A survey of spiders ( Araneae ) inhabiting the euedaphic soil stratum and the superficial underground compartment in Bulgaria

In 2005 a team of Bulgarian zoologists started a project aiming to study the invertebrates inhabiting the deeper soil stratum (euedaphon) and the Superficial Underground Compartment (SUC) in Bulgaria. In the course of a four-year sampling, a total of 52 species of spiders were caught from 19 collecting sites and 9 geographical regions. They belong to the following families: Scytodidae (1), Segestriidae (1), Dysderidae (8), Nesticidae (1), Anapidae (1), Theridiidae (1), Linyphiidae (20), Agelenidae (3), Cybaeidae (1), Dictynidae (2), Amaurobiidae (2), Liocranidae (3), Corinnidae (1), Zodariidae (1), Gnaphosidae (5), and Salticidae (1). The family Anapidae, with the species Zangherella relicta (Kratochvíl, 1935) is recorded from three sites in the Pirin and Slavyanka mountains, and this represents the first record of the family, genus and species in Bulgaria. In spite of the active investigations of the epigean and cave spiders in these regions over the years Z. relicta was not found and it seems it occurs only in deeper subterranean habitats and nowhere else. Comparative study of almost topotypic specimens of Z. relicta from Montenegro with those collected from Bulgaria showed no variation in the shape of palp and female vulvae. Until the true identity of Z. apuliae (Caporiacco, 1949) from Italy is revealed, it remains unclear whether Z. relicta and Z. apuliae are conspecific, as it remains unclear whether the older records of Z. apuliae from the Balkan Peninsula refer to this species or to Z. relicta. Pelecopsis mengei (Simon, 1884) (Linyphiidae) and Scotolathys simplex Simon, 1884 (Dictynidae) are also reported from Bulgaria for the first time, the latter being also new to FYR of Macedonia. A faunistic overview of the spiders found in these underground environments is made, along with remarks on the distribution and ecology of some rare and interesting species. The presence of cave-dwelling and superficial spiders in the sampled sites indicates that SUC and euedaphon are inhabited by different ecotypes, e.g. litter(tanathostromic), soil(edaphic) and cave-(troglobitic) which at some places co-occur.

There are various subterranean spaces that invertebrates inhabit. Depending mostly on their origins, size and distance from the surface one can distinguish different types of subterranean habitats, e.g. caves, lava tubes, microspaces in stony debris and screes, deep undreground micro-and macrospaces in the deeper gravel layers, etc. Each habitat has specific characteristics comprising temperature fluctuations, humidity, aeration, organic carbon levels, soil texture, etc. For instance daily temperature fluctuations exist in the surface of scree slopes and stony debris, while they cease completely at one-meter depth and after several dozens of metres inside large caves (RŮŽIČKA 1999). Organic carbon levels are higher in the Superficial Underground Compartment (SUC) than in caves (CULVER & PIPAN 2009). Animals that are adapted to living in complete darkness and with limited energy supplies usually acquire a specific appearance that includes depigmentation, micro-or anophthalmy, loss of wings, elaboration of extra-optic sensory structures, elongation of appendages (in cases of troglobionts) or shortening of appendages (in case of geobionts), cuticle thinning, etc. These morphological alterations known as troglomorphy are widespread in subterranean animals (CHRISTIANSEN 1962). Some species that inhabit SUC and deeper soil strata and have adapted to dwelling in these aphotic environments may have given rise to species occurring in caves (CULVER & PIPAN 2009). The vertical distribution of arthropods in the soil depends on the porosity of soil, soil type, temperature and humidity, as well as the amount of organic matter (LAŠKA et al. in press). JUBERTIE et al. (1980) defined and described (in French) as the "Milieu Souterrain Superficiel" (MSS) one of the subterranean habitats where they found several troglomorphic invertebrates. In publications in English this particular environment is referred to as the "Superficial Underground Compartment" (SUC) or "Mesocavernous Shallow Stratum" (MSS) (Figs. 1-2). In addition to its geomorphologic structure, the SUC has specific microclimatic and hydrological characteristics. According to JUBERTHIE & DECU (1994) the SUC exists as interconnected microspaces in valley versant screes or similar fissures in the superficial zone of maternal rocks. It connects with other profound subterranean environments like caves by way of deep fissures (NEGREA & BOITAN 2001). It is also believed that the SUC represents an intermediate stratum where litter-(tanathostromic), soil-(edaphic) and cave-(troglobitic) species may occur together. Furthermore, several SUC specialists highly adapted to subterranean manner of life inhabit the SUC and never penetrate into epigeic habitats. An example of such species is the beetle Speonomus hygrophylus Jeannel, 1907(cf. CULVER & PIPAN 2009).
The most serious contribution to the study of subterranean spiders and in particular those living in the stony debris and screes in Central Europe was made by Vlastimil RŮŽIČKA, who studied the underground spider assemblages in the Czech Republic, Romania, Montenegro and Austria (see e.g., RŮŽIČKA 1989RŮŽIČKA , 1992RŮŽIČKA & THALER 2002). However, this subject received very little attention in the other parts of Europe, and yet our knowledge of the underground spider assemblages in biodiversity-rich regions such as the Balkan Peninsula remains perfunctory. A profound review of spiders living in caves and hypogeic environments can be found in RIBERA & JUBERTHIE (1994).
The spiders living in superficial, hemiedaphic and cave environments in Bulgaria have been studied

