Batillipes ( Tardigrada , Arthrotardigrada ) from the Portuguese coast with the description of two new species and a new dichotomous key for all species

Five species of Batillipes Richters, 1909 were collected from subtidal sediments of the Portuguese coast. Two of them, B. algharbensis sp. nov. and B. lusitanus sp. nov., are new to science. Batillipes algharbensis sp. nov. differs from all the other Batillipes species in having the middle toes 3 on the fourth feet longer than middle toes 4 and by the presence of rounded lateral body projections between legs III and IV. Batillipes lusitanus sp. nov. has the middle toes of the fourth feet equal in length, but it exhibits a dorsal cuticular ornamentation, constituted by large pillars, similar to the cuticle of B. adriaticus grimaldi de Zio, Morone De Lucia, D’Addabbo gallo & grimaldi, 1979 and B. roscoffensis Kristensen, 1978. however, contrary to B. adriaticus, the caudal apparatus of B. lusitanus sp. nov. is a roundish cuticular expansion and B. roscoffensis lacks caudal apparatus. Batillipes adriaticus and B. phreaticus Renaud-Debyser, 1959 are new records for Portugal. Based on the examination of specimens of B. phreaticus collected at the Portuguese coast and their comparison with type material of this species and also of B. littoralis Renaud-Debyser, 1959, the toe arrangement patterns in species of Batillipes are clarified and a new identification key to species of this genus is provided. European Journal of Taxonomy 425: 1–32 (2018) 2

Regarding marine tardigrade surveys, the Northeast Atlantic coast is considered one of the best-known regions of the world (Kaczmarek et al. 2015).unfortunately, within this region, the Iberian Atlantic coast, mainly the Portuguese coast, has been barely explored.In Portugal, until the beginning of this century, excluding the Azorean Sea, only four species of marine tardigrades were recorded (Rubal et al. 2013b): Batillipes similis Schulz, 1955; Echiniscoides sigismundi sigismundi Schultze, 1865; Echiniscoides sigismundi hispaniensis Kristensen & hallas, 1980 and Prostygarctus aculeatus Rubal, Veiga, Fontoura & Sousa-Pinto, 2013. Recently, Rubal et al. (2016, 2017) added seven new records to the marine Portuguese tardigrade fauna, three of which belong to the genus Batillipes (B.minius Rubal, Veiga, Fontoura & Sousa-Pinto, 2017, B. pennaki Marcus, 1946and B. tubernatis Pollock, 1971) although the only Portuguese record of B. similis was questioned.This scenario justifies the urgent need for species inventories on this previously neglected region, aiming at contributing to a deeper knowledge of the Portuguese marine fauna and at understanding the peculiarities of the tardigrade biogeography.
In this paper, five species of Batillipes are reported from shallow subtidal sediments of sandy beaches, two of which, B. algharbensis sp.nov.and B. lusitanus sp.nov., are new to science and another two constitute new records for the Iberian Peninsula, B. adriaticus grimaldi de Zio, Morone De Lucia, D' Addabbo gallo &grimaldi, 1979 andfor Portugal B. phreaticus Renaud-Debyser, 1959.Moreover, the presence of B. adriaticus on the South Atlantic coast of the Iberian Peninsula is the first record of this species outside the Mediterranean Sea.
At each site, five sediment samples, each with 100 cm 3 , were collected separated from each other about 5 m.Later they were pooled into a single sample of 500 cm 3 .Samples were fixed in 4% buffered formaldehyde for further study.At the laboratory, sediments were washed and sieved through a 40 µm mesh.Then tardigrades were sorted under a dissecting microscope (50× Wild M5A, Wild-heerbrug) and transferred to microscope slides.Some specimens were permanently mounted in polyvinyl alcohol (PVA) and others in glycerol that, after allowing a period of several days to evaporate to glycerine, were sealed with nail varnish.Microscope slides are deposited in the collection of tardigrades in the Department of Biology, Faculty of Sciences, university of Porto, Portugal.both equipped with digital cameras and using Zen Imaging Software for light microscopy (from Zeiss).one specimen of B. lusitanus sp.nov.was prepared for scanning electron microscopy (SEM); this has been dehydrated through an ethanol / acetone series (50-100%), freeze-dried, then sputter coated with a thin layer of gold and examined under high vacuum in a JEoL JSM 6301F / oxford INCA Energy 350 microscope at the Materials Centre of the university of Porto (CEMuP).
For comparison, the holotype and a paratype (slides AA1287, AA1288) of B. littoralis, the holotype (slide AA1289) of B. phreaticus, all of them from Arcachon Bay (France), and a specimen of the latter species from Scotland (slide AR607), collected by Pollock in 1981, deposited in the collection "Méiofaune" at the Muséum national d'histoire naturelle de Paris (MNhN), France, were examined.A specimen of B. phreaticus from Firemore Bay, Lock Ewe, Scotland from the Pollock's collection (kindly provided by Paul J. Bartels, Warren Wilson College, uSA) was used for the same purpose.Finally, specimens of B. tubernatis, B. minius and misidentified B. similis (Rubal et al. 2016) from Portugal as well as Batillipes sp. 1 (Veiga et al. 2009) from galicia (NW Spain) from the collection of the Department of Biology, Faculty of Sciences, university of Porto were also examined.Comparisons with other species were based on their descriptions in the literature (Marcus 1946;Schulz 1955;Renaud-Debyser 1959;de Zio 1962;Pollock 1970aPollock , 1971;;McKirdy 1975;Kristensen 1978;grimaldi de Zio et al. 1979;Villora-Moreno & de Zio grimaldi 1993;gallo D'Addabbo et al. 2005).

