A karyological study of four European species of Roncus ( Pseudoscorpiones : Neobisiidae )

We present the results of the first study on the karyotypes of four European species of Roncus: Roncus alpinus L. Koch, 1873, Roncus lubricus L. Koch, 1873, Roncus transsilvanicus Beier, 1928 and Roncus sp. The diploid number was 2n = 23 in Roncus sp., 2n = 43 in R. alpinus and R. transsilvanicus and 2n = 45 in R. lubricus. Telocentric autosomes predominate in species with a high chromosome number and metacentric autosomes in Roncus sp. We assume that the ancestral situation for this genus is a high number of chromosomes. A low number of chromosomes is very likely a consequence of centric fusions, which have possibly played a very important role in karyotype evolution in the genus Roncus. All the species analyzed have the X0 sex chromosome system. The X chromosome is metacentric and is the smallest element in the karyotypes of all the species analyzed.


Chromosome preparations
The chromosome preparations were made from gonads using the spreading technique described in Šťáhlavský & Král (2004).Briefly, the gonads were dissected and placed in a hypotonic solution of 0.075 M KCl for 15 min and then fixed in a fresh solution of glacial acetic acid : ethanol (1 : 3) for at least 20 min.The fixed material was then transferred to a drop of 60% acetic acid on a clean microscope slide using a tungsten needle.The slide was placed on a warm histological plate (temperature 40-45°C) and the drop then spread on the slide with the aid of a needle until it evaporated.The chromosome preparations were stained in a 5% Giemsa solution in Sörensen phosphate buffer (4.75 g Na2 HPO4 •12 H2O and 4.54 g KH2PO4 in 1,000 ml H2O, pH = 6.8) for 35 min.
The chromosomes were photographed using a Jenaval microscope and Kodak Technical Panfilm and then digitized using a Nikon Super Coolscan 5000 ED scanner.The chromosome morphology was classified according to Levan et al. (1964) and the measurements of the chromosomes were taken from photographs using the software ImageJ 1.45 (http://imagej.nih.gov/ij) with the Levan plugin (Sakamoto & Zacaro, 2009).The sister metaphase II cells with well-marked centromeres were used to obtain measurements of chromosomal arms and assemble the karyograms.At least ten measurements were used for the characterization of the karyotype in all species.We calculated the relative chromosome length as a percentage of the total length of the diploid set, including the sex chromosome.

Roncus alpinus
The male diploid complement comprises 43 chromosomes in all four populations (Fig. 1a).The male karyotype of specimens from Castelo Toblino comprises two metacentric (Nos 12, 20), two submetacentric (Nos 16, 21), six subtelocentric (Nos 2,5,9,14,17,19) and eleven telocentric pairs of autosomes.The autosomes gradually decrease in size from 3.76% to 1.10% of the diploid set.The sex chromosome X is metacentric (arm ratio 1.26) and it is the smallest chromosome within the karyotype (0.79% of the diploid set).Similar characteristics were also recorded for the specimens of R. alpinus collected from other Italian localities -Egna and Aprica.
The odd diploid number of males indicates that the sex chromosome system is X0.In meiosis from leptotene the sex chromosome is clearly visible as a knob showing positive heteropycnosis (Fig. 3a).Starting from the diplotene phase, the structure of the X chromosome is isopycnotic and during diplotene easily recognized as a small univalent (Fig. 3b).In the following metaphase II (Fig. 1a) and anaphase II (Fig. 3c) the X chromosome is still isopycnotic.Low chiasma frequency was recorded in this species.Predominance of bivalents with one chiasma (81% cases) was observed at diplotene (N = 59).Rarely, one or two bivalents with two chiasmata were observed.The mean chiasma frequency was 21.31 ± 0.68 SD per cell, which equates to 1.02 ± 0.03 SD per bivalent.

