The diploid set consists of 21 chromosomes in male (Fig. 1a) and 22 chromosomes in female. The male karyotype comprises ten pairs of acrocentric autosomes and one metacentric X sex chromosome. The first three acrocentric pairs of autosomes are significantly longer (RCLs 13.23%, 12.49% and 10.92%) than the remaining autosome pairs that gradually decrease in RCL from 7.98% to 2.97%. The X represents the largest chromosome in the karyotype reaching the length of 27.36% of the haploid set.

Figure 1.

Karyotypes of Chthonius (Chthonius) males based on postpachytene and metaphase I. AC. (C.) alpicola (2n = 21, X0) (large black spot on the first autosome pair represents overlap with the sperm) BC. (C.) carinthiacus (2n = 35, X0) CC. (C.) ischnocheles, cytotype I (2n = 31, X0) DC. (C.) ischnocheles, cytotype II (2n = 35, X0) EC. (C.) raridentatus, cytotype I (2n = 29, X0) FC. (C.) raridentatus, cytotype II (2n = 29, X0) GC. (C.) rhodochelatus, cytotype II (2n = 35, X0). Asterisks indicate chromosome overlaps. Scale bar = 10 µm.

Seven individuals from central and eastern parts of the Alps displayed 2n = 35 in all cases (Fig. 1b). The karyotype of this species comprises 17 pairs of acrocentric autosomes and one metacentric X sex chromosome. The RCL of autosomes gradually decreases from 7.29% to 2.34%. The RCL of the X chromosome is 23.30%.

Variability in chromosome number and morphology was detected in this species; two different cytotypes were distinguished. Cytotype I was detected only in two males from two geographically proximate localities in Switzerland and France. The diploid set of this cytotype comprises 31 chromosomes (Fig. 1c). There are 13 pairs of acrocentric and two pairs of metacentric autosomes (pairs No. 1 and 13) and one metacentric X chromosome. Autosome RCLs decrease gradually from 8.47% to 3.66%. The RCL of the X is 17.85%. Cytotype II was detected from seven different localities. The diploid number of chromosomes of this cytotype is 35 (Fig. 1d). The karyotype comprises 14 pairs of acrocentric autosome pairs, two metacentric pairs (pairs No. 1 and 13), and one submetacentric pair (pair No. 3), with the X chromosome metacentric. The RCLs of the autosomes decrease gradually from 7.47% to 1.45% and the last autosome is significantly shorter than the previous pair. The RCL of the X is 17.12%.

The diploid number of chromosomes in all analysed individuals was 29 (Fig. 1e). Detailed analyses detected the existence of two cytotypes within this species. Cytotype I was found in most of the males, comprising 12 acrocentric autosome pairs, one metacentric pair (pair No. 2), and one subtelocentric pair (pair No. 5), and one metacentric X chromosome. Cytotype II was detected only in two individuals from different localities in northwest Slovenia. The karyotype is composed of 12 pairs of acrocentric and two pairs of metacentric autosomes (pairs No. 1 and 2), and one metacentric X (Fig. 1f). The karyotypes in both cytotypes showed a length differentiation of the autosomes; the first three pairs of autosomes are longer (roughly 3–3.5 ×) than the remaining chromosomes in the nucleus. In cytotype I, the first three autosomes are also considerably longer (RCLs 10.91%, 10.12% and 9.84%) than the remaining autosomes that gradually decrease in size, and X sex chromosome is the longest chromosome in the karyotype (Fig. 2). In cytotype II, RCLs of the three longest autosomes is 10.91%, 9.55% and 9.39% of the haploid set. The RCLs of the remaining autosomes gradually decrease from 7.39% to 2.90%, and the RCL of the X is 20.01%.

Figure 2.

