Insights into the Karyotype Evolution of Charinidae, the Early-Diverging Clade of Whip Spiders (Arachnida: Amblypygi)

Simple Summary Whip spiders (Amblypygi) are spectacular arachnids characterized by powerful raptorial pedipalps and exceptionally elongated forelegs. Although cytogenetic data from amblypygids might be, given their phylogenetic position, important for the reconstruction of arachnid ka-ryotype evolution, cytogenetics of this order is still largely understudied. Here, we applied conventional and molecular cytogenetics to describe the karyotype patterns in Charinidae—the family placed almost at the base of amblypygid phylogeny, thereby providing insights into the ancestral amblypygid karyotype traits. We surveyed four Charinus and five Sarax species and found a wide range of diploid chromosome numbers (2n) in both genera. Representatives with lower 2n possessed generally a higher proportion of biarmed (metacentric and submetacentric) chromosomes. Our results indicate the important roles of chromosome fusions and pericentric inversions in the karyotype differentiation of Charinidae, similarly to that suggested previously for neoamblypygids. Our data, gathered from C-banding, fluorescent banding, and chromosomal mapping of ribosomal DNA and telomeric repeats, bring evidence for the action of these rearrangements and suggest the general trajectory towards 2n reduction from ancestral high chromosome counts. However, we cannot rule out the contribution of chromosome fissions. Abstract Whip spiders (Amblypygi) represent an ancient order of tetrapulmonate arachnids with a low diversity. Their cytogenetic data are confined to only a few reports. Here, we analyzed the family Charinidae, a lineage almost at the base of the amblypygids, providing an insight into the ancestral traits and basic trajectories of amblypygid karyotype evolution. We performed Giemsa staining, selected banding techniques, and detected 18S ribosomal DNA and telomeric repeats by fluorescence in situ hybridization in four Charinus and five Sarax species. Both genera exhibit a wide range of diploid chromosome numbers (2n = 42–76 and 22–74 for Charinus and Sarax, respectively). The 2n reduction was accompanied by an increase of proportion of biarmed elements. We further revealed a single NOR site (probably an ancestral condition for charinids), the presence of a (TTAGG)n telomeric motif localized mostly at the chromosome ends, and an absence of heteromorphic sex chromosomes. Our data collectively suggest a high pace of karyotype repatterning in amblypygids, with probably a high ancestral 2n and its subsequent gradual reduction by fusions, and the action of pericentric inversions, similarly to what has been proposed for neoamblypygids. The possible contribution of fissions to charinid karyotype repatterning, however, cannot be fully ruled out.

Cytogenetic data on amblypygids are scarce, with the first record dating back to a study of Millot and Tuzet [16], which provides information on the diploid chromosome number (2n) and sex chromosome constitution in Sarax sarawakensis (Charinidae). The 2n and telomere sequence mapping was reported for Damon medius (Phrynichidae) by Vítková et al. [17]. Later, Paula-Neto et al. [18] shed light on the karyotype of Heterophrynus longicornis (Phrynidae), including the distribution of heterochromatin and the number and position of nucleolar organizer regions (NORs). Recently, the karyotype data of one Phrynus and six Paraphrynus species (Phrynidae) were reported by Seiter et al. [7], who evidenced a wide range of 2n (from 24 to 68). Karyotype differentiation of Paraphrynus involved centric fusions and pericentric inversions.
In the present work, we aim to elucidate karyotype differentiation in the family Charinidae, the earliest-diverging lineage among euamblypygids. Charinids consist of three genera (Charinus, Sarax, and Weygoldtia) and 132 species [10,11,[19][20][21]. The charinid lineage is sister to the remainder of the euamblypygids, which form the so-called neoamblypygid clade [10,22]. It is presumed that the radiation of charinids and neoamblypygids was initiated before the fragmentation of the Pangean and Gondwanian supercontinents, which might explain their recent distribution [1,22]. Due to their basal phylogenetic position within whip spiders, charinids are an important target for analyses of karyotype evolution in whip spiders and Tetrapulmonata. We conducted both conventional (Giemsa staining, C and fluorescent banding, silver staining of NORs) and molecular cytogenetic analyses [chromosomal mapping of (TTAGG) n telomeric sequence and 18S ribosomal DNA (rDNA) sites by fluorescence in situ hybridization (FISH)].