Material and Methods
The investigation was carried out in the period April 2006 -June 2009. Forty-three traps were set in the mountains of Vitosha, Pirin, Slavyanka, Belasitsa, Ruj, Western Rhodopes, Ichtimanska and Sashtinska Sredna Gora, and the Derventsky Heights (Fig. 3, Table 1). The trap was made of a PVC pipe with a diameter of 8 cm and two different lengths: 60 and 80 cm, respectively (Fig. 4). 108 holes with a diameter of 8 mm were made in the pipe, at a distance of 10 cm from its lower end (Fig. 4) and covering nearly 1/3 of its total length. A hole of 60 to 80 cm depth was dug in the ground where the pipe was placed vertically. After the proper positioning of the pipe, the space between the pipe and the hole was filled up, initially with gravel to the upper level of the holes and subsequently with soil and other particles up to the top of the pipe. A 10-centimetre plastic pot tied to a polythene rope was put at the bottom of the pipe. The pot was filled with etylenglycol with a few drops of formalin. The trap was covered tightly with a solid plastic cover and additionally with soil, tufts of grass and leaves to prevent penetration by the superficial fauna and infiltration of water during pouring rain. Description of each collecting site and the traps is given in Table 1. Usually, traps were checked and emptied once every three months, sometimes once or twice a year. All material was sorted down to a species level and put into 70% spirit. The entire collection is preserved in the Institute of Zoology, Bulgarian Academy of Sciences (IZ). Almost topotypic material of Zangherella relicta (Kratochvíl, 1935) was obtained for study from the Senckenberg Museum Frankfurt (SMF). All drawings were made with a camera lucida mounted on a Wild stereoscope. Nomenclature follows PLATNICK (2010).

Dysderidae
Dysderids are frequent in underground habitats and in leaf-litter, and prefer warm and humid environmental conditions. Whilst there are very few cave records in Bulgaria, dysderids are quite often found in caves elsewhere and some species are even cave specialists, which have become adapted to this particular environment (e.g. CHATZAKI & ARNEDO 2006). In Bulgaria only Harpactea babori (Nosek, 1905) has been recorded from caves . Nevertheless, the family is well represented in the SUC and euedaphic stratum (Table 2), whereby species like, e.g., Harpactea srednogora, H. deltshevi and Dysdera longirostris, were found at several sites and were among the most abundant species in the traps. Besides depigmentation, none of the dysderids found in the underground habitats possess obvious troglomorphic features to be categorized as representing strictly geo-or troglobiontic ecomorphotypes.

Nesticus cellulanus
Members of the family Nesticidae are prone to colonize shallow or deep subterranean spaces. KRA-TOCHVÍL (1933) described several morphological alterations in nesticids resulting from their subterranean manner of living. Out of the three species of Nesticus hitherto known from Bulgaria, in the SUC and euedaphic stratum we have only found N. cellulanus. This species is widespread throughout the country, and is among the most common spiders in Bulgarian caves , DELTSHEV & PETROV 2008. In the Czech Republic it occurs in shallow subterranean spaces in screes, cave entrances, and buildings (BUCHAR & RŮŽIČKA 2002).

