Distribution of 18S rDNA clusters in Central European harvestmen of the suborder Eupnoi (Arachnida: Opiliones)

rDNA clusters are an important cytogenetic marker for studying karyotype evolution and chromosomal changes. The variability of this cytogenetic characteristic is, however, still almost unknown in the karyotypes of the entire class Arachnida (Arthropoda: Chelicerata). This situation is particularly evident in harvestmen (Arachnida: Opiliones), with 97 species studied cytogenetically, for which there is information on the number and position of rDNA clusters for only 13. Moreover, previous studies indicate that the number of rDNA loci is highly variable in the species analysed, ranging from one to fi ve pairs of rDNA clusters. Based on this fragmentary information, which is for rare species mainly from the limits of the distribution of their families, it is still not possible to reconstruct the ancestral state for this important cytogenetic feature in this order. Building upon recent research in Central Europe, we analysed the number and position of 18S rDNA in 13 species belonging to the suborder Eupnoi. This revealed that their karyotypes were variable in terms of the diploid number (2n = 16–36) and number of 18S rDNA clusters (from one to seven pairs). For the fi rst time, an 18S rDNA cluster was detected on B chromosomes in harvestmen. Our study sheds new light on the karyotype evolution and 18S rDNA distribution in harvestmen and provides an improved understanding of the ancestral state of karyotypes in the order Opiliones. * Corresponding author; e-mail: hirmanm@natur.cuni.cz INTRODUCTION The Eupnoi is one of the four suborders of the order Opiliones (Arthropoda: Arachnida), which together with the suborders Dyspnoi, Cyphophthalmi and Laniatores, constitute the third-largest order of the class Arachnida (e.g. Giribet & Kury, 2007; Pinto-da-Rocha et al., 2007). Despite their high diversity (6500 described species: Kury, 2017), only 97 species of harvestman have been cytogenetically analysed (Tsurusaki et al., 2020). Despite this paucity of data, some harvestmen are very variable in their basic cytogenetic characteristics, such as chromosome number (2n = 10–109), degree of differentiation between the sex chromosomes, the presence of B chromosomes and possible polyploidy (see Tsurusaki & Cokendolpher, 1990; Tsurusaki, 2007). The most general ancestral state indicate morphologically undifferentiated sex chromosomes in harvestmen (e.g. Tsurusaki, 2007; Svojanovská et al., 2016). The XY sex chromosome system is reported only in the harvestman Sabacon makinoi Suzuki, 1949 (Dyspnoi: Sabaconidae) (Tsurusaki, 1989) and 13 species of the Sclerosomatidae Eur. J. Entomol. 117: 282–288, 2020 doi: 10.14411/eje.2020.032


Chromosome preparations and karyotype evaluation
We used the "plate-spreading" technique (Traut, 1976) for chromo some preparations according to Šťáhlavský & Král (2004). During this procedure, dissected gonads were incubated in a hypotonic solution of 0.075 M KCl for 20 min then fi xed in methanol : glacial acetic acid (3 : 1) for at least 20 min. Finally, the tissue was dissolved in a drop of 60% acetic acid on a microscope slide and spread on the surface of the microscope slide and left to evaporate. The chromosome preparations were stained with 5% Giemsa solution in Sörensen phosphate buffer for 15 min. The chromosomes were photographed using an ORCA-AG monochromatic camera (Hamamatsu Photonics Europe, Herrsching, DE) attached to an Olympus IX81 microscope. The karyotypes were described based on at least fi ve sister metaphase II (the morphology of the chromosomes is unclear in the mitotic metaphases) nuclei using the software ImageJ 1.45r (Schneider et al., 2012) with the plugin Levan (Sakamoto & Zacaro, 2009).

FISH detection of 18S rDNA
The probe for 18S rDNA was prepared according to Šťáhlavský et al. (2018) and labelled with biotin-14-dUTP using the Nick Translation Kit (Abbott Molecular, Chicago, Illinois, USA). The overall hybridisation procedure followed the protocol described by Fuková et al. (2005) including the initial treatment with RNase A (200 μg/ml in 2 × SSC) for one hour (37°C). Denaturation of chromosomes was done at 68°C for 3 min and 30 s in 70% formamide in 2 × SSC. Then a probe mixture (20 ng of the probe, 25 μg of salmon sperm DNA, 10 μl of 50% formamide, 20% dextran sulphate in 2 × SSC) was put on each slide and allowed to hybridize overnight (37°C). Then the probe was detected using Cy3-conjugated streptavidin, followed by the application of biotinylated antistreptavidin and another dose of Cy3-conjugated streptavidin. Chromosomes were counterstained with DAPI (FluoroshieldTM; Sigma-Aldrich, St. Louis, Missouri, USA) and observed under an Olympus IX81 microscope equipped with an ORCA-AG monochromatic charge-coupled device camera (Hamamatsu). The images were pseudo coloured and superimposed using CellˆR software (Olympus Soft Imaging Solutions GmbH).