Results
A total of 440 specimens from five species of Batillipes were found in sediment samples at six of the surveyed sites.Fifty-five specimens of Halechiniscus greveni Renaud-Mornant & Deroux, 1976 were also found.Tardigrades were not found in sediment samples from two of the surveyed sites: Torreira Beach and Vieira Beach.

Remarks
The specimens clearly correspond to the original description of grimaldi de Zio et al. (1979).The main characteristic of this species is its dorsal cuticle sculpture constituted by large pillars, appearing as large tubercles (diameter about 2-3 µm); ventrally the cuticle exhibits the fine punctuation as do the majority of Batillipes species.Cephalic appendages have a lance-like tip and papillary secondary clavae are present.Between legs III and IV, an acute sculptured conical body projection is present.Lateral body projections between the first three pairs of legs are not referred to by Grimaldi de Zio et al. (1979).however, in some Portuguese specimens, small blunt ventro-lateral body projections are visible.The caudal appendage is a long spine inserted on a cylindrical base.Middle toes (3 and 4) on feet of legs IV are of similar length.Sensory organs are present on all legs, considerably long on legs IV.
The specific characters of the species: dorsal sculpture, conical lateral body projection between legs III-IV and spine-like caudal appendage are already present in the four-toed larva.

Distribution
up to now, this species had only been reported from the Mediterranean Basin (Adriatic Sea, Ionian Sea and Tyrrhenian Sea, see Kaczmarek et al. 2015).This is its first record for the Atlantic Ocean.
Batillipes algharbensis sp.nov.urn:lsid:zoobank.org:act:005ABE3C-1A30-4E9C-8C29-2AD0F8AA3B3B Figs 2-3, Table 1 Diagnosis Batillipes with distinct head, separated from the body by a neck constriction followed by lateral processes.Primary clavae very thin and undivided.Strongly developed secondary clavae present.Cephalic cirri with spatula-like distal tips.Sensory spines on all legs.Sensory spine on leg I with spatula-like distal tip separated from basal portion by a van der Land's organ.on fourth feet, middle toes 3 and 4 of different lengths, with toe 3 longer than 4. A dorsal papilla present at distal extreme of all legs.Blunt lateral body projections present between all leg pairs: faint between legs I-III and very well-developed between legs III-IV.The caudal apparatus consists of a sharp and long spine inserted in a slightly elongated swollen base.Dorsal cuticle coarsely punctated.Rosette-like female gonopore.

Etymology
The name refers to the region where the species was found, the Portuguese region of Algarve, from the Arabic 'Al Gharb' (Al = the + Gharb = West); algharbensis = inhabiting Algarve.