Ideograms of Chthonius (Chthonius) raridentatus cytotype I from Roče (y axis - % of the chromosome length of the haploid set). Comparison of different meiotic (ppach - postpachytene (white), met I - metaphase I (light grey), met II - metaphase II (dark grey)) and mitotic (mit - mitotic metaphase (black)) stages with examples of chromosomes 2 and X. Ideograms include min. - max. values and the centromeres are indicated only in metacentric chromosomes (all other chromosomes are acrocentrics).

In cytotype I, we tested differences of the chromosome lengths and also arm ratio in biarmed chromosomes during distinct spiralization of several mitotic (mitotic metaphase (N = 44)) and meiotic stages (postpachytene (N = 14), metaphase I (N = 42), metaphase II (N = 29). We detected significant differences in two thirds of the comparisons among chromosomes (Suppl. material 1). The most considerable difference is noticed in the X chromosome during different stages (Fig. 2). It is probably an effect of different spiralization states of the X during meiosis and mitosis, also visible as different degrees of heteropycnosis (see paragraph below). The arm ratio of biarmed chromosomes (pair No. 2 and X) is significantly different only in few cases (Suppl. material 1) and the metacentric morphology has been detected during all analyzed stages.

During the meiosis X chromosome undergoes changes in condensation. During early prophase (leptotene-zygotene), the X forms a prominent spherical body and exhibits positive heteropycnosis (Fig. 3a). The body starts expanding during pachytene. The individual positively heteropycnotic arms of the X chromosome become visible, however they are still connected by their ends (Fig. 3b). During postpachytene, the sex chromosome becomes slightly negatively heteropycnotic (Fig. 3c). During metaphase I, all the chromosomes become isopycnotic except for prominent knobs in centromeric regions (Fig. 3e). These knobs are visible on the chromosomes from pachytene to metaphase I, but cannot be detected during metaphase II (Fig. 3f) and anaphase II. C-banding and analysis of cells at metaphase II of this species confirmed that constitutive heterochromatin is exclusively localized in these prominent knobs that correspond to the centromere regions (cf. Fig. 3c and 3d).

Figure 3.

Meiotic chromosomes of Chthonius (Chthonius) raridentatus, cytotype I (A–F) and Chthonius (Ephippiochthonius) tetrachelatus (G, H). A zygotene B pachytene C, D postpachytene E metaphase I F metaphase II G, H postpachytene. Standard chromosomes stained with Giemsa (A–C, E–G) and the chromosomes after C-banding stained with DAPI (inverted) (D, H). Arrowheads indicate X sex chromosomes, arrows indicate metacentric autosomes. Scale bar = 10 µm.

Variability in chromosome morphology was detected in this species, resulting in two distinguishable cytotypes. The diploid number of chromosomes in cytotype I is 35. The karyotype comprises 15 acrocentric and two metacentric autosome pairs (pairs No. 2 and 13), and one metacentric X chromosome. No significant length differentiation of autosomes was detected. The RCLs of the autosomes gradually decrease from 6.25% to 3.24%. The RCL of the X is 22.43%. Cytotype II was detected in one male from a locality in the central Alps. This individual had a diploid set of 35 chromosomes (Fig. 1g). The karyotype comprises 15 acrocentric pairs, one metacentric pair (pair No. 13), and one submetacentric pair of autosomes (pair No. 14), with the X chromosome metacentric. The RCLs of the autosomes gradually decrease from 6.73% to 2.92%. The RCL of the X is 20.38%.