Family Phalangiidae
We analysed seven species belonging to following genera: Egaenus, Lacinius, Mitopus, Oligolophus, Opilio, an 18S rDNA probe, frequently reveals one or two pairs of 18S rDNA clusters in different families of the suborder Cyphophthalmi and Parapurcellia amatola de Bivort & Giribet, 2010 (Cyphophthalmi: Pettalidae), with the number of such clusters sometimes being up to fi ve pairs (Svojanovská et al., 2016). An increase in the number of 18S rDNA clusters from two to seven and the variability of the heterozygosity in the number and size of such clusters, have also been identifi ed in fi ve species of phalangiid from South Africa (Šťáhlavský et al., 2018). All previously analysed species have considerably limited dispersal abilities, which has resulted in distinct genetic differentiation (e.g. Giribet et al., 2016) and most likely in rapid changes in the distribution of NORs. FISH, using the 5S rDNA probe, revealed that in the harvestman Psathyropus tenuipes L. Koch, 1878 (Eupnoi: Sclerosomatidae) there is only one pair of this minor cluster of the rRNA gene (Watanabe et al., 2009). However, the position of this 5S rRNA gene cluster evolved independently of that of the major rRNA gene clusters (see Sochorová et al., 2018) and, therefore, this characteristic cannot help us understand the evolutionary dynamics of NORs in harvestmen. This is the reasons why we are focusing on the distribution of 18S rDNA in phalangiids in Central Europe in the present study. Species from this area are generally widely distributed and gene fl ow in these taxa is usually not limited, unlike in the previously analysed groups. The objective of this analysis was to better identify the ancestral state of the NORs and the main evolutionary changes in this cytogenetic marker in this suborder.

Material
We analysed 13 species belonging to the families Phalangiidae and Sclerosomatidae from different localities in Central Europe (Table S1). The harvestmen were collected individually and transported alive to the laboratory for subsequent cytogenetic analyses. Analysed specimens are deposited in the public collection of the Faculty of Science, Charles University in Prague.  1A). In this species, we detected one pair of 18S rDNA clusters at the ends of the seventh pair of chromosomes (Fig. 1B). The karyotypes of Egaenus convexus (C.L. Koch, 1835) (Fig. 1C), Opilio canestrinii (Thorell, 1876) (Fig. 1E) and Phalangium opilio (Linnaeus, 1761) (Fig.  S1A) consisted of 2n = 24 with one pair of 18S rDNA clusters in the males of these species. These clusters were close to the centromere of the fi rst (E. convexus, Fig. 1D) and third (P. opilio, Fig. S1B) pair of chromosomes and at a subterminal position on the fi rst pair of chromosomes (O. canestrinii, Fig. 1F). Several 18S rDNA gene clusters occurred in the last three species analysed. Karyotype of Lacinius epiphiatus (C.L. Koch, 1835) (2n = 30) included fi ve pairs of these clusters at terminal positions on the eighth, ninth, twelfth, thirteenth and fourteenth pair of chromo-somes ( Fig. 1G-H). In Mitopus morio (Fabricius, 1799) (2n = 32) there were seven pairs of clusters of 18S rDNA also always at a terminal position (Fig. 1I-J). Karyotype of the species Rilaena triangularis (Herbst, 1799) (2n = 36) included four pairs of 18S rDNA clusters at subterminal or terminal positions (Fig. 1K-L). For the last two mentioned species it was not possible to specify the chromosome pairs bearing 18S rDNA clusters in their karyotype.

