A new species of Pseudosinella Schäffer, 1897 (Collembola, Entomobryidae) from Altamira Caves (Cantabria, Spain)

Abstract This paper describes Pseudosinella altamirensissp. nov. from the Altamira Caves, municipal district of Santillana del Mar (Cantabria, Spain), and five other caves near the coast of Cantabria (northern Spain). Its taxonomic position is discussed and differences and similarities among morphologically and geographically close species are highlighted. The new species can be identified by its five eyes, the basal and small inner paired teeth on the claw, and the length of the uncrenulated part of the distal dens.


Introduction
During sampling work to increase the knowledge of the Collembolan cave fauna of the Cantabrian Mountains, we have captured a considerable number of undescribed species of Collembola. Some of these, mainly Pseudosinella Schäffer, 1897, are new species, and it is necessary to describe them. In this paper, a new species of the genus Pseudosinella is described, found in some limestone caves within the municipal districts of Miengo, Santillana del Mar, Reocín, and Cabuérniga (Fig. 1). A complete review of specimens collected in Altamira and other nearby caves (see Luque and Labrada 2016 for details) has shown that this new species was erroneously attributed to Pseudosinella superoculata Gisin & Gama, 1969 by Luque and Labrada. Dorsal macrochaetotaxy and other morphological characters have been used here for species identification. Christiansen et al. (1983) produced a catalogue of world Pseudosinella species and established a code for dorsal macrochaetotaxy characters. Christiansen et al. (1990) designed a computer-assisted identification key (Delta key) of the species of Pseudosinella with more characters. This electronic key is now available on the Web and regularly updated (Jordana et al. 2020). The new species was easily detected using this electronic key by the combination of chaetotaxy formula and other characters used in Pseudosinella species description.

Abbreviations used in the description and figures:
Abd abdominal tergite accp accessorial p-sensilla Ant antennal segment a.s.l.

Terminology
Dorsal head and body chaetotaxy follows Gisin (1965Gisin ( , 1967a, Szeptycki (1979), and Soto-Adames (2010). Equivalence between the notation proposed by Gisin and the AMS system sensu Soto-Adames 2010 is given in Table 1. The characters defined by Christiansen et al. (1990) for Pseudosinella and those used by Christiansen (2013) and Jordana et al. (2020) in the Delta key have been used for identification. Some characters proposed by Mateos (2008) and Winkler and Mateos (2018) have also been considered.