Anapidae
Zangherella relicta  Pseudanapis relicta Kratochvíl, 1935: 18, pl. 1, f. 7-12. Type locality: Montenegro: 1 , 1 . =?Pseudanapis apuliae sensu BRIGNOLI 1974BRIGNOLI , 1977BRIGNOLI , 1978BRIGNOLI , 1984 (BRIGNOLI 1981) retracted the synonymy of the genus Zangherella with Pseudanapis Simon, 1905, and assigned to it the species Z. algerica, Z. apuliae and Z. relicta. Having at their disposal specimens from Greece, THALER & KNOFLACH (1998) revised the genus Zangherella and its distribution and stated that according to the original descriptions the main difference between relicta and apuliae concerned the number of spines on tarsus I (4 in relicta vs. 6 in apuliae). The authors studied several transitional cases where distal spines were only weakly developed and considering the completely identical shape of palps they argued that both species might actually be conspecific (THALER & KNOFLACH 1998). They also provided new illustrations of palps of specimens from Greece. The whereabouts of the type material of P. relicta are unknown and it is believed to have been lost during the Second World War (cf. BRIGNOLI 1968).

4
The almost topotypic material of Zangherella collected in Herceg Novi, Montenegro and the abundant material amassed using subterranean traps in Bulgaria made it possible to compare vis-a-vis the available specimens. We were not able to find any reliable differences between the Montenegrin and Bulgarian specimens when studying the structure of the vulva and the male palp (compare Figs. 5-6, 9-10 with 7-8, 11-12). They also correspond well with the illustrations of specimens of Z. apuliae reported by THALER & KNOFLACH (1998) from Greece. We were not able to obtain comparative material of Z. apuliae from Italy nor to examine the type material and could not decide whether the two species, relicta and apuliae are conspecific, as presumed by BRIGNOLI (1968). Combined molecular and morphological analysis is required to determine whether Z. apuliae is a valid species of a possibly restricted distribution in the Apennines or whether there is only one valid species, Z. relicta, distributed from the Apennines to Asia Minor.

Antrohyphantes rhodopensis
This species was hitherto known only from caves of the Western Rhodope Mts. and from the orophyte zone of the Rila and Pirin mountains (DELTSHEV 1996, DELTSHEV & PETROV 2008. Its presence in subterranean environments in southern Pirin adds very little to its geographical distribution but shows that the species is a subterranean specialist which is likely to be restricted to colder microhabitats and suitable humidity.

Centromerus milleri
This species was hitherto known only from Dupnitsa cave in the Strandzha Mts., the European part of Turkey (anophthalmic population, Deltshev, unpublished), Maronia cave in north-eastern Greece, several caves in the Eastern Rhodope Mts. and Stapalkata Cave in the Western Rhodope Mts., Bulgaria (DELT-SHEV & PETROV 2008). The new record from the Ruj Mts. significantly extends its range in north-western direction and indicates a possibly wider distribution. The anophthalmic population in the cave Dupnitsa is of special interest, as it might represent a new, closely related troglobitic species. A molecular study of the different populations may reveal the routes of colonization, specialisation and evolution of the species in the different parts of its distribution area. The Ruj population does not show any obvious morphological difference from the other Bulgarian population.

Centromerus acutidentatus
The species belongs to the sylvaticus-group of the genus Centromerus Dahl, 1886 and is currently known from caves in Bulgaria, FYR Macedonia and Serbia (DELTSHEV & ĆURČIĆ 2002). As well as in caves, the species has also been found in the detritus of forests, and now also in the Vitosha Mts. at a depth of 40-60 cm.  1994, as well as from other subterranean soil habitats. It occurs among detritus and deep under stones in beech and spruce forests, in void systems, and in screes (LASKA et al. in press). It is also quite common in caves in the Czech Republic and Slovakia (cf. RŮŽIČKA 2007).

Lepthyphantes centromeroides
This species is comparatively widespread on the Balkan Peninsula and can be considered as an example showing the process of cave colonization and subterranean adaptation. It occurs in caves, but also in the humus and ground detritus (DEELEMAN-REINHOLD 1978: 196-200).