Individuals
Twenty-one individuals belonging to four Charinus and five Sarax species were analyzed (for details, see Table 1). Voucher specimens are mostly deposited in the Laboratory of Arachnid Cytogenetics, Department of Genetics and Microbiology (Faculty of Science, Charles University, Prague). Specimens of S. seychellarum are placed in the collection of the Department of Zoology (Faculty of Science, Charles University, Prague) and the samples of C. pescotti are deposited in the collection of the Western Australian Museum, Perth (Australia).

Chromosome Preparations and Evaluation of Karyotypes
Mitotic and meiotic chromosomes were obtained from gonadal tissue of preferentially adult or subadult specimens using a spreading technique described originally for butterflies by Traut [23], with several modifications (detailed in [24]). Briefly, the tissue was hypotonized in 0.075 M KCl for 20 min, and then fixed in three rounds of freshly prepared ethanol: acetic acid (3:1; v/v) for 6, 10, and 20 min. A piece of fixed gonad was suspended in a drop of 60% acetic acid placed on a clean slide. The cell suspension was spread on a slide placed on a histological plate heated to 40 • C, using a tungsten needle. The chromosome preparations were subsequently stained in 5% Giemsa solution in Sörensen buffer (pH 6.8) for 30 min. To evaluate the chromosome morphology and to construct the karyotypes, at least five mitotic metaphases (C. dominicanus, C. pescotti, S. aff. batuensis, S. seychellarum) or meiotic metaphases II (C. cavernicolus, C. neocaledonicus, S. huberi, Sarax sp.) were analyzed. In S. ioanniticus, the quality of metaphase spreads only permitted the identification of the 2n and an approximate determination of chromosome morphology. We obtained just a limited number of chromosome plates due to the small size of the gonads and the low number of specimens collected. Therefore, selected techniques of chromosome banding and molecular cytogenetics could be applied only to some species. The sequential use of the slides for more techniques was met with varied success (see Table 1).
Classification of chromosome morphology was based on the position of the centromere according to Levan et al. [25], but modified as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a). To calculate the FN value (number of chromosome arms, fundamental number [26]), metacentric and submetacentric chromosomes were scored as biarmed, while subtelocentric and acrocentric chromosomes were scored as monoarmed.

Chromosome Banding Techniques
The protocol for chromosome preparation was modified for C-banding. Specifically, the tissue was fixed in methanol: acetic acid (3:1), and the temperature of the histological plate was reduced to 35 • C for better preservation of the chromatin. The distribution of the constitutive heterochromatin was analyzed using the C-banding method following Sumner [27]. Preparations were dried and stained with 5% Giemsa in Sörensen buffer (pH 6.8) for 75 min. In C. pescotti, the chromosomes were stained with 4 ,6-diamidino-2-phenolindole (DAPI) (FluoroshieldTM; Sigma-Aldrich, St. Louis, MO, USA) and the pictures were were inverted for better contrast. This protocol modification provides a better resolution of the C-bands and, hence, allows visualization of the tiny blocks of constitutive heterochromatin. Although it has already been repeatedly used in some other arachnids (e.g., [28,29]), we are aware that it might, on the other hand, underestimate GC-rich heterochromatin blocks.
The composition of constitutive heterochromatin was determined in C. neocaledonicus using the GC-specific fluorochrome chromomycin A 3 (CMA 3 ) and the AT-specific fluorochrome DAPI (both Sigma-Aldrich) following the method of Sola et al. [30] and Mayr et al. [31].
Silver staining of transcriptionally active NORs [32,33] was performed according to Dolejš et al. [24], with a modified staining time ranging from 5 to 7 min.

Telomeric FISH
A telomeric probe corresponding to the insect telomeric motif (TTAGG) n was generated by non-template PCR according to Ijdo et al. [34], following the protocol modification of Sahara et al. [35]. The probe was labeled with biotin-14-dUTP by nick translation using a Nick Translation Kit (Abbott Molecular, IL, USA). The FISH was carried out according to the protocol described above for rDNA FISH, and the probe was also detected with Cy3-conjugated streptavidin. The chromosomes were counterstained with DAPI (Fluoroshield™; Sigma-Aldrich).