Description
holotype, female with a body length 121.1 µm (150.6 µm including the caudal apparatus) and 57.9 µm wide between the third and fourth pair of legs (Fig. 2A-B; Table 1 for morphometrics).Eye spots not observed in mounted specimens.Trapezoid head (Fig. 3A-B) separated from the body by a neck constriction followed by lateral processes (body projection 1).Internal cephalic cirri inserted dorsally, bearing cirrophores (cirrophores ca 3.4 µm long) (Fig. 3A).External cephalic cirri, with indistinct cirrophores, inserted more ventrally, near the common pedestal bearing lateral cirri A and primary clavae (Fig. 3B).The median cirrus with a cirrophore (ca 3.5 µm long).Lateral cirri A located dorsally in relation to the unconstricted primary clavae (Fig. 3A).The primary clavae, with a van der Land's organ at the base, particularly thin (thinner than lateral cirri) and sharpened at the tip (Fig. 3B).In the frontal edge of the head, conspicuous and strongly developed dome-shaped secondary clavae (Fig. 3B, D) also present (major diameter 3.8 µm).All cephalic cirri, have long spatula-like swollen tips (Fig. 3A-B).ovoid pharyngeal bulb 14 µm long and 16.6 µm wide.Ventral mouth opening in a protruded circular cone.Stylets and placoids not visible after slide mounting.
Blunt ventro-lateral body projections (body projections 2 to 4, respectively from legs I to IV) present between all leg pairs: very small and difficult to see between the first three pairs of legs and welldeveloped between legs III-IV (Fig. 3C).The caudal apparatus is constituted by a long and sharp spine inserted in a slightly elongated swollen base (Figs 2A-B, 3E).
Sensory spines present on all legs, decreasing in length from leg I to leg III.First (Fig. 3D) and fourth (Fig. 3E) leg sensory organs with spatula-like tips similar to the ones of cephalic appendages and both divided into two parts, separated by a van der Land's organ.Basal and distal parts are respectively 4.8 µm and 2.5 µm long for the sensory organ on legs I and 12.6 and 7.6 µm long for the sensory organ  on legs IV.The large spatula-like tips of sensory organs on legs IV are 3.7 µm long.The entire fourth leg sensory organ is 20.2 µm long.Sharply pointed cirri E with small cirrophores present.
Telescopic legs with toes having the distal stalk enlarged distally (about 2.1 µm wide), ovoid suction discs (diameter 2.9 µm measured on toes of leg IV) with conspicuous braces.On the first three pair of legs, toe 2 is the shortest (considering toe 1 the most cephalically), toes 3 and 5 are the longest and toes 1, 4 and 6 are medium sized.on feet of the fourth pair of legs, the middle toes 3 and 4 are of different lengths with toe 3 longer than toe 4; toes 2 and 5 are the longest, and toes 1 and 6 of intermediate size (Fig. 3F).A dorsal papilla (dark when observed under PCM) is present at the distal extreme of each leg.
Dorsal cuticle uniformly and coarsely punctated, about 9-10 pillars / 10 µm (each pillar about 1.4 µm high), with barely visible transversal folds.Ventrally the cuticle punctation is more delicate.Body with a considerable amount of adherent detritus, mainly near the lateral body projections.
Rosette-shaped gonopore separated from the anus by a weakly defined groove.Other details of the gonoporal apparatus are not visible.

Differential diagnosis
only one known species of Batillipes, B. tubernatis, originally described from the Northeast Atlantic ocean (Scotland, germany and England), shares with the new species the same toe arrangement pattern in the fourth feet, characterized by having toes 3 and 4 of different lengths and toe 3 longer than 4. In the original description, the caudal region of B. tubernatis is described as being round without appendages (Pollock 1971).however, this character can be very variable and should be used with caution in species comparisons (gallo D'Addabbo et al. 2000)

Remarks
The four-toed larva (morphometry in Table 1) is similar to the adult.
Specimens collected in Galicia (NW Spain) and previously identified by Veiga et al. (2009)

Diagnosis
Batillipes with tubular undivided primary clavae with a wrinkled surface.Conspicuous papillar secondary clavae.Cephalic cirri with lance-like distal tips.Sensory spines on all legs.A small rigid process is present in the distal extreme of legs IV.Toes 3 and 4 on feet of legs IV equal in length.A distinct head separated from the body by a neck constriction followed by well-developed lateral processes.Ventro-lateral body projections between all leg pairs present: small, often indistinct, between legs I-III and well developed, semicircular or slightly pointed, between legs III-IV.The caudal apparatus consists of a semicircular cuticular expansion.Dorsal cuticle constituted by large pillars (ca 3.2 µm high), appearing as large tubercles (diameter about 2-3 µm).Ventral cuticle finely punctated.Rosettelike female gonopore separated from the trilobed anus by a groove.