Pelecopsis mengei
The species is widespread in the Holarctic region (PLATNICK 2010), and this represents the first record from Bulgaria. So far it is known only from the region of Panagyurishte, where it was found in the soil at a depth of 40-90 cm.

Histopona tranteevi
So far this species was known only from a few caves in the Western Rhodope Mts. (DELTSHEV & PETROV 2008). The new records from subterranean habitats in Ruj, the Western Rhodopes and Slavyanka mountains suggest that it is much more widespread than previously thought. The species is obviously inclined to dwell in semiaphotic and aphotic environments. The Ruj locality lies quite apart from the other two and suggests that the species also occur in similar habitats in Serbia.

Cicurina cicur
The majority of the species of the genus Cicurina

Discussion
Twenty-five per cent of the spiders (13 species) found in the SUC and euedaphic stratum are also known from caves. All of them show one or more troglomorphic traits (Table 3) and thus they could be considered true hypogeicolous animals (subterranean specialists). The remaining species are either surface dwelling (e.g. Eurocoelotes jurinitschi, Gnaphosa modestior) species or they had accidentally fallen in the traps when they were laid (Euophrys frontalis).
In a survey of the spiders living in deeper soil layers in the Czech Republic, LASKA et al. (in press) found 48 species of spiders, of which five, Palliduphantes alutacius, Centromerus cavernarum, Diplostyla concolor, Nesticus cellulanus, and Cicurina cicur, the authors considered more or less bound to subterranean environ-ments. P. alutacius was found to occur at a maximum depth of 95 cm, C. cavernarum at 75 cm, D. concolor at 15 cm, and both N. cellulanus and C. cicur at 85 cm. These species were also found during our study, as D. concolor was found at 8 collecting sites. Another frequently collected species was Harpactea srednagora, which was found at 11 sites.
Of all the species found in the SUC and euedaphon only Antrohyphantes rhodopensis, Centromerus milleri and Zangherella relicta have a distinct troglomorphic appearance. All three are also known from caves. If we adopt the recent classification of subterranean biota (SKET 2008), where troglobionts are defined as "strongly bound to hypogean habitats", then the species mentioned above should be considered troglobionts. However, there are some alternative classifications of the subterranean animals, e.g. that of DECU et al. (2006) who distinguish between hypogeicolous and cavernicolous fauna, and further subdivide the former category into colluviotroglobitic, eluviotroglobitic, cleitrotroglobitic and volcanotroglobitic, based on the origins and texture of the substrate (colluvic, eluvic, cleitric and volcanic MSS, respectively). According to the latter classification Zangherella relicta, which was found only at a depth of 40-50 cm, could be considered a true colluviotroglobite, if it is proved that in Bulgaria it occurs only in the SUC and is absent in caves or superficial soil layers. Histopona tranteevi and Centromerus milleri were hitherto known only from a limited number of caves (DELTSHEV & PETROV 2008) but turned out to also live below 30 cm depth in the SUC and euedaphon. It is evident that the boundary between true troglobionts and true geobionts is very vague and there is no unequivocal tool for identifying to which of these two categories an animal belongs. Table 3 summarizes the distribution of the species found in subterranean habitats in Bulgaria and provides information on their troglomorphic traits. There are several highly adapted cavernicolous species such as Centromerus bulgarianus and Troglohyphanthes drenskii, which are so far unknown from soil strata and the SUC and might thus represent true cave-dwellers. Other troglomorphic species, which are usually considered either eutroglophiles or troglobites, such as Centromerus cavernarum, C. lakatnikensis, C. milleri, Lepthyphantes centromeroides, Palliduphantes istrianus, P. spelaeorum and P. trnovensis have been recorded from both caves and deeper soil strata and their habitat preferences are in need of more profound studies. for this study. Vlastimil Růžička (Institute of Entomology, České Budějovice) provided valuable papers on the study of subterranean spiders in the Czech Republic and commented on the earlier version of the manuscript. We are also grateful to Mrs. Ekaterina Ivanova for providing illustrations 1 and 4. S.L. and C.D. are especially grateful to Peter Jäger and Julia Altman (SMF) for their overall support