Microscopy and Image Analysis
Standard preparations stained by Giemsa were inspected under a BX 50 microscope (Olympus, Tokyo, Japan) and images were photographed under immersion objective 100× using a DP 71 CCD camera (Olympus). Pictures from FISH and C-banding were captured by an IX81 microscope (Olympus) equipped with an ORCA-AG CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). The digital images from the FISH and fluorescent banding were pseudocolored (red for Cy3 and CMA 3 , green or blue for DAPI) and superimposed with the CellˆR software (Olympus Soft Imaging Solutions GmbH, Muenster, Germany). Fluorescent banding was evaluated under immersion objective 100× using a Provis AX70 Olympus microscope with an appropriate fluorescence filter set. Images were photographed by a black and white DP30W CCD Olympus camera for each fluorescent dye using Olympus Acquisition Software. Karyotypes were arranged in Corel PHOTO-PAINT X4 software (Corel, Ottawa, ON, Canada). Composed images were optimized and arranged using Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA).

Distribution and Composition of Constitutive Heterochromatin
C-banding was performed in two Charinus species-C. neocaledonicus ( Figure 5a) and C. pescotti (Figure 1b). In both species, the blocks of constitutive heterochromatin were mostly confined to the terminal parts of the chromosomes. The (peri)centromeric region of all monoarmed chromosomes was formed by heterochromatin. Moreover, acrocentric pairs nos 6,16,19,22,27,32, and 33 displayed an additional band at the end of their long arms. In C. pescotti, approximately one-third of the chromosomes possessed constitutive heterochromatin in their middle or subterminal part. These blocks corresponded to the (peri)centromeric region of biarmed chromosomes (Figure 1b). The metacentric pair no. 25 exhibited (peri)centromeric heterochromatin and two terminal bands, each at one chromosome arm. A remarkable interstitial block of constitutive heterochromatin was identified in one acrocentric pair (possibly pair no. 1) of C. neocaledonicus (Figure 5a). Additional analysis by fluorescent banding revealed that this block is composed of two segments differing considerably in their base composition (Figure 5b). While one region was GC-rich (i.e., CMA 3 -positive), the other one was AT-rich (i.e., DAPI-positive). CMA 3positive signals were also localized at one end of most chromosomes, while DAPI-positive regions were less pronounced. They were often placed at the opposite chromosome ends.

Distribution and Composition of Constitutive Heterochromatin
C-banding was performed in two Charinus species-C. neocaledonicus ( Figure 5a) and C. pescotti (Figure 1b). In both species, the blocks of constitutive heterochromatin were mostly confined to the terminal parts of the chromosomes. The (peri)centromeric region of all monoarmed chromosomes was formed by heterochromatin. Moreover, acrocentric pairs nos 6,16,19,22,27,32, and 33 displayed an additional band at the end of their long arms. In C. pescotti, approximately one-third of the chromosomes possessed constitutive heterochromatin in their middle or subterminal part. These blocks corresponded to the (peri)centromeric region of biarmed chromosomes (Figure 1b). The metacentric pair no. 25 exhibited (peri)centromeric heterochromatin and two terminal bands, each at one chromosome arm. A remarkable interstitial block of constitutive heterochromatin was identified in one acrocentric pair (possibly pair no. 1) of C. neocaledonicus (Figure 5a). Additional analysis by fluorescent banding revealed that this block is composed of two segments differing considerably in their base composition (Figure 5b). While one region was GC-rich (i.e., CMA3-positive), the other one was AT-rich (i.e., DAPI-positive). CMA3-positive signals were also localized at one end of most chromosomes, while DAPI-positive regions were less pronounced. They were often placed at the opposite chromosome ends

Chromosomal Distribution of Telomeric Repeats
The FISH with the telomeric probe (TTAGG)n was performed only in S. seychellarum. Since this species exhibits the lowest 2n in our sampling, it is the one with a possible high incidence of chromosome rearrangements, herein particularly fusions, which may produce interstitial telomeric sites (ITSs). The probe revealed signals at the ends of all chromosomes, and a single ITS on one chromosome (Figure 4c).

Chromosomal Distribution of Telomeric Repeats
The FISH with the telomeric probe (TTAGG) n was performed only in S. seychellarum. Since this species exhibits the lowest 2n in our sampling, it is the one with a possible high incidence of chromosome rearrangements, herein particularly fusions, which may produce interstitial telomeric sites (ITSs). The probe revealed signals at the ends of all chromosomes, and a single ITS on one chromosome (Figure 4c).