Material examined
Holotype PORTUGAL: ♀ adult, collected at Meia-Praia Beach, Lagos, Algarve, 37°7′1″ N, 8°38′37″ W, mounted in glycerol (slide C.IX-1).B) separated from the body by a neck constriction followed by well-developed lateral processes (body projection 1).head with eleven cephalic appendages: Internal cephalic cirri with cirrophores (ca 3.4 µm long), are inserted dorsally on the frontal edge of the head (Fig. 5A).External cephalic cirri, with indistinct cirrophores, inserted more ventrally, near the lateral cirri A and primary clavae (Fig. 5B).The median cirrus with cirrophore.The lateral cirrus A located dorsally in relation to the unconstricted tubular primary clava.These two appendages share a common pedestal.A van der Land's organ present at the base of primary clavae, which have a wrinkled surface and exhibit a terminal hole (Figs 5A-B).An indentation present in the frontal edge of the head between the external cephalic cirrus and the pedestal with the primary clava and lateral cirrus.Papillar secondary clavae (Fig. 5A) well visible (major diameter ca 7.5 µm long).Cephalic cirri, including the external cirri and the lateral cirri with swollen tips.Ventral mouth opening in a protruded circular structure.ovoid pharyngeal bulb, 28.1 µm long and 25.5 µm wide, with placoids (ca 17.2 µm long).
Sensory spines present on all legs, increasing in length from leg I to IV.First and fourth leg sensory organs with swollen tips.The sensory organ on leg IV divided into a cirrophore (not measurable in the holotype but observed in paratypes) and a basal portion (7.1 µm long) separated from a distal portion (14.5 µm long) by a van der Land's organ (Fig. 5D).Sharply pointed cirri E with small cirrophores present.
Telescopic legs with toes with the distal stalk enlarged distally (ca 3.5 µm wide), ovoid suction discs (5.2 × 5.1 µm) and conspicuous braces (Fig. 6A).On feet of the first three pairs of legs, toe 2 is the shortest, toes 3 and 5 are the longest and toes 1, 4 and 6 are medium sized.on feet of the fourth pair of legs, (Fig. 6A-B) the medial toes 3 and 4 are equal in length.Toes 1 and 6 are medium sized; toe 2 and especially toe 5 are the longest.A small rigid process (ca 3 µm long) is present in the distal extreme of legs IV (Fig. 6B).
The punctation of the dorsal cuticle is very peculiar.It is constituted by large pillars (about 5 pillars / 10 µm, each pillar with ca 3.6 µm high) that, when observed under PCM and DIC, appear as large tubercles, especially if observed laterally (Fig. 7A-B).When observed under SEM, tubercles showed to be depressions that correspond to the wide top of pillars, surrounded by a epicuticular fold Rosette-shaped gonopore separated from the anus by a well-defined groove (Fig. 6C).

Differential diagnosis
Excluding the new species, there are 26 species of Batillipes with the same toe arrangement pattern on feet of fourth legs as B. lusitanus sp.nov., which has the middle toes (3 and 4) on the fourth feet equal in length.however, only two of those species, B. roscoffensis Kristensen, 1978 and B. adriaticus, have the dorsal cuticular ornamentation, constituted by large pillars (ca 3.0 µm high).Batillipes roscoffensis clearly differs from the new species in having scattered and randomly distributed dots ("small pox" according the terminology of Kristensen 1978).Those dots lack in all the other known Batillipes species and they were described by Kristensen (1978)