Variability in chromosome number and morphology was detected in this species, resulting in five distinguishable cytotypes (Fig. 4). Most individuals displayed cytotype I: 2n = 35 with 16 pairs of acrocentric and one pair of metacentric autosomes (pair No. 9), and one metacentric X chromosome (Fig. 4a). The RCLs of the autosomes gradually decrease from 6.41% to 2.65%. The RCL of X sex chromosome is 28.66%. Cytotypes II–IV have 2n = 33 and differ among themselves in the morphology of some autosome pairs. Cytotype II comprises 15 pairs of acrocentric and one pair of metacentric autosomes (pair No. 1) (Fig. 4b). The first autosomes pair (RCL 10.95%) is roughly two times longer than the rest. The RCLs of the remaining autosomes gradually decrease from 6.46% to 2.90%. Cytotype III comprises 13 pairs of acrocentric, two pairs of metacentric (pairs No. 1 and 11), and one pair of subtelocentric autosomes (pair No. 4) (Fig. 4c). The first pair of autosomes is significantly longer (RCL 11.18%) than the remaining autosomes whose RCLs gradually decrease from 5.95% to 3.13%. Cytotype IV comprises 16 acrocentric autosome pairs (Fig. 4d). The first autosome pair is significantly longer (RCL 10.61%) than the remaining autosomes whose RCLs gradually decrease from 6.59% to 2.72%. In all cytotypes with 2n = 33, the X is metacentric and the largest chromosome in the karyotype, with RCLs 22.87%, 20.44%, and 22.86% for cytotype II, III, and IV, respectively. Two male individuals of C. (C.) tenuis from Liguria showed a different chromosome number in the karyotype, namely 2n = 21. This cytotype V comprises seven pairs of acrocentric and three pairs of metacentric autosomes (pair No. 1, 3, and 4), and one metacentric X chromosome (Fig. 4e). In cytotype V, the two chromosomes of the first and the second pair are almost five times longer (RCLs 19.06% and 16.28%) than the other pairs. The two following autosome pairs are of medium size (RCLs 9.44% and 7.56%), whereas the remaining autosomes gradually decrease in RCL from 4.27% to 2.88% of the haploid set. The RCL of the X chromosome is 26.78%.

Figure 4.

Ideograms of Chthonius (Chthonius) tenuis cytotypes (y axis - % of the chromosome length of the haploid set) and examples of chromosomes in postpachytene and metaphase I. A cytotype I (2n = 35, X0) B cytotype II (2n = 33, X0) C cytotype III (2n = 33, X0) D cytotype IV (2n = 33, X0) E cytotype V (2n = 21, X0) F distribution of cytotypes. Arrows indicate X sex chromosome and extra-large autosomes.

All examined individuals displayed 35 chromosomes in the diploid set (Fig. 5a). The karyotype of this species comprises 16 pairs of acrocentric and one pair of subtelocentric autosomes (pair No. 2), and one metacentric X sex chromosome. The autosomes gradually decrease in RCL from 7.38% to 2.43%. The RCL of the X is 19.99%.

Figure 5.

Karyotypes of Chthonius (Ephippiochthonius) and Chthonius (Globochthonius) males. AC. (E.) boldorii (2n = 35, X0), postpachytene BC. (E.) fuscimanus (2n = 35, X0), metaphase I CC. (E.) tetrachelatus (2n = 35, X0), postpachytene DC. (E.) nanus (2n = 25, X0), sister metaphases II EC. (G.) poeninus (2n = 25, X0), mitotic metaphase. Asterisks indicate overlaps of chromosomes. Scale bar = 10 µm.

Figure 6.

The proportion of biarmed (metacentric and submetacentric) and one-armed (subtelocentric and acrocentric) autosomes in karyotypes of the genus Chthonius. Data from Table 1.

The diploid set of this species consists of 35 chromosomes (Fig. 5b). The karyotype comprises 17 acrocentric autosomes pairs and one metacentric X chromosome. The autosomes gradually decrease in RCL from 6.70% to 3.33%, and the RCL of the X is 18.86%.

The diploid set of this species comprises 35 chromosomes (Fig. 5c). They are 15 pairs of acrocentric, one pair of subtelocentric (pair No. 7), and one pair of submetacentric autosomes (pair No. 9); the X chromosome is metacentric. The autosomes gradually decrease in RCL from 7.45% to 2.96%, and the RCL of the X is 19.59%. C-banding confirmed the exclusive localization of constitutive heterochromatin in the centromere region (cf. Fig. 3g and 3h).