Distribution of 18S rDNA/NOR
A single NOR-bearing chromosome pair was detected in C. neocaledonicus (Figure 5c), C. pescotti (Figure 5d), and C. seychellarum (Figure 4b). In C. neocaledonicus and C. pescotti, the NOR occupied the terminal chromosomal region (Figure 5c,d). In contrast, the NOR of C. seychellarum was placed interstitially, more specifically, in the middle of one arm of the smallest metacentric chromosome pair (no. 11) (Figure 4b).

Patterns of Karyotype Differentiation
The cytogenetic features of amblypygids are highly underexplored. Apart from an early report on the charinid S. sarawakensis [16], they are confined to two derived families, Phrynidae and Phrynichidae [7,17,18]. With nine newly analyzed species, the present study increased the number of cytogenetically analyzed amblypygids to 18.
Available cytogenetic reports on amblypygids point to a striking diversity in 2n [7,[16][17][18] and chromosome morphology [7]. Moreover, our results on Charinidae, an early diverging amblypygid lineage, showed a wide range of 2n in Sarax (2n =  and Charinus (2n = 42-76). The karyotypes with higher 2n were composed predominantly of monoarmed chromosomes, while the karyotypes with low 2n consisted exclusively of biarmed elements. These observations suggest the major role of chromosome fusions or fissions in the karyotype evolution of charinids. To infer the direction of this process and, hence, reconstruct the ancestral charinid karyotype, the obtained karyotype patterns were plotted onto the recent charinid phylogenetic hypothesis [10], as seen in Figure 6.
Given the considerable karyotype diversity in both analyzed genera, we are currently unable to propose a coherent scenario regarding the main trajectory of the karyotype reshuffling. We cannot exclude similar mechanisms of karyotype evolution (i.e., centric fusions and pericentric inversions) as described in the derived amblypygid lineage Phrynidae, wherein the comparably wide range of 2n (24-68) has been reported [7]. While the available data do not allow a conclusive assessment about ancestral karyotype pattern in charinids and amblypygids in general, there are some indications supporting high ancestral 2n. We found very similar karyotypes in Charinus and Sarax with high chromosome counts (C. cavernicolus, 2n = 76; C. pescotti and C. neocaledonicus, 2n = 74; S. ioanniticus, 2n = 72 and Sarax sp., 2n = 74). These species display a predominance of monoarmed chromosomes. It is much less probable that a series of independent chromosome fissions would convergently lead to such a similar chromosome count; hence, it is more likely that the process took the opposite trajectory, which has led to the reduction in high ancestral 2n. Unfortunately, we have no information about the degree of conserved synteny among the charinid karyotypes and, therefore, the real interspecific karyotype differences remain unknown. We may only suppose (bearing in mind also the supportive results from other methods, see below) that chromosomal fusions leading to the reduction in high ancestral 2n represent one of the main mechanisms of karyotype differentiation in Charinidae. This notion is corroborated by the comparably high 2n in two derived amblypygid lineages, specifically in Damon medius (2n = 70; Phrynichidae) [17] and three species belonging to Phrynidae (Heterophrynus longicornis, 2n = 66; Paraphrynus robustus, 2n = 64; Phrynus marginemaculatus, 2n = 68) [7,18]. The high ancestral 2n of amblypygids may also be consistent with the recently proposed theory regarding the whole genome duplication in the common ancestor of scorpions and tetrapulmonate arachnids [39][40][41]. The conception of high 2n in ancestral tetrapulmonates is supported by high chromosome counts in most basal spiders, mesothelids [42]. However, our current data do not fully rule out the opposite scenario, i.e., low ancestral 2n in Charinidae and its subsequent convergent elevation within Sarax and Charinus by fissions. This alternative hypothesis is consistent with the basal phylogenetic position of C. dominicanus, a species with low 2n (Figure 6). study increased the number of cytogenetically analyzed amblypygids to 18.
Available cytogenetic reports on amblypygids point to a striking diversity in 2n [7,[16][17][18] and chromosome morphology [7]. Moreover, our results on Charinidae, an early diverging amblypygid lineage, showed a wide range of 2n in Sarax (2n = 22-74) and Charinus (2n = 42-76). The karyotypes with higher 2n were composed predominantly of monoarmed chromosomes, while the karyotypes with low 2