Study area
The Saja River catchment is in the central sector of the Cantabrian mountain range and flows to the Cantabrian Sea after following a course of 67 km in which it descends approximately 1700 m in altitude. It follows a practically rectilinear south-north course in its middle and upper sections, and in its lower one it flows in a west-east direction, which changes to a south-north direction in its final reach at the mouth of the San Martín de la Arena estuary between the municipalities of Miengo and Suances (Fig. 1). This course coincides with the main fracturing and folding lines which characterise the area. In the two municipalities which compose the middle course (Ruente and Cabuérniga), a dismantling of Cretaceous sandstones and clays has taken place, promoting the rising to the surface of Jurassic carbonate materials (Fig. 1). Among the few cavities known over these Jurassic limestones are the caves of Poyo and Cobezo (also the caves located in the Saja-Besaya Natural Park) and the flooded Fuentona de Ruente cave. The first Creaceous limestone outcrops are in the largest municipalities (Cabezón de la Sal, Reocín, Santillana, and Miengo), which are located along the middle-lower course of the Saja River. In the geologic context, this coastal area is distinguished by the abundance of lower to upper Cretaceous carbonates, which favours karst development. Among the abundant explored cavities in these limestone rocks are two (the caves of Peña Caranceja and La Venta del Cuco) that harbor the new species described here. Cudón cave, which has prehistoric paintings, is near the town of the same name at the opening of the Saja River that forms the San Martín de la Arena estuary; Cudón cave is approximately 9 km from Altamira Cave (Fig. 1).
The Altamira Cave is in the central region of Cantabria (northern Spain), in one of the limestone hills that surround the small valley in Santillana village. On the whole, the geological and structural characterisation of Altamira Cave (270 m in length) indicates the evolution of a karstic complex from the first prehistoric human occupation until present. It is one of the many caves in the upper vadose area of the tabular polygenic karstic system that developed on Cretaceous calcarenite limestones (Sánchez et al. 2007). This area is known as Santa Olaja, although it is also known by the name of Planes (Santillana del Mar), where the medieval chronicles place the site of the "Sant[a] Illana" monastery (Luque and Labrada 2016). The small plain that extends over the Altamira Cave has an elevated position (159 m a.s.l.) below the Mount Santa Olaja hill range (168 m a.s.l.), and is protected between the mounts of Vispieres (226 m a.s.l.) and Cildad (287 m a.s.l.). This area separates the Saja River catchment from the Santillana physiographic basin (66 m a.s.l.). Altamira Cave, lying 4 km from the sea, is little more than 2 km from the nearby Saja River. It is situated on a topographical high point (152 m a.s.l.) and is only 3-22 m (averaging 8 m) below the surface. Having a length of 270 m, the cave features a main passage 2-12 m high, and 6-20 m wide (Fig. 2). It was discovered in 1868; exploration began in 1875, but it was not until 1879 when the first paintings, one of the first to be cataloged as Palaeolithic, were discovered by Marcelino Sanz de Sautuola (Lasheras 2009). Regarding the cave biology, Ignacio Bolívar was the first entomologist to explore the Altamira Cave in July 1883 Labrada 2014, 2016). Since then, entomological visits to the cave have been rather sporadic. For example, in the early 20 th century, Charles Alluaud (28 June 1913) and Cándido Bolívar (28 August 1915) explored this cave Labrada 2014, 2016). On 7 May 1924, the cave and its surrounding area was protected by a resolution of the Government of Spain. Since 1985, the cave and its paintings have been included in the UNESCO list of World Heritage Sites (SC-85/Conf. 008/9 1985).
Furthermore, a small cave with stalactites was discovered in the summer of 1928 when stone was being quarried out for the construction of a road to Altamira Cave. It is purely of geological interest, with beautiful stalactites and stalagmites, as no cave art has been found within. In October 1935, Hans Jürgen Stammer was the first entomologist that explored the Stalactites Cave (Luque and Labrada 2016). This cave also hosts the new species described here. (2) areas where soil contained a rat cadaver (Rattus rattus); (3) areas where soil contained a bat cadaver (Rhinolophus hipossideros); (4) areas where walls had insect cadavers with visible fungal; (5) areas where water drips or flows towards the cave (hygropetric habitats); (6) areas where water had flooded the surface with small rim stone dams or gours; (7) areas with high clay soil content in the water; (8) areas of continuous monitoring of radon. Plan of the caves with its location courtesy of the National Museum and Research Centre of Altamira.