Family Sclerosomatidae
We analysed six species belonging to the genus Leiobunum and one to the genus Nelima. The karyotypes (2n = 20-24) and the number and positions of 18S rDNA differ slightly between genera. In all species biarmed chromosomes predominate and gradually decrease in size (Table  1).
In one male of L. blackwalli (Meade, 1861), we identifi ed 2n = 20 ( Fig. 2A) same as in the species L. rotundum (Latreille, 1798). However, in one male of L. rotundum from České Budějovice we identifi ed a variable 2n caused by the presence of 0-3 B chromosomes (Fig. 2C). Three other species, L. limbatum L. Koch, 1861 (Fig. 2E), L. rupestre (Herbst, 1799) (Fig. 2G) and L. gracile Thorell, 1876 (Fig.  2I) had 2n = 22. The number and position of 18S rDNA is very consistent in all of the fi ve species of Leiobunum analysed. We identifi ed only one pair of 18S rDNA signals always at a terminal position and mostly on one of the fi rst three chromosome pairs, see Table 1 ( Fig. 2B, D, F, H, J). Interestingly, we identifi ed an 18S rDNA cluster on all three B chromosomes in the species Leiobunum rotundum (Fig. 2D). Compared to that in the species Nelima semproni (Szalay, 1951) (2n = 24) (Fig. 2K) two pairs of 18S rDNA clusters were detected by FISH on terminal parts of the third and fi fth chromosome pair (Fig. 2L).

DISCUSSION
NORs are one of the most commonly observed markers in animal karyotypes. Previously, this region was identifi ed mainly by silver staining, which can underestimate the number of NORs (Miller et al., 1976). FISH is currently a better and more frequently used tool for mapping the major ribosomal RNA (i.e. 45S: 18S, 5.8S and 28S) gene clusters (e.g. Nguyen et al., 2010;Mattos et al., 2014;Štundlová et al., 2019). Recent reviews of the literature and an online database indicate that, despite the wide range in numbers of 45S rDNA loci (from one to 54 loci/2C) in animals, about 60% of the karyotypes have only a single 45S locus (Sochorová et al., 2018) and 45S rDNA is most frequently distally located (Sochorová et al., 2018). These characteristics correspond with the predicted ancestral state of 45S rDNA in arachnids in general (Forman et al., 2013) and harvestmen in particular (Svojanovská et al., 2016). Only a few species of harvestmen, from only two suborders, have been analysed, the Cyphophthalmi (Svojanovská et al., 2016;Hiřman et al., 2018) andthe Eupnoi (Šťáhlavský et al., 2018). These studies reveal numbers of rDNA clusters that differ from the predicted ancestral state; for example, Parapurcellia amatola (Cyphophthalmi) has 10 signals of 18S rDNA (Svojanovská et al., 2016) and Rhampsinitus leighi (Pocock, 1903) (Eupnoi) has seven (Šťáhlavský et al., 2018). However, Miopsalis sp. (Styllocelidae), which has a basal position in the harvestmen phylogeny, with a diploid number 2n = 30, has one pair of 18S rDNA clusters in a terminal position and no acrocentric pairs of chromosomes in the karyotype (Svojanovská et al., 2016).
Our study has increased the knowledge based on the number and position of rDNA clusters in the Suborder Eupnoi (Šťáhlavský et al., 2018). We detected one pair of 18S rDNA clusters in most of the species analysed, whilst showing that there is some difference in localisation on chromosome; for example, pericentromeric in Egaenus convexus, subterminal in Opilio canestrinii and terminal in Rilaena triangularis (all family Phalangiidae). Also, we detected 18S rDNA multiplication in three species of the family Phalangiidae: Lacinius epiphiatus (2n = 30, fi ve pairs), Mitopus morio (2n = 32, seven pairs) and Rilaena triangularis (2n = 36, four pairs), and in one species of the family Sclerosomatidae: Nelima semproni (2n = 24, two pairs). These results indicate that, despite the recent fi nding of high variability in 18S rDNA clusters in species from South Africa (Šťáhlavský et al., 2018), 18S rDNA may be conserved in some species of the Suborder Eupnoi. High numbers of 18S rDNA clusters are frequently recorded in specimens inhabiting regions with extreme conditions and may thus represent an adaptation to such conditions, as has previously been postulated for other taxa, such as fi sh (Symonová et al., 2013;Sember et al., 2015). However, we need more data to confi rm this in harvestmen. B chromosomes are already reported in some species of the Suborder Eupnoi (Tsurusaki, 2007;Watanabe et al., 2009), but here, for the fi rst time, 18S rDNA was detected on B chromosomes in harvestmen, specifi cally in the species Leiobunum rotundum. B chromosomes carrying rDNA (active or inactive) have already been detected in vertebrates, such as rats (Stitou et al., 2004) and fi sh (Baroni et al., 2009;Poletto et al., 2010), and also in invertebrates, such as the grasshopper Dichroplus pratensis Bruner, 1900 (Bidau et al., 2004). Further investigation is necessary to obtain a better understanding of the function of rDNA on B chromosomes in harvestmen.  Table S1. Sex, location and date collected for the species included in this study.