The diploid set consists of 25 chromosomes (Fig. 5d). The karyotype possesses 11 acrocentric autosome pairs and one metacentric pair (pair No. 1), and one metacentric X chromosome. The metacentric pair of autosomes is significantly longer (RCL 20.91%) than the remaining autosomes whose RCLs decrease from 6.26% to 2.96%. The RCL of the X is 31.03%.

The diploid set of this species consists of 25 chromosomes (Fig. 5e). The karyotype comprises of 11 pairs of acrocentric and one pair of metacentric autosomes (pair No. 2), and one metacentric X sex chromosome. The autosomes gradually decrease in RCL from 8.04% to 3.37%, and the RCL of the X is 33.74%.

Only 11 species of Chthoniidae have been studied so far, eight of them belonging to the genus Chthonius from Romania, Czech Republic, and Greece (Šťáhlavský and Král 2004) (Table 1). Analyses of 11 species from the Alps fully confirm previously detected cytogenetic characteristics of the genus. Pseudoscorpions from the Alps present monocentric chromosomes, similar to other representatives of the genus as well as all other pseudoscorpion taxa that have been cytogenetically analysed (e.g. Šťáhlavský and Král 2004, Šťáhlavský et al. 2006). Achiasmatic meiosis has been confirmed in males of the genus Chthonius, which is probably characteristic for the entire family Chthoniidae (Šťáhlavský and Král 2004). This meiosis type is otherwise known within the class Arachnida only in scorpions (e.g. Schneider et al. 2009), spiders from the families Dysderidae and Segestriidae (Benavente and Wettstein 1980) and mites from the superfamily Hydrachnellae (Oliver 1977). Given that groups presenting achiasmatic meiosis are not closely related (e.g. Sharma et al. 2014), multiple independent origins of achiasmatic meiosis within arachnids could be assumed. Another characteristic specific to the genus Chthonius is the sex chromosome system. The typical sex chromosome system in the family Chthoniidae is X0 (Šťáhlavský and Král 2004), which was detected in all species analysed in the present study. The X sex chromosome is always metacentric and is the longest chromosome in the karyotype for all species in the genus Chthonius. The sex chromosome system with a large metacentric chromosome X has been detected in most pseudoscorpion families that have been cytogenetically analysed (Chernetidae, Geogarypidae, Garypinidae, Olpiidae, Atemnidae) (Šťáhlavský et al. 2005, 2006, 2012). Metacentric morphology and large size of X sex chromosomes have been documented within arachnids also in different species from different groups of spiders (e.g. Král et al. 2006, 2013). Furthermore, extremely large metacentric X sex chromosomes is also known in some beetles from the family Chrysomelidae (Insecta, Coleoptera) (e.g. Almeida et al. 2009). It is evident that this type of X chromosome would have originated independently by different evolutionary mechanisms. However, there are only few exceptions to the morphology of the X chromosome in pseudoscorpions, namely one population of the species Olpium pallipes (Lucas, 1849) (Olpiidae) (Šťáhlavský et al. 2006) and two neotropic species Semeiochernes armiger (Balzan, 1892) and Cordylochernes scorpioides (Linnaeus, 1758) (Chernetidae) (Šťáhlavský et al. 2009). This evidence supports the assumption that the X0 sex chromosome system with large metacentric X is the plesiomorphic state in pseudoscorpions (e.g. Troiano 1990, 1997, Šťáhlavský et al. 2012).

The C-banding analyses performed in this study represent the first time that this procedure is applied in pseudoscorpions. Constitutive heterochromatin was only detected in the centromere regions. Blocks of heterochromatin located on different parts on the chromosome, known from some araneomorph spiders (Král et al. 2006), have not been detected. The concentration of constitutive heterochromatin in the centromere region could represent an ancestral state in this group, similar to that hypothesized in spiders (Rodríguez-Gil et al. 2007). The C-banding also confirmed the hypothesis that the prominent heteropycnotic blocks on chromosomes, visible in the early stages of meiosis, correspond to centromeres (Šťáhlavský et al. 2006). The results also indicate that the X is formed mainly of euchromatin outside of the centromere region, and the positive heteropycnosis during the early phases of meiosis is caused by intensive condensation, similarly as reported in wolf spiders (Dolejš et al. 2010).