Remarks
Measurements of structures obtained from specimens of the new species are provided in Table 2. Sexual dimorphism is not evident.Males are similar to females in both qualitative and quantitative characters, except for their circular gonopore with a cuticular crescent shaped fold and located nearer the anus (Fig. 6D).Juveniles, with six toes on each leg but without a visible gonopore, are also similar to adults (Table 2 for morphometrics).In the holotype, ventro-lateral body projections between legs I-III (body projections 2-3, respectively) are indistinct, but in some paratypes, although small, they are visible between all leg pairs (Fig. 5C).This variability could be attributed to the effect of microslide preparation highlighted by the ventral position of lateral projections.Among adults, the size and morphology of the caudal apparatus and lateral body projections, especially those between legs III and IV, show some variation, being perfectly semicircular, as in the holotype, or slightly pointed.In four-toed larvae, caudal apparatus and lateral projections are clearly conical.
The variability in shape of lateral projections and caudal apparatus was responsible for the misidentification of three specimens collected in 2011 in Portugal at Memória Beach, Matosinhos, (41°13′49″ N, 8°43′21″ W) (Rubal et al. 2013b).These specimens were wrongly attributed to B. similis on account of the conical-shaped cuticular projections, evidenced by microslide preparation.A deeper examination showed that the middle toes on legs IV are equal in length and that the cuticle exhibits the same peculiar pattern of B. lusitanus sp.nov.Therefore, as suggested by Rubal et al. (2016Rubal et al. ( , 2017)), the presence of B. similis on the Portuguese coast cannot be assumed.

Distribution
A common, worldwide distributed intertidal species that can occur in subtidal zones (for a more detailed geographic distribution see Kaczmarek et al. 2015).Batillipes pennaki was already known for Portugal, but up till now, only recorded in the northern region (Rubal et al. 2016) both on sandy beaches as well as on rocky shores.

Short description
Distinct head separated from the body by a neck constriction followed by well-developed lateral processes (Fig. 8A).Eyes not observed in mounted specimens.Caudal apparatus constituted by one major pointed spine surrounded, at its base, by a crown of small accessory spines (2 to 6) (Fig. 8B).Paired internal and external cephalic cirri and median cirrus all with lance-like tips.Surface of primary clavae with black punctations, sharing the same pedestal with the lateral cirri A, that also have a lancelike tip.Papillar secondary clavae present.Dorsal cuticle shows fine punctuation, with pillars uniformly distributed.Single sharp conical body projections between legs III and IV (Fig. 8B).Lateral body projections between the first three pairs of legs often indistinct.When present, they are blunt between legs I-II and triangular between legs II-III.Dorsal cirri E present.Sensory spines on all legs, longer on legs IV (Fig. 8B).Middle toes (3 and 4) on feet of legs IV (Fig. 8C) equal in length.

Distribution
Interstitial species recorded in the Eastern Atlantic region (Atlantic ocean, North Sea, Irish Sea and Celtic Sea) and in the Mediterranean Basin (Balearic and Ionian Seas) where it has been recorded, mostly intertidally, but also subtidally (Kaczmarek et al. 2015).Batillipes phreaticus was recorded from the galician coast, NW Spain (Veiga et al. 2009), but this is the first record from Portugal.

