The mesovoid shallow substratum (“milieu souterrain superficiel”; MSS or terrestial SSHs) (Juberthie et al., 1980, 1981; Uéno, 1980, 1981; Pipan & Culver, 2012) is a terrestrial habitat that consist of a network of voids and interstices located above the deep subterranean domain and immediately below the soil, lightless and highly humid (Mammola et al., 2016). It includes talus and scree slopes in both carbonate (soluble) and non-carbonate rocks, including volcanic rocks. Also are an aquatic SSHs that include epikarst and the hypotelminoheic realm (Pipan & Culver, 2012). The epikarst, uppermost layer of karst, may be air or water filled and occupies a similar vertical position to that of the MSS. The perched aquifers (isolated wetlands) (hypotelminorheic by Mestrov, 1962) are the most superficial of SSHs and together with epikarst and MSS expand the scope of subterranean habitats (Pipan & Culver, 2012). The characteristics of these environments are very different as different are the faunas that can inhabit them (Pipan & Culver, 2012). The common characteristics are absence of light, high relative humidity and attenuated fluctuations in temperature throughout the year. In the MSS there is a rich and diverse terrestrial fauna composed of both hypogean species with different degrees of adaptation to the subterranean environment as well as epigean and endogean species that transit between the surface and the subterranean environment (Pipan & Culver, 2012; Ortuño et al., 2013; Mammola et al., 2016). The MSS (terrestrial SSHs) has been hardly studied, mostly in the French Pyrenees and other (noncalcareous) areas from Europe, Japan and China (Gers, 1992; Juberthie & Decu, 1994; Ruzicka et al., 1995). Medina & Oromí (1990) extended the habitat to include volcanic terrain in the Canary Islands (López & Oromí, 2010; Pipan et al., 2011).

Here, we report for the first time the presence of Bathynellacean crustaceans in the MSS. These aquatic animals live exclusively in groundwater (stygobionts). To date they have been found in caves (gours, puddles, ponds, lakes, rivers, etc.), seepage springs, sources, artificial wells, aquifers (mine bores, bore holes, irrigation waters) and in the hyporheic habitat associated to epi- and hypogean rivers. Never before had they been found in a terrestrial environment like the MSS as defined.

Bathynellacea were collected with traps set to sample terrestrial subterranean fauna in the Sierra de Guadarrama National Park (Madrid, Spain) (Fig. 1). The study area is located in the Central System mountain range of the Iberian Peninsula, which peaks from 1200 to 2428 m above sea level (a.s.l). Orthogneiss of metamorphic quartz-feldspathic origin are the geological substratum predominant in the Park (Vialet et al., 1987), and appears as colluvial and moraine deposits whose subsoil has been sampled as described elsewhere (Baquero et al., 2017; Ledesma et al., 2019; Ortuño et al., 2019). Thirty-three subterranean sampling devices (SSD) set in 33 scree slopes covered most of the Park area (Fig. 1). Each SSD consisted of a PVC tube 1 m long and 11 cm in diameter with perforations of 8 mm in diameter arranged along 40 cm from midway of the cylinder to its base. The cylinders were inserted vertically into a previously excavated hole. A pitfall trap baited with very smelly cheese and filled with 1,2-propanediol was deployed within each cylinder and the whole set covered as shown (see Fig. 2A-B). The sampling period, as a whole, covered from May 20, 2015, to October 14, 2016, but the SSD-32 (Fig. 2C) worked from July 9, 2015 to October 28, 2016 (Fig. 3). In this SSD, 12 specimens of the family Parabathynellidae were collected (7 ♂♂, 4 ♀♀ and a juvenile specimen displaying only 4 pairs of thoracopods) (Fig. 4A) during the first sampling period (09/07/2015 to 22/10/2015) (Fig. 3). They were preserved in 90% ethanol to carry out morphological and molecular studies.

We selected three whole specimens and the abdomen of another five to extract DNA (see Table 1). We succeed to extract DNA from six animals, and 18S rRNA gene sequences from three of them. We failed to sequence the COI gene in all the extracts. DNA extraction and amplification methods appear described in Camacho et al. (2018). The extracted DNA was deposited in the Tissues and DNA Collection of the MNCN (voucher numbers of the specimens shown in Table 1).

For the morphological study, nine specimens were completely dissected (Table 1) and the appendages preserved as permanent slides (special metal slides, glycerine-gelatine stained with methylene blue and paraffin as mounting medium; see Perina & Camacho, 2016). The morphological examination was performed using an oil immersion lens (at 1000x magnification) with a Zeiss interference contrast microscope equipped with a drawing tube. Photographs were taken with a Leica camera (LEICA MC170 HD) attached to the microscope with 400x magnification and 1000x. The specimens prepared on permanent slides are deposited in the Collection of Arthropoda of the Museo Nacional de Ciencias Naturales-ARTP/MNCN-, Madrid, Spain (see voucher in Table 1).