Overall, the pseudoscorpions are represented by a great variety of chromosome numbers from 7 in Olpiidae to 143 in Atemnidae (Šťáhlavský et al. 2012). However, there is a much narrower range within individual families. For example, the typical range of the diploid number for a specific family is: 2n = 7–23 in Olpiidae, 2n = 15–23 in Geogarypidae, 2n = 16–67 in Neobisiidae, 2n = 47–73 in Chernetidae, and 2n = 65–143 in Atemnidae (see Šťáhlavský 2016). The results of this study confirm that the variability in chromosome number in the genus Chthonius is in agreement with previous findings (Šťáhlavský and Král 2004). The diploid number in males ranged from 21–35 (most frequently 35) and the acrocentric chromosomes are the most common morphology in the karyotype (see Table 1). Šťáhlavský and Král (2004) suggested that this chromosome number, acrocentric morphology of the chromosomes, and their gradual decrease in size are the ancestral conditions for species of this genus. The findings of this study confirm the assumption in C. (C.) carinthiacus and C. (E.) fuscimanus with 2n = 35 species presenting only acrocentric chromosomes. In other species with a diploid number 2n = 35 and a majority of acrocentric chromosomes, the presence of different morphological types of autosomes has also been detected, probably as a result of pericentric inversions. Šťáhlavský and Král (2004) also hypothesized that centric and tandem fusions play an important role in the karyotype evolution of the genus Chthonius, leading to a decrease in the number of chromosomes and a change of their morphology from uniarmed to biarmed. The exact mechanism and direction of the karyotype evolution of pseudoscorpions remain unknown. However, the abundant frequency of karyotypes with 35 chromosomes (Table 1) could indicate that this number corresponds to the ancestral state. Similar changes of the number, morphology, and size of the chromosomes linked to both centric and tandem fusions can be observed in the different cytotypes of C. (C.) tenuis (Fig. 4a–e). We detected several cytotypes (II, III and IV) with the same diploid number of 33 in this species. The number presumably decreased from the hypothetic ancestral state of 2n = 35 (cytotype I, Fig. 4a) by means of centric (cytotype II, III, Fig. 4b, c) or tandem (cytotype IV, Fig. 4d) fusions. Independent (centric or tandem) fusions or subsequent pericentric inversions may produce a different morphology of extra large autosomes. Accumulation of chromosome fusions occurred in cytotype V decreasing the number to 2n = 21 and leading to differentiation of autosomes into three categories: large, medium, and small (Fig. 4e). In C. (C.) alpicola species, with a chromosome number lowered to 2n = 21 and presence of only acrocentric chromosomes in the karyotype (Fig. 1a), multiple tandem fusions presumably played a key role in the process of lowering the chromosome number. From the comparison of the frequency of morphologic types of chromosomes in the karyotypes of the genus Chthonius (Fig. 6), it is apparent that the frequency of metacentric and submetacentric chromosomes does not vary considerably, but the number of acrocentric and subtelocentric autosomes decreases in karyotypes with lower chromosome numbers. This phenomenon could be explained because of tandem fusions, or potentially as a result of a centric fusion subsequently accompanied by a pericentric inversion. However, for precise understanding of these mechanisms, the use of additional cytogenetic tools would be needed in for the exact identification of homologous segments of the chromosomes and detection of particular chromosome rearrangements leading to changes in both chromosome morphology and size (e.g. Nie et al. 2012). Unfortunately, the exact direction of karyotype evolution within the genus Chthonius is also not possible to determine without the knowledge of the phylogenetic relationships among the Chthonius species, which are currently unknown.