Specimen collection and measurement
The specimens were obtained by direct capture. Additionally, baited pitfall traps were used in the parts of the cave that were considered favourable for the presence of fauna (Fig. 2) (see Luque and Labrada 2016 for details). The specimens were captured using a manual aspirator and then preserved in vials containing 70% ethyl alcohol. Each vial was labelled with the following data: collection site, coordinates, date of capture, name of the organisation, and person involved in the capture. The specimens were mounted in Hoyer medium, and optical observations were made under an Olympus BX51-TF microscope with a multiviewing system and phase contrast, and an Olympus BX50-F4 microscope with differential interference contrast (DIC). For the measurements, a U-DA drawing attachment UIS (Universal Infinity System) and a scale calibrated with a slide by Graticules Ltd (1 mm divided in 100 parts) was used. For electron microscopy, three specimens were fixed with 4% (v/v) glutaraldehyde in 0.  Diagnosis. 5 + 5 ocelli. Antennae moderately long. Ant III sense organ with two paddle-or leaf-shaped and two additional sensilla. Area not ringed of dentes nearly five times the length of mucro. Claw elongate, with two paired basal teeth; tenent hair acuminate. Reduced formula: R 0 R 1 R 2 000/00/0101+2/s, -aBq 1 q 2 , M 1 m 2 R*el 1 l 2 (* 1/3 to 2/3 of M; sometimes M 1 smooth and L 2 ciliated, and usually asymmetrically).
Description. Habitus (Figs 3, 4A). Body length up to 3.1 mm (holotype: 2.3 mm). Colour: blue pigment laterally on body from Th II to Abd IV, but extended to dorsal part in Th II, coxae I-III, first third of femur III, laterally on head and vertex and Ant I-III. Abd IV paler. Eyes and a spot on central vertex strongly pigmented.
Head. With five eyes (ABDEF or ABCDF; C and F smaller, almost imperceptible in some specimens). Intraocular chaetae p, r, and s present. Only A 0 , A 2 , A 3 , An 1 , An 2 , An 3a , and An 3 as Mc (Fig. 7C). Ratio antenna/cephalic diagonal 1.64-2.16. Antennal segments I/II/III/IV ratios 1/2-2.7/2.3-2.7/3.5-4.1. Ant IV proximal area in Fig. 5A; apical vesicle absent (Fig. 7A); in this segment there are up to four special leaf-shaped sensilla on the distal third, two of them aligned, and at least five other different types of sensilla (some described by Beruete et al. 2002): smooth and cylindrical, some with short fringes, clearly different from the normal chaetae (Fig. 5B); smooth, shorter and narrowed in its distal third (Fig. 5C); and leaf-shaped, similar to the sensorial chaetae 's' of sensory organ of Ant III (Fig. 5D). Ant III sense organ with a peculiar configuration: two paddle-shaped sensilla (individually encased in a pit and more or less one above the other), another two similar ones but in a dorso-internal position, and two small, rounded, spiny guard sensilla on both sides of the first one (Fig. 7B). Apical region of the Ant II-III with pseudopore in internal-ventral position, far from chaetae line. Antennae without scales. Prelabral chaetae ciliated; labral row a, m, and p all smooth (distal row (a) on papillae). Formula of the labial base M 1 m 2 Rel 1 l 2 ; M 1 sometimes smooth, asymmetrical in some specimens; R ciliated, 1/3 to 1/2 length of the neighbouring smooth Mc m 2 ; l 1 occasionally ciliated; the remaining chaetae smooth, but with minute fringes or ciliation seen only in SEM (Fig. 5E). Bifurcate maxillary palp with three smooth sublobal chaetae. Labial papilla (l.p.) E with finger-shaped process just reaching the base of apical appendage. Maxilla in Fig. 5F.
Body. Th II without Mc; pseudopore of this tergite in Fig. 6A. Th III without Mc. Abd II: chaetae a, q 1 and q 2 as ciliated mic, chaeta B as broad ciliated Mc (Fig. 7D). Abd III chaetotaxy shown in Fig. 7E. Accessory chaeta 's' in the anterior trichobothrial   complex of Abd IV present. Medial chaeta B 5 below the level of the trichobothrium T 4 . Pseudopore between B 5 and B 6 . Legs scales only on coxae. Trochanteral organ with ca 30 chaetae (Fig. 4C). Remaining chaetae clearly visible on all legs, acuminate and largest on leg III. Differentiated supraempodial inner chaeta on hind tibiotarsus well differentiated and acuminate. Dorsal tibiotarsal tenent hairs acuminate, 0.3 times the length of inner margin of claw. Claw with only three internal teeth: dental plate occupying 35% of the basal internal edge, with the basal paired teeth of different sizes (posterior one larger and slightly more distal than anterior); unpaired tooth well developed, approximately 40% from base; lateral tooth, anterior, less frequently posterior, present in some specimens on leg I and in basal positions; dorsal tooth basal, observed only in one specimen at SEM. Empodium appendage acuminate, basally swollen, externally smooth, with a minute tooth in some specimens (Figs 4B, 6B). Retinaculum with 4 + 4 teeth and one ciliated chaeta. Ventral tube without scales; lateral flap with a maximum of eight smooth and six ciliated chaetae. Manubrium and dens with scales only ventrally (anteriorly); two internal and 7-12 external chaetae related to two distal pseudopores of manubrial plate; area not ringed of dentes nearly five times the length of mucro; mucro with distal tooth longer than the anteapical; basal spine reaching the tip of distal tooth ( Fig. 6C-F). Chaetotaxy from head to Abd V illustrated in Fig. 7C-G.
Biology. This species is always found over organic matter accumulation. In Cudón cave, it has been found over the residue of rotten and wet wood; in the other caves it was found over old, mouldy rat and bat faeces and generally in insect cadavers with visible fungi. Although this species reaches to the dark zone of the caves near very wet areas, it has been found in deep zones on walls, roofs, and soils where the environmental humidity is near the saturation point (Fig. 2). The species appears to be restricted to the karst systems associated with the Saja River and within the municipal districts of Miengo, Santillana del Mar, Reocín, and Cabuérniga (Luque and Labrada 2016) (Fig. 1).
Etymology. The name is toponymical and refers to the type locality, the Altamira Cave, one of the most important Palaeolithic art sites in Europe.