We identified two species of the Parabathynellidae family in the collected material: Hexaiberobathynella mateusi (Galhano, 1967) (9 specimens; 5 ♂♂ and 4 ♀♀) and Hexabathynella nicoleiana Camacho, 1986 (3 specimens; 2 ♂♂ and 1 juvenile) (Fig. 4B; Table 1).

Morphologically both genera are very different, but the differences are only observed in the dissected specimens and not when they are observed under the stereo-microscope (Fig. 4A). The species belong to the only two genera characterized by the display of only six pairs of thoracopods. The rest of known genera (85 currently; Camacho, 2019) display seven pairs of thoracopods. Hexaiberobathynella Camacho & Serban, 1998 display a 7-segmented antennule (AI) and a 3-segmented antenna (AII) (Fig. 4B); while Hexabathynella Schminke, 1972 has a 6-segmented AI and a 5-segmented AII, in addition to many other relevant differences (Table 2).

The molecular results have confirmed the morphological identification of both genera. We have succeeded in sequencing the 18S gene in extracts corresponding to three specimens (see Table 1). Comparison with sequences we have in our database (Hexabathynella sevillaensis Camacho, 2005, Hexaiberobathynella hortezuelensis Camacho & Serban, 1998 and Hi. mateusi) has shown that the new sequences correspond, without any doubt, to these two genera.

The confirmation of the identification to species of the studied material was not possible based on gene sequences since we do not have sequences of specimens from the type localities of both taxa: Douro River mouth (for Hi. mateusi) and Jarama River (for H. nicoleiana; Table 3). Furthermore, we failed to get COI sequences of the two species studied. Since cryptic species are frequent among Bathynellacea (Camacho et al., 2011) we cannot discard that although we have morphologically identified the specimens as belonging to these two species, it could be that they are sister species. What is unquestionable is the generic ascription of both species.

Both species had previously been found in Madrid, in the Jarama River Basin in Torrelaguna, Patones, and Talamanca del Jarama (Camacho, 1986, 1987), and Hi. mateusi also at Cueva del Reguerillo (Patones) and in other sites of the Iberian Peninsula (Soria, Guadalajara, Teruel, Toledo, Granada and Jaén, plus in Portugal (Galhano, 1967; Camacho & Serban, 2000; Camacho et al., 2000, 2014, 2017; Guil & Camacho, 2001; Camacho, 2003). Both species were recently discovered to occur at different points of the interstitial medium of the Tajuña river in the provinces of Madrid and Guadalajara (Camacho, 2019) (Table 3, Fig. 5). It is remarkable that in many occasions both species appear together. The type locality of Hi. mateusi is the hyporheic of the Duero River, 9km from the sea, near Oporto. However H. nicoleiana has not been found outside the provinces of Madrid and Guadalajara. Hexaiberobathynella is an endemic genus of the Iberian Peninsula that comprises only two species: Hi. mateusi and Hi. hortezuelensis (Soria). Hexabathynella, on the contrary, is the only cosmopolitan genus of Parabathynellidae and includes 23 species (Camacho, 2019). In the Iberian Peninsula, in addition to H. nicoleina, three more species are known to occur: H. minuta (Noodt & Galhano, 1969), found in several localities of Spain and Portugal (Table 3); H. valdecasasi Camacho, 2004 and H. sevillaensis Camacho, 2005, which are only known from their respective type localities, Arroyo el Torcón (Toledo) and Cueva de Santiago el Grande (Sevilla) respectively (Camacho, 2019) (Fig. 5).

It is interesting to remark the mutability of some MSS habitats, that can appear as a terrestrial underground environment and, temporarily, also as an aquatic underground environment. Thus, while the flooding process occurs, the MSS may also contain species which are typically aquatic. Therefore, similarly as to in the epikarst, the same spaces can, at different times, serve as a habitat for terrestrial and aquatic fauna, both epigean and hypogean (Ortuño et al., 2013). As seen in Figure 3, the SSD-32 collected aquatic fauna in two of the three sampling periods. In the first period, the aquatic fauna was limited to Bathynellacea (7% of the collection), and in the second period to Copepoda and Nematomorpha (50% and 3% of the collection, respectively). During the third period, the MSS was not flooded, and only terrestrial fauna was collected.

This discovery broadens the sampling horizon for this peculiar group of aquatic crustaceans. It will be necessary to consider looking for Bathynellacea in areas where there are shallow aquifers, more or less confined, and not as deep as those that have yielded such a large number of species as in the mining arid and remote areas of Australia (Perina et al., 2018, 2019).