Remarks
In the original description of B. phreaticus from Arcachon Bay, France (Celtic Sea), the presence of lateral body projections between the first three pair of legs is not referred (Renaud-Debyser 1959).
Concerning toes, Renaud-Debyser (1959) states that they are similar to those of B. littoralis (also from Arcachon Bay), which are described in the same publication as having stalks of different lengths.however, the relative size of toes and details of the arrangement among feet were not referred by Renaud-Debyser (1959).Specimens from England, Filey Beach and Stoup Beck Beach, Yorkshire, collected by Pollock (1971) and from germany, Elbe Estuary (Riemann 1966) attributed to B. phreaticus, differed from the original description in some morphometric aspects and in having lateral body projections often present (Pollock 1971).Moreover, Pollock (1971) noticed that in English specimens, the middle toes (toes 3 and 4) on feet of legs IV were equal in length.Later, Villora-Moreno & de Zio grimaldi (1993), based on specimens collected on sandy beaches from the Mediterranean Sea (Balearic Sea), provided a redescription of the species, confirming the differences mentioned by Pollock (1971), regarding the original description: presence in adult specimens of a ventro-lateral body projection between legs II and III, and middle toes on feet of legs IV equal in length (toe pattern group A, Pollock 1970a).Despite his own observations, Pollock (1971) did not correct his proposal from 1970 of three different patterns of toe arrangement within Batillipes species, having included B. phreaticus in group B, which considers middle toes on feet of legs IV of different length and toe 1 equal to 3, while B. littoralis have been included in group C (middle toes on feet of legs IV of different length and toe 2 equal to 4).
The system of toe arrangement patterns on the fourth feet of species of Batillipes species was modified by Kristensen & Mackness (2000), who proposed a fourth group (group D, constituted by species with middle toes of different length and also different from all the other toes).As previously proposed by Pollock (1970a), B. phreaticus and B. littoralis were kept in groups B and C, respectively, by Kristensen & Mackness (2000).
Taking into account the taxonomic importance of those characters (gallo D'Addabbo et al. 2000), the examination of type material of those two species deposited in the collection of the MNhN was of primordial importance.This examination showed that the toe pattern of the fourth feet of B. littoralis perfectly matches the group D proposed by Kristensen & Mackness (2000).Concerning B. phreaticus, despite the poor condition of the specimen, the middle toes of the fourth feet seem to be equal in length and shorter than all the other, from which, toes 2 and 5 are the longest and about the same length, and toes 1 and 6, also similar in size and of intermediate length (toe pattern of group A). one of the examined specimens from Scotland collected by Pollock in 1981 also exhibits the middle toes of the fourth feet equal in length (Fig. 8D).unfortunately, this character was not visible in the other Scottish specimen deposited in the MNhN (slide AR607).
Therefore, specimens from the Portuguese coast match with observations of Pollock (1971) and Villora-Moreno & de Zio grimaldi (1993).Ventro-lateral body projections between legs I-III were not observed in the type specimen, neither in the Scottish specimens.however, according to Pollock (1971) and as observed in Portuguese specimens, these lateral projections can be absent in some specimens or, as stated by Villora-Moreno & de Zio grimaldi (1993), are indistinct as a result of slide preparation.It is important to remark that in a few adult specimens from Portugal (three specimens from Vasco da gama Beach and six from Portinho da Arrábida Beach) the main caudal spine was inserted on a wide conical base, lacking the basal crown of small accessory spines as it occurs in juveniles.This ontogenetic variability of the morphology of the caudal appendage was also referred by Villora-Moreno & de Zio grimaldi (1993).