Discussion
According to the dorsal macrochaetotaxy (R000/00/0101+2), the presence of chaeta 's' in the anterior trichobothrial complex of the Abd IV, the Abd II chaetotaxy (-aBq 1 q 2 ), and the formula of the labial base (M 1 m 2 Rel 1 l 2 ; M 1 or m 1 ; L 1 o l 1 ), this species is similar to Pseudosinella goughi Gama, 1972, P. suboculata Bonet, 1931, and superoculata (with more constant labial base formula: M 1 m 2 Rel 1 l 2 except in the case of P. goughi with M 1 or m 1 ). Nevertheless, these three species have six eyes (A, B, C, D, E, and F). Pseudosinella suboculata can have five eyes, but its dorsal tibiotarsal tenent hair is clavate and its claw is clearly different, with longer paired teeth, impaired tooth distal (more than 60% from base of inner claw), and the empodium appendage not basally swollen. Pseudosinella superoculata has the paired teeth of the inner claw at the same level, claw approximately 30 % longer than in the new species, manubrial plate with three chaetae (two in one specimen) internal to pseudopore, and sensorial chaetae 's' of sensory organ of Ant III rod-like (after Gisin and Gama 1969 and observed also in some specimens from the Cantabria and Navarra caves). Pseudosinella goughi exceptionally has up to seven eyes and only two internal teeth on claw. In addition, the Table 2. distance distal paired claw tooth from base/total claw, %. C20: distance distal unpaired claw tooth from base/total claw, %. C21: Antennae/head ratio. C22: differentiated inner chaeta on hind tibiotarsus: (1) unclear or absent, (2) clear, acuminate, (3) clear, truncate or clavate. D: accumulated differences. "*", difference with P. altamirensis sp. nov. "U", unknown. "-", not applicable. Table 1. Equivalence table of Gisin (1965Gisin ( , 1967aGisin ( , 1967b notation formulae for chaetotaxy to the modern notation of Soto-Adames 2010 (AMS) as presented in the figures. four special leaf-shaped sensilla present on Ant IV in the new species separate it from its most similar species. With the same dorsal macrochaetotaxy, but without chaeta 's' on Abd II, there are six species: P. alpina Gisin, 1950 (one eye); P. astronomica Gisin & Gama, 1970 (one eye); P. christiani Stomp, 1986 (without eyes); P. mucronata Gouze & Deharveng, 1987 (five or six); P. thibaudi Stomp, 1977 (two eyes); and P. vandeli relicta Gisin, 1964 (no eyes). The differences between these nine species and the new species are shown in Table 1.