Morphological and cytogenetic characteristics of Neobisium (Blothrus) slovacum Gulička, 1977 (Pseudoscorpiones, Neobisiidae), the northernmost troglobitic species of the subgenus Blothrus in Europe

Abstract A redescription is provided of the adult, tritonymph and deutonymph life stages of the troglobitic Neobisium (Blothrus) slovacum Gulička, 1977, which is known from Slovakia and Hungary. Material examined included 35 previously deposited museum specimens and 15 newly collected specimens. In addition, the karyotype and distribution of 18S rDNA clusters are described, using fluorescence in situ hybridization (FISH). The male karyotype of N.slovacum comprises 69 chromosomes, with a predominance of biarmed chromosomes, and an X0 sex chromosome system. Two pairs of signals for 18S rDNA on biarmed chromosomes (submetacentric and metacentric) of different sizes were identified. The present study provides the first information about the distribution of these clusters in the arachnid order Pseudoscorpiones. The geographic distribution of the species is summarized and mapped. Neobisiumslovacum is endemic to the Slovak and Aggtelek Karst area in southern Slovakia and north-western Hungary, where it has been recorded from 16 caves. One of these, Hačavská cave (in Slovakia), is the northernmost locality known for any species of the subgenus Blothrus.

In Slovakia, the sole species of the subgenus Blothrus is Neobisium slovacum Gulička, 1977, which has clear troglomorphic characters (Gulička 1977a, b). This species is endemic to the Western Carpathians (Gulička 1977a, Ducháč andMlejnek 2000a), with a distribution limited to the Slovak and Aggtelek Karst, a well-known karst area on the border between Slovakia and Hungary. This species was first collected by Lysenko in 1966 in the Diviačia chasm (Lysenko 1972), but it was not identified at that time. The specimens were rediscovered in Dr P.H. Verner's collection thirty years later and correctly assigned to this species by Ducháč (Ducháč and Mlejnek 2000a, b).
Neobisium slovacum is a strictly troglobitic species, with specific morphological adaptations to the cave environment. Compared to epigean relatives, the body of N. slovacum is pale, the legs and pedipalps are elongated, and eyes are absent. The remarkably elongated appendages provide an advantage in locating and capturing prey in the cave environment. The specimens are usually found on walls, in stony debris with clay sediments and near organic material (bat guano) in caves (Kováč et al. 2010).
The species was originally described from Stará brzotínska cave by Gulička (1977a), based on an adult holotype (sex not indicated) and two paratype nymphs. The description of the type material was incomplete because it omitted many important features necessary for proper species delimitation and identification. The depository of the type material is not known. Ducháč (1996) examined an additional specimen from the type locality and added some characteristics that were not reported in the original description. However, some morphological features useful for identification remained unknown. Ducháč (2002) summarized all previous collections of N. slovacum in his Ph.D. thesis, which contained descriptions of 53 adults, two tritonymphs and one deutonymph. However, these were still incomplete, with characters such as the chaetotaxy of sternites, morphometric data of legs I and IV, and the number of teeth on the cheliceral and palpal fingers not being specified.
Later, Ducháč (2004a, b) described in detail the male genitalia and the chaetotaxy of the genital opercula of N. slovacum and provided partial morphological descriptions of deutonymphs and tritonymphs, but the drawings and measurements were inadvert- ently omitted by the journal. Intraspecific variability of N. slovacum from the Slovak Karst was discussed by Ducháč (1999), who compared it with all Carpathian species of the subgenus Blothrus.
After Stará brzotínska cave, the species was found at 14 other localities in the Slovak Karst (Fig. 2, Table 1). Of these, Šingliarova chasm was the northernmost locality for the species, as well as for subgenus. Outside Slovakia, N. slovacum has been recorded from Meteor cave in the Aggtelek Karst in Hungary (Ducháč and Mlejnek 2000b). The distribution of N. slovacum in caves of the Slovak and Aggtelek Karst was discussed by Ducháč and Mlejnek (2000a, b). More recent findings of N. slovacum have been published by Vlk (2001) from Natrhnutá chasm, by Christophoryová (2009) from Šingliarova chasm, and by Kováč et al. (2010) from Slnečná, Vlčia and Veľká Peňažnica chasms.