Roncus lubricus
The male diploid complement of specimens of R. lubricus from all three locations comprised 45 chromosomes.The karyotype based on sister plates of metaphase II from the Czech Republic (Fig. 1b) includes two pairs of metacentric (No. 18,22)  gradually decrease in size from 3.70% to 1.16% of the diploid set.The last two pairs of autosomes are small (0.83% and 0.67%).The sex chromosome X is metacentric (arm ratio 1.61) and is the smallest element in the karyotype (0.6% of the diploid set).A similar characteristic was recorded for the population from the United Kingdom, over 1,200 km away.
The diploid number of chromosomes is 45 in males and 46 in females (Fig. 3d).Additionally, one univalent was found in late metaphase I (Fig. 3f) and a different number of chromosomes in the cells of the sister metaphase II (22 and 23).The combination of these factors indicates that the males of this species have an X0 sex chromosome system.As in the previous species, the X chromosome is positively heteropycnotic during leptotene, zygotene (Fig. 3e) and pachytene, but starts to be isopycnotic from diplotene.We recorded a low chiasma frequency despite almost half of the observed diplotene, diakinesis and metaphase I included one or two bivalents with two chias-mata (Fig. 3f).The mean chiasma frequency was 22.55 ± 0.69 SD per cell, which is equivalent to 1.02 ± 0.03 SD per bivalent (total N = 36).

Roncus transsilvanicus
The diploid chromosome number is 43 in males (Fig. 2a) and 44 in females (Fig. 3g).The male karyotype based on sister plates of metaphase II of specimens from the Retezat Mts contains one pair of metacentric (No. 10), one pair of submetacentric (No. 6), five pairs of subtelocentric (Nos 9, 14, 16, 20, 21) and 14 pairs of acrocentric (Nos 6,23,26,32,33,34) autosomes (Fig. 2a).Autosomes gradually decreased in size from 3.80% to 1.15% of the diploid set.This species has an X0 sex chromosome system.The sex chromosome X is metacentric (arm ratio 1.60) and is the smallest element in the karyotype (1.43% of the diploid set).The X chromosome is positively heteropycnotic during early prophase (leptotene, zygotene, pachytene) (e.g.Fig. 3h) but starts to become isopycnotic at diplotene (Fig. 3i) and stays this way until the end of meiosis.A similar low chiasma frequency as in R. alpinus was recorded.Bivalents with one chiasma (81% cases, total N = 69) also predominate in R. transsilvanicus.A bivalent with two chiasmata was rarely recorded.The mean chiasma frequency was 21.18 ± 0.39 SD per cell or 1.01 ± 0.02 SD per bivalent.

Roncus sp.
The diploid chromosome complement is 23 chromosomes in males (Fig. 2b) and 24 in females (Fig. 3j).In the karyotype of males there were only biarmed chromosomes: nine pairs of metacentric, two pairs of submetacentric (Nos 8 and 11) autosomes and one metacentric sex chromosome.The length of the chromosomes gradually decreases in size from 6.34% to 3.68% of the total diploid set.Only the last pair of chromosomes (No. 11) is considerably shorter than the previous pairs, forming only 2.46% of the total diploid set.The X chromosome is metacentric (arm ratio 1.3) and is the shortest element of the karyotype, forming only 1.51% of the diploid set.Like all previous species, the X chromosome is positively heteropycnotic during early prophase (leptotene, zygotene and pachytene) (e.g.Fig. 3k) and again starts to become isopycnotic from diplotene to the end of meiosis (Fig. 3l).Despite the marked reduction in the diploid number and larger size of the metacentric chromosomes, a low chiasma frequency was also recorded in this species.The mean chiasma frequency was 11.30 ± 0.72 SD per cell or 1.03 ± 0.07 SD per bivalent (total N = 98).