Karyotypes of pseudoscorpions show considerable differences among species within all analysed families (see Šťáhlavský 2016), and therefore cytogenetic data have great potential for taxonomic application in the order. The usefulness of karyology has been demonstrated in the genus Roncus L. Koch, 1873. This mainly European group, with more than 140 described species (Harvey 2013), usually exhibits similar external morphology (e.g. Gardini 1991). However, the karyotypes of very morphologically similar species differ in diploid number, chromosome morphology, and sex chromosome systems (X0 and XY) (e.g. Troiano 1990). Substantial differences in karyotypes were used to distinguish R. montsenyensis Zaragoza and Šťáhlavský, 2008 (2n = 16) from R. cadinensis Zaragoza, 2007 (2n = 38), which are morphologically very similar (Zaragoza and Šťáhlavský 2008). Unfortunately, the use of cytogenetics in cryptic species detection in pseudoscorpions is complicated due to the limited amount of data available for comparison among different species. Furthermore, different degrees of intraspecific karyotype variability can represent a problem to determine and distinguish the existence of cryptic or isomorphic species. These challenges, observed in the genus Chthonius from the Alps, are also known from various arachnid groups (e.g. Tsurusaki 1985, Řezáč et al. 2007, Schneider et al. 2009), and many other organisms (e.g. Duffy et al. 2008, Severns and Liston 2008, Dincǎ et al. 2011, Sadílek et al. 2013, Sember et al. 2015).

Our data completely agree with described karyotypes of C. (E.) fuscimanus and C. (C.) carinthiacus (misidentified see Christophoryová et al. 2012) from Central Europe (both species 2n = 35, only acrocentric autosomes) (Šťáhlavský and Král 2004). Small differences between karyotypes of C. (E.) tetrachelatus from Central Europe (Šťáhlavský and Král 2004) and from the Alps (present study) may be an artefact of the quality of the preparations. Furthermore, the precision of visualizing the centromere position, using C-banding in this study, was likely better. Karyotype similarity from distant localities within these three species suggests that the use of these approaches may also contribute to characterize pseudoscorpion species very well.

In contrast to these findings, we identified different cytotypes in more abundant material of four species of the subgenus Chthonius from the Alps (Table 1). Only in C. (C.) ischnocheles and C. (C.) rhodochelatus, different cytotypes were complemented by visible morphological differences in surface granularity and pedipalp size. These particular morphological differences would potentially allow us to treat the cytotypes as new cryptic species. Nevertheless, standard morphological characteristics overlap substantially among the cytotypes in both C. (C.) raridentatus and C. (C.) tenuis. The interpretation of the variability in this case is not trivial. The Chthonius (C.) tenuis cytotypes may present both intraspecific variability and a mix of different levels of speciation. Specific cytotypes from different parts of the Alps have been reported in species with large distribution (e.g. Capanna and Riscassi 1978, Zima et al. 1996), but the difference between the highly derived cytotype V (2n = 21) and the remaining cytotypes most likely represents a strong reproductive barrier between them. Thus, cytotype V may represent a cryptic species without evident morphological differentiation. The fact that cytotype I spatially overlaps cytotype V (Fig. 4f) coupled with the fact that no anomalies during meiosis have been detected in the studied material, suggests an absence of hybridization between them and further supports the cryptic species status of cytotype V (e.g. King 1993). It should be noted that material used in the present study is probably not substantial enough to rule out the existence of hybrids and more investigation is needed before any taxonomic changes can be made. The putative chromosomal speciation of Chthonius in the Alps may be owed to the dispersal limitation of the genus. Chromosomal rearrangements may be fixed in certain areas with limited gene flow and consequently cause hybrid sterility among different cytotypes. Alternatively, the environments of the Alps may influence chromosome rearrangement that favours higher fitness for specific environmental conditions (see e.g. Faria and Navarro 2010). Additional sampling, comprehension of phylogenetic relationships among the analysed species, and knowledge of the genetic structure of their populations are needed for the full understanding of karyotype variability and its role in the speciation of the genus Chthonius in the Alps.