pairs of claws. These ratios are similar to or a little larger than values for M. hufelandi. In contrast, ratios for M. ariekammensis and M. hyperonyx differ largely.
N Large lunules, those of the fourth pair larger than others and with more or less clear dentations: character present in all species of the ''tenuis group'' yet common to other Macrobiotus species (e.g. Macrobiotus walteri Biserov, 1999 andMacrobiotus crenatus Maucci, 1987a). Tumanov (2005Tumanov ( , 2007 defined the tenuis-type claw as: ''slender claws with main and secondary branches well differentiated … long thin common tract (secondary branch diverging at about half of the claw length) … distal part of basal portion typical of genus Macrobiotus, and … the stalk, connecting the claw base with the lunules, forming a prominent frontal appendage on the claw base''. This ''frontal appendage'' is not clearly evident in any of the pictures or figures provided by this author. It is therefore hard to recognize in M. ciprianoi n. sp. or other species of the tenuis group examined here.
Although some morphometric characters (large size, short ventral lamina, stylet support insertion distance, hind claws larger than the rest) and morphological features (slanting cuticular bars between the apophyses and the first macroplacoid, large lunules on the fourth pair with clear dentations) seem to be common to almost all species of the ''tenuis group'', this group of species is far from homogeneous, with many of these characters also occurring in other species which may or may not belong to the genus Macrobiotus. As pointed out by Tumanov (2005), these characters can be considered of small phylogenetic significance according to current knowledge. Indeed, the morphometric and morphological characters mentioned above are also evident in Macrobiotus species that do not have characteristics associated with the claw shape of the ''tenuis group'' (Tumanov 2005(Tumanov , 2007. Moreover, certain features such as cuticular pearls, microplacoids, and a large tooth in the buccal tube are not common characters within the ''tenuis group''. The presence of long, thin double-claws with much shorter secondary branches than primary ones, which roughly form at right angles to them, is a feature that better characterizes the ''tenuis group'' of species, but is also present in other species such as Macrobiotus ciprianoi n. sp., Macrobiotus bondavallii, and Macrobiotus caelicola ( Figure 4A-J). Nonetheless, these three species (Macrobiotus ciprianoi n. sp., Macrobiotus bondavallii, and Macrobiotus caelicola) share other characteristics with species from the ''tenuis group'' (see taxonomic remarks, original descriptions, and Table II). Thus, Macrobiotus bondavalli could be assigned to the ''tenuis group'' (as defined in Maucci 1987b). Macrobiotus ciprianoi n. sp. and M. caelicola show independent characters that, if incorporated in this group, would render the ''tenuis group'' even more heterogeneous. Macrobiotus ciprianoi n. sp. has a peculiar claw structure (with the secondary claw branch forming a true right angle with the primary branch; Figures 2D, E, 3C, 4A) compared to other Macrobiotus species and to those of the ''tenuis group'' (in which the angle is more acute, such as in M. willardi; Figure 4B). Macrobiotus caelicola has a particular sclerified bar below the claws on the first three pairs of legs and cuticular pearls, which are lacking in most ''tenuis group'' species. In effect, within the ''tenuis group'', only M. ariekammensis and M. hyperonyx have cuticular pearls. Moreover, these two species and M. caelicola have several common characters that distinguish them from other species of the ''tenuis group'' and from other Macrobiotus species: cuticular pearls, a large dorsal tooth on the inner surface of the buccal tube, a long ventral lamina, and notably long claws, which are even longer on the hind legs. Tumanov (2005) proposed a new group of species within Macrobiotus designated the ''ariekammensis'' group, based on claw structure. This ariekammensis group is composed of M. ariekammensis and Macrobiotus kirghizicus Tumanov, 2005. Macrobiotus kirghizicus shares with M. hyperonyx, M. ariekammensis, and M. caelicola the features of a long ventral lamina, long claws and longer claws on the hind legs compared to the other legs, but differs in that it lacks cuticular pearls and a large dorsal tooth on the innner surface of the buccal tube.
Our findings indicate that the ''tenuis group'', as defined by Maucci (1987aMaucci ( , 1987b, does not represent a homogeneous group of species. Further taxonomic revisions and phylogenetic analyses of Macrobiotus species are needed to define and clarify the status of the ''tenuis'' (Maucci 1987b;Tumanov 2005Tumanov , 2007 and ''ariekammensis'' (Tumanov 2005) groups of species. Moreover, the entire genus would have to be taxonomically revised and analysed to understand and clarify the phylogenetic relationships among these taxa, and discover how and why they evolved.