Discussion
Fourteen marine tardigrade species are recorded from the Portuguese coast, of which 7 belong to the genus Batillipes: B. adriaticus, B. algharbensis sp.nov., B. lusitanus sp.nov., B. minius, B. pennaki, B. phreaticus and B. tubernatis (see also Rubal et al. 2016Rubal et al. , 2017)).Three of these species, the two newly described and B. minius, can be considered endemic to this region up to now.The distribution of B. adriaticus, up till now only known from the Mediterranean Sea, was considerably extended to the Atlantic ocean.Batillipes phreaticus and B. tubernatis are typical of the NE Atlantic, while B. pennaki is considered cosmopolitan (Kaczmarek et al. 2015).
our study clearly shows that the diversity within the genus Batillipes is very high.Species of the genus (raised to 37 with this study) represent about 17% of all known marine heterotardigrade species (Guidetti & Bertolani 2005;Degma & guidetti 2007;Degma et al. 2009Degma et al. -2017;;Jørgensen et al. 2014) and 50% of the diversity of Portuguese marine tardigrades.This number will probably increase in the near future (see Bartels et al. 2016).As shown in this research, many species are very similar to each other and this is problematic for the taxonomy of the genus.Difficulties are increased by the small number of taxonomic characters available and by the marked variability observed in some characters (e.g., McKirdy 1975;gallo D'Addabbo et al. 2000).on the other hand, the use of morphometric data for taxonomic purposes must be carefully used because measurements can be biased by specimen orientation or by the physical slide mounting processes (see Mcginty & higgins 1968;Pollock 1971;Kristensen 1978;Morone De Lucia et al. 1988;Villora-Moreno & de Zio grimaldi 1993;gallo D'Addabbo et al. 2000;Kristensen & Mackness 2000;Zawierucha et al. 2015;Fontoura et al. 2017;Santos et al. 2017).For these reasons, some of the old descriptions of species of Batillipes are incomplete and they may represent complexes of similar species, e.g., cosmopolitan B. mirus and B. pennaki as suggested by Zawierucha et al. (2013Zawierucha et al. ( , 2015)).In order to solve the problems that affect the taxonomy of the genus, research should focus on characters previously neglected for marine tardigrades (gallo D'Addabbo et al. 2000).Toe arrangement patterns, especially on feet of fourth legs (Pollock 1970a;Kristensen & Mackness 2000;Santos et al. 2017), toe disc shape (McKirdy 1975) and cuticle sculpture (gallo D'Addabbo et al. 2000) are examples of such taxonomic characters, considered very reliable, especially if used in combination with other attributes.
As also shown in this study and confirmed with the examination of the type material of B. phreaticus and B. littoralis deposited in MNHN (Paris), inaccuracies on the definition of toe arrangement patterns on the fourth feet proposed by Pollock (1970a) and later modified by Kristensen & Mackness (2000) were detected.Therefore, new modifications of this indicator are proposed.To apply this indicator, toes must be correctly numbered: Toe 1 being inserted on the tarsus most cephalically, then the other toes numbered sequentially in a clockwise direction, being toe 6 inserted on the tarsus most caudally (Fig. 9).This terminology avoids confusions introduced by using "external" and "internal" toes that is affected by the position of legs IV.Three major toe arrangement patterns can be recognized in Batillipes as follows (Table 3, Fig. 9): -Group A: on the feet of legs IV, the middle toes 3 and 4 are similar in length; toes 2 and 5 are the longest ones and toes 1 and 6 of intermediate size.Toes 1 and 6 can be similar to middle toes 3 and 4, but not much shorter (Fig. 9A).This is the major group, constituted by 27 species.In three of these species, toes can be so short that suction discs resemble sessile (that is, the toe stalks are so short that they seem to be absent and, consequently, the discs seem to be directly inserted on the feet), especially in the middle toes (Fig. 9B).-Group B: on the feet of legs IV, the middle toes 3 and 4 are clearly different in length; toes 2 and toes 5 are the longest and toes 1 and 6 of intermediate size (or similar but not shorter than middle toes 3 and 4).This group is subdivided into two subgroups: Subgroup B1: middle toe 3 shorter than middle toe 4 (Fig. 9C); Subgroup B2: middle toe 3 longer than middle toe 4 (Fig. 9D).-Group C: on the feet of legs IV, the middle toes 3 and 4 are clearly different in length; toes 2 and toes 5 are the longest and toes 1 and 6 are the shortest (much shorter than middle toes 3 and 4) (Fig. 9E).
This clarification on toe arrangement patterns on feet of legs IV justifies the proposal of a new identification key to the species of the genus Batillipes (see below).
In addition, this study confirms the particular biogeographical position of the Portuguese coast which is supported by the first record outside the Mediterranean Sea of B. adriaticus.Similarly, Rubal et al. (2016) reported the first occurrence out of the Mediterranean Sea of Styraconyx sardiniae   and Styraconyx (i.e., southern limit for boreal and northern limit for warmer water species) is in agreement with observations on other marine groups such as macroalgae (Ardré 1971) or gastropods (Rubal et al. 2013a).This study also supports the need for more extensive and deeper surveys.Despite the interesting biological information that they can provide (de Zio grimaldi & gallo D'Addabbo 2001; Faurby et al. 2011Faurby et al. , 2012)), biogeographical studies on marine tardigrades, and especially on Batillipes, are scarce, often restricted to generalist lists (e.g., Kaczmarek et al. 2015;Miller & Perry 2016) and sometimes leading to contradictory interpretations.Actually, results of recent studies suggested that geographic / ecological factors can act as barriers, promoting speciation (e.g., Faurby et al. 2011Faurby et al. , 2012;;Zawierucha et al. 2015;Faurby & Barber 2015) contrary to hypotheses in favour

A dichotomous key to the identification of species of Batillipes
Traditionally, keys to the identification of species of Batillipes used the caudal apparatus (e.g., Ramazzotti & Maucci 1983) as the main distinguishing trait.however, after the discussion of the variability of taxonomic characters within the genus (McKirdy 1975;gallo D'Addabbo et al. 2000), this trait, unless peculiar for some species or when combined with other characters, is no longer valid.
Recently, a comprehensive key to all known species of Batillipes was published by Menechella et al. (2017).This key used the toe arrangement pattern, suggested by Kristensen & Mackness (2000), as the first differentiating character.However, this key is no longer practical on account of the inaccuracies on the definition of toe patterns referred to in the present study.Therefore, a new key is provided, using a new proposal of toe arrangement patterns, shape of primary clava, ventral body projections between legs III-IV and details of cuticular sculpture as main taxonomic characters, allowing an easier identification based on observations under light microscopy (particularly PCM or DIC).
The shape and development of lateral body projections, as well as the relative size of leg sensory organs, also secondarily considered in the key, are ontogenetically variable characters (Villora-Moreno & de Zio grimaldi 1993;gallo D'Addabbo et al. 2000), for this reason only adult specimens should be identified using the proposed key and more than one character should be used.
For a more accurate identification of some species, several characters are given in the last identification step.unfortunately, for some old described species, some reliable attributes are unknown (e.g., details of punctation) and its presence / absence cannot be assumed, making the construction of identification keys for species of Batillipes an ongoing process.