Although N. slovacum seems to be restricted to the cave systems of the Slovak and Aggtelek Karst area, the possibility exists that the isolation of populations in separate cave systems could have led to diversification and cryptic speciation in this region. The detection of cryptic species diversity has recently been based mainly on the analysis of mitochondrial and nuclear gene sequences in terrestrial (e.g. Opatova and Arnedo 2014) and subterranean (e.g. Harrison et al. 2014) arachnid groups. However, cytogenetic techniques can also be useful for the identification of cryptic species within various orders of arachnids, such as harvestmen (Šťáhlavský et al. 2018a), spiders (Řezáč Figure 2. Distribution of Neobisium slovacum in karst in Slovakia and Hungary. Green star indicates the type locality and red spot the northernmost locality of the subgenus Blothrus. The karyotype is recorded for two localities. Localities numbered as in Table 1. et al. 2018), scorpions (Šťáhlavský et al. 2018b) and pseudoscorpions (Zaragoza and Šťáhlavský 2008). This is especially notable in some species of edaphic pseudoscorpions characterized by limited dispersal ability, for which distinct karyotypes may be found even in very close localities (Kotrbová et al. 2016). It is therefore useful to combine descriptions the external morphology with karyotype analysis (Plíšková et al. 2016), as is done here.
Summarizing the previously published data, 61 adults and six nymphs have been collected from Slovakia. Despite this, knowledge of the morphology of N. slovacum remained incomplete. All previous descriptions were deficient in important details. Consequently, the aims of the present study are to: (1) complete and examine previously studied material of N. slovacum, (2) describe newly collected specimens and provide additional information on the variability of morphometric and morphological characters, (3) analyse the N. slovacum karyotype and (4) analyse the distribution of N. slovacum in caves of the Western Carpathians.

Methods
To locate previously studied material of N. slovacum, the natural history museums in Vienna, Bratislava and Berlin, and the zoology departments of Charles University in Prague and Comenius University in Bratislava were contacted. In total, 35 specimens from eight localities in the Slovak Karst were obtained, including one specimen from the type locality. These specimens have been deposited in the Department of Zoology of Charles University in Prague and the Natural History Museum in Vienna (NHMW). The recently collected specimens of N. slovacum (15 specimens) were obtained at three localities of the Slovak Karst: Šingliarova chasm, Hačavská cave and Zvonivá chasm. The new material from Hačavská cave and Šingliarova chasm was deposited in the Natural History Museum in Vienna (NHMW) and material from the Zvonivá chasm in the Department of Zoology, Charles University in Prague.
Recently collected specimens were identified using the identification keys in Beier (1963), Christophoryová et al. (2011) andNovák (2014). Morphological terminology follows Chamberlin (1931) and Judson (2007); nomenclature follows Harvey (2013). All specimens were studied as temporary slide mounts using lactic acid as the medium, then rinsed in water and returned to 70% ethanol. Morphological and morphometric analyses were performed using a Leica DM1000 compound microscope with ICC50 Camera Module (LAS EZ application, 1.8.0). Measurements (in mm) were taken from digital images using the AxioVision 40LE application. Reference points for measurements follow Chamberlin (1931).

Cytogenetic analysis
One male from Zvonivá chasm (locality 2: Table 1 and Fig. 2) and two males from Šingliarova chasm (locality 7: Table 1 and Fig. 2) were used for the cytogenetic analysis.
The chromosomes were prepared by the "spreading" method described in Šťáhlavský and Král (2004), slightly modified as noted below. The dissected gonads were hypotonized in 0.075 M KCl for 30 min and then fixed in a methanol:glacial acetic acid (3:1) solution for at least 20 min. The tissue was then dissociated in a drop of 60% acetic acid on a clean microscope slide and the suspension was moved with tungsten needles on the surface until the fluid evaporated. The chromosomes were stained with 5% Giemsa solution in Sörensen phosphate buffer for 30 min. Chromosomes were documented using an Olympus IX81 inverted microscope equipped with a Hamamatsu ORCA-AG monochromatic camera. In total, ten metaphases II were measured using the LEVAN plugin (Sakamoto and Zacaro 2009) for IMAGEJ 1.47 software (http://imagej.nih. gov/ij/). For the identification of rDNA clusters, FISH with an 18S rDNA probe was used for all three males. This probe was prepared from the scorpion Euscorpius sicanus (Koch, 1837), as described by Šťáhlavský et al. (2018a). The probe was labelled by PCR with biotin-14-dUTP (Roche) using a Nick Translation Kit (Abbott Molecular) following the manufacturer's guidelines. The FISH protocol was performed following Forman et al. (2013), on the same chromosome preparation used for the standard karyotype analysis after washing Giemsa in methanol:glacial acetic acid (3:1) solution. During the FISH procedure, the slides were treated with RNase A (200 µg/ml in 2× saline-sodium citrate) (60 min, 37 °C). The chromosomes were then denatured in 70% formamide in 2× saline-sodium citrate (3 min 30 s, 68 °C). Finally, the probe mixture (20 ng of probe, 25 ng of salmon sperm DNA (Sigma Aldrich), 10 µl of 50% formamide, 10% dextran sulphate in 2× saline-sodium citrate) was applied to each slide and hybridization was performed overnight in a black box at 37 °C. The following day, the probe was detected with Cy3-conjugated streptavidin. The signal was intensified by application of biotinylated anti-streptavidin and Cy3-conjugated streptavidin. Chromosomes were counterstained with DAPI (Fluoroshield, Sigma-Aldrich) and documented using an Olympus IX81 microscope equipped with a Hamamatsu ORCA-AG monochromatic CCD camera. The images were pseudocoloured (red for Cy3 and blue for DAPI) and superimposed with Cell^R software (Olympus Soft Imaging Solutions GmbH).