DISCUSSION AND CONCLUSIONS
Our study provides further information on the cytogenetics of the pseudoscorpion genus Roncus (Neobisiidae) based on a study of another four species, which extends the previous work on this genus that was based on eight species (Troiano, 1990(Troiano, , 1997;;Zaragoza & Šťáhlavský, 2008).As in the previous study we found differences between the karyotypes that may be useful for future cytotaxonomic studies (Table 1).The great variability in karyotype characteristic of Roncus has also been recorded for several other pseudoscorpion families, e.g.Atemnidae, Chthoniidae, Chernetidae, Geogarypidae and Olpiidae (e.g.Šťáhlavský & Král, 2004;Šťáhlavský et al., 2005, 2006, 2009, 2012).In addition, our analysis detected some similar features in the diploid number and morphology of the chromosomes of R. alpinus, R. lubricus and R. transsilvanicus.The karyotypes of these species include some of the highest numbers of chromosomes in the genus Roncus, which consist of mainly subtelocentric and acrocentric autosomes.Moreover, all three species have the X0 sex chromosome system with a metacentric X chromosome, which is the smallest element in the karyotypes.This finding contrasts with other species of this genus from Italy (Liguria) (Troiano, 1990(Troiano, , 1997) ) and Spain (Catalonia) (Zaragoza & Šťáhlavský, 2008).The karyotypes of these species are even more variable.Even species from nearby localities differ significantly in their chromosome number and morphology; furthermore they have different sex chromosome systems (X0 and XY).These major differences, especially in the number and morphology of autosomes, are accounted for in terms of centric fusions or fissions (Troiano, 1990;Zaragoza & Šťáhlavský, 2008).Considering that R. alpinus, R. lubricus and R. transsilvanicus probably represent different phylogenetic lineages (they are from different European regions and exhibit different external morphology) (e.g.Beier, 1963), we assume that similar features of the karyotypes of these species reflect an ancestral state of the genus Roncus.This hypothesis is also supported by their having the same sex chromosome system X0 in which the size of the X chromosome is similar and same morphology in all three species.The X0 sex chromosome system is generally supposed to be the ancestral one for pseudoscorpions (e.g.Troiano, 1990).This sex system and metacentric morphology of the X chromosome are recorded in Chthoniidae (Šťáhlavský & Král, 2004), one of the most basal lineages of the pseudoscorpions (Murienne et al., 2008) and in other families (e.g.Šťáhlavský et al., 2005, 2006, 2012).The XY sex chromosome system is considered to be a derived state in R. ingaunus, R. tuberculatus (Troiano, 1990), R. belluatii (Troiano, 1997), R. cadinensis and R. montsenyensis (Zaragoza & Šťáhlavský, 2008), which is also supported by the different size and morphology of the sex chromosomes in these species.The sex chromosomes are nearly homomorphic and both biarmed in R. inguanus, R. cadinensis and R. montsenyensis (Troiano, 1990;Zaragoza & Šťáhlavský, 2008).On the other hand the X is large and metacentric and the Y about a third of the size of X and is acrocentric in R. tuberculatus (Troiano, 1990).The main trend in karyotype evolution in the genus Roncus is probably in the reduction in the number of chromosomes as a result of centric fusions (Troiano, 1990).This type of chromosomal rearrangement leads to an increase in the number of biarmed chromosomes (metacentric and submetacetric).The reduction in the number of chromosomes (Fig. 4a) leads to full saturation via this type of rearrangement.In contrast, during the reduction in the number of chromosomes, the number of one armed chromosomes (subtelocentric and acrocentric) decreases (Fig. 4b).The results from the current study support this hypothesis.The karyotype of Roncus sp.(2n = 23) from the Slovak Republic contains only biarmed chromosomes but this species is morphologically similar to R. lubricus (2n = 43) in which there is a predominance of acrocentric chromosomes.However, in order to answer the question, how often has the reduction in the diploid number occurred in the evolution of Roncus, an independent phylogenetic analysis is needed.

TABLE 1 .
, one pair of submetacentric Summary of the cytogenetic data for the genus Roncus.