Fig. 7 .
Fig. 7. Batillipes lusitanus sp.nov. A. Posterior portion of the body, showing the dorsal sculpture.B. Caudal apparatus, showing details of cuticular pillars.C. SEM photo showing the dorsal (white arrowhead) and ventral (black arrowhead) aspect of the cuticle.Pillars are also visible (black arrow).Scale bars = 10 µm.

Fig. 8 .
Fig. 8. Batillipes phreaticus Renaud-Debyser, 1959.A-C.Portuguese specimens.A. Anterior region of the body.B. Posterior region of the body, black arrow indicates fourth leg sensory organ.C. Detail of the fourth feet.D. Detail of the fourth feet showing the toe arrangement pattern of a specimen from Scotland in Pollock's collection.Scale bars = 10 µm.

Table 1 .
Measurements (µm) of selected morphological structures from the holotype and paratype, a fourtoed larva, of Batillipes algharbensis sp.nov.from Portugal, and specimens from galicia (NW Spain) (SD -Standard deviation; Range refers to the smallest and largest measured specimen / structure; N -number of specimens / structures measured).
as Batillipes sp. 1 with similarities with B. spinicauda also exhibit the unique characters of B. algharbensis sp.nov.(Table1).Therefore, they are assigned to B. algharbensis sp.nov.
Descriptionholotype, female of robust body 236 µm long (248.4µmincluding the caudal apparatus) and 100.9 µm wide between the third and fourth pair of legs (Figs 4A-B, Table2for morphometrics).Eyes not observed in mounted specimens.Trapezoid head (Figs 5A- under TEM (transmission electron microscopy), as elevations (of spongy electron dense mass, seeKristensen 1978: figs 13, 17).Moreover, in B. roscoffensis the caudal apparatus totally lacks and the lateral cirri A is much longer than in B. lusitanus sp.nov.(cirrus A ca 100 µm long, corresponding to 40% of body length in B. roscoffensis, and less than 50 µm long, corresponding to ca 20% of body length in the new species).
lusitanus sp.nov. shares with B. adriaticus, the most similar species, the peculiar dorsal cuticle punctation constituted by large pillars, appearing as large tubercles.These two species can be clearly distinguished by the shape of the caudal apparatus that consists in a roundish cuticular expansion in B. lusitanus sp.nov.and in a sharp and long spine in B. adriaticus.The lateral body projection between legs III-IV is also blunt or slightly pointed in the new species while it is sharply pointed in B. adriaticus.Moreover, contrary to B. adriaticus, in the new species, a small rigid process is present in the distal extreme of legs IV and the surface of the primary clava is wrinkled.In addition, although with overlapping values, in adults of B. lusitanus sp.nov. the sensory organ on legs IV is slightly shorter than in B. adriaticus (range: 9-27 µm long in B. lusitanus sp.nov.and 20-40 µm long in B. adriaticus).

Table 3 .
Toe arrangement patterns on the fourth feet in Batillipes species (* indicates species with very short middle toes).D'Addabbo gallo, Morone De Lucia & de Zio grimaldi, 1989.Therefore, these results suggest that the Portuguese coast is the occidental boundary for some Mediterranean tardigrade species.Additionally, Rubal et al. (2016) also reported a new southern limit of B. tubernatis and S. haploceros Thulin, 1942 at the Portuguese coast.These boundary patterns suggested by the genera Batillipes Rubal et al. 2016)ck , 1989;;Kristensen & Mackness 2000;Faurby et al. 2012)habitats such as different depths (e.g., hansen 2005), diverse environmental gradients (e.g.,Pollock 1970bPollock , 1989;;Kristensen & Mackness 2000;Faurby et al. 2012)or types of substrates (e.g.,Rubal et al. 2016), would enhance our understanding of tardigrade distribution, and, particularly, could be pivotal to our understanding of the biogeography of the genus Batillipes.