Holotype. Adult of undetermined sex collected from stony debris on clay sediment on 26 September 1974. Apparently lost. Paratypes. 2 nymphs, from type locality, collected on 10 August 1975. Apparently lost.
Note. The depository of the type material is unknown. Searches for the holotype and paratypes in various institutions (listed in Methods section) were unsuccessful.
Material examined (see Table 1  Remarks. Faunistic data and descriptions of the 35 specimens listed above were provided by Ducháč (1996Ducháč ( , 1999Ducháč ( , 2002Ducháč ( , 2004a and Ducháč and Mlejnek (2000a, b). It was generally not possible to correlate specimens with an individual literature source, because only the numbers of individuals and variability of some characters were mentioned. Of these specimens, 27 were used in the present study to add information on previously ignored characters. The remaining eight specimens were not examined in detail, either because the locality was unknown or because they were not in a suitable condition. Remarks. The newly collected specimens are described here, except for those used in the cytogenetic analyse. The record of the four specimens (NHMW 28659) from Šingliarova chasm was previously published by Christophoryová (2009). Hačavská cave represents a new locality for the species in Slovakia and is the northernmost locality of any member of the subgenus Blothrus (Figs 2, 4). In total, 14 adults and one Redescription. In total, 40 adults, 1 tritonymph and 1 deutonymph were examined in the present study. Measurements of adults are given in Table 2.
Adults. Body yellowish; carapace, chelicerae and pedipalps light brown. Vestitural setae of body and pedipalps long and pointed. Carapace rectangular, without granulation, longer than broad, epistome and eyes absent, a subocular seta usually present (sometimes missing on one side). Tergites and sternites undivided. Chelicera with 5 setae on hand, 1 seta on movable finger, spinneret well developed in female, weak in male, rallum of 8 blades, 2 distal blades dentate. Anterolateral process of coxa I long, broad and apically pointed, medial process prominent, rounded, with strong denticles. Pedipalps slender, chelal fingers with normal number of trichobothria (8 on fixed and 4 on movable finger), sensillum p 1 slightly distal of st, p 2 nearer to st than to sb, situated close to dental margin. Palpal trochanter without tubercles. Legs elongated, I-IV with 2 tarsal segments (basitarsus and telotarsus separated). Subterminal setae of telotarsi I and IV with short, smooth, ventral rami and small dorsal denticles in distal part. Telotarsus IV with 2 long tactile setae, telotarsus I without tactile seta. Claws with a small dorsal denticle about one-third from base.

Discussion
The original description of N. slovacum by Gulička (1977a) included only a few morphological and morphometric features, but it was sufficient for recognition of the species. The recently examined specimens fit well with this description, except for minor variability in the chaetotaxy of the tergites and measurements of chelicera. Ducháč (2004b) provided descriptions of the deutonymph and tritonymph, but the figures and table of measurements are missing on the web site of the journal and no printed version of the paper is available. According to the editor of the journal (Dr Eduard Stloukal, pers. com.), the figures and table were never published.
The redescription of previously studied and newly obtained material in the present paper gives a better assessment of intraspecific variability and adds some previously unstudied characters, such as the chaetotaxy of sternites, measurements of legs and the number of teeth on cheliceral and palpal fingers, presence of subocular setae, anterolateral and medial processes of coxa I, positions of chelal sensilla p 1 and p 2 , form of subterminal seta, tactile seta of legs I and IV and denticulation of leg claws. The individuals show great variability in some characters, such as palpal femur and trochanter length, palpal chela length/width ratio, femur I and telotarsus I length/depth ratio, tibia IV and telotarsus IV length/depth ratio, femoropatella IV length/depth ratio in males, number of teeth on chelal and palpal fingers, chaetotaxy of sternites, and chaetotaxy of the male genital operculum. However, no significant differences were observed in chaetotaxy or measurements between populations of different caves. The greatest variability was observed in the palpal femur length/width ratio, with a range of 5.69-8.81 mm. Novák (2014) separated Blothrus species of the Carpathian Mountains in couplet 1 of his identification key between those with a palpal femur ratio of up to 6 and those, including N. slovacum, with a ratio of 6.5 or more. According to our measurements, this couplet will not always be reliable for identifying N. slovacum.
Concerning the karyotype, no differences were observed between the three males analysed. It should be noted that the material used for cytogenetic analysis comes only from two localities (Fig. 2) near each other in the northern part of the Plešivecká Plateau (Slovak Karst). In this case, dispersion of individuals between caves and hence gene flow between populations cannot yet be excluded. The number of chromosomes in N. slovacum (2n = 69) is similar to that of Neobisium (Neobisium) carcinoides (Hermann, 1804) (2n = 67; Sokolow 1926) and falls within the known range (2n = 30-71) reported in a preliminary analysis of species of this genus (Šťáhlavský et al. 2012b). Other cytogenetic features of N. slovacum are consistent with previous findings for pseudoscorpions. The chromosomes gradually decrease in length and their morphology is variable, which is typical for karyotypes of pseudoscorpions with higher number of chromosomes (Šťáhlavský et al. 2009, 2012a). The sex chromosome system is X0, which is assumed to be the ancestral state for pseudoscorpions (Troiano 1990). However, the morphology of the X chromosome is usually metacentric in this order (Šťáhlavský et al. 2012a(Šťáhlavský et al. , 2013(Šťáhlavský et al. , Kotrbová et al. 2016, whereas an acrocentric X was identified in N. slovacum. This morphology of the X chromosome was previously documented in only one population of Olpium pallipes (Lucas, 1846) (Olpiidae) within pseudoscorpions (Šťáhlavský et al. 2006). It was supposed to be an effect of pericentric inversion from Greece in the population concerned. This type of chromosomal rearrangement can also be presumed for N. slovacum, because all other neobisiids thus far karyotyped possess biarmed X chromosomes (Šťáhlavský et al. 2013). Interestingly, two pairs of 18S rDNA clusters were identified in N. slovacum, even though one pair is considered to be the ancestral state for arachnids (Forman et al. 2013). However, a multiplication of the 18S rDNA clusters seems to be frequent, at least in arachnids with limited dispersal ability (Svojanovská et al. 2016(Svojanovská et al. , Šťáhlavský et al. 2018a). In such groups the variability in the number and position of rDNA clusters suggests intensive chromosomal changes that may also be expected in N. slovacum, in view of its subterranean habitat. It should be noted that no information is available about the number and position of rDNA clusters in any other pseudoscorpions, and the presence of two pairs of rDNA clusters might be typical for this order.
Summarizing all known distributional records, N. slovacum is currently known from 15 localities in the Slovakia and one locality in Hungary (Table 1 and Fig. 2). Based on this distribution, it can be expected to occur in the karst areas between the localities and near the border of the two countries. There are no valid records of species of the subgenus Blothrus in countries lying north of Slovakia in Europe or elsewhere. Beier (1936) described Neobisium (Blothrus) vulpinum Beier, 1936 from Krkonoše, western Sudetenland (a mountain range now within parts of the Czech Republic and Poland). However, Beier (1963) later stated that the locality details of the types were erroneous and synonymized this name with N. (B.) minutum (Tömösváry, 1883). The size of the caves and chasms occupied by N. slovacum range from small (Fialová cave, length 21 m; Vlčia chasm, length 29 m) to large (Zvonivá chasm, length 494 m; Diviačia chasm, length 468 m), situated at elevations between 258 and 795 m a.s.l. Air temperature in the caves, measured at the time of collection (using a digital thermometer), ranged from 5.4 °C (Zvonivá chasm) to 9.7 °C (Stará brzotínska cave). Air relative humidity during the collection of specimens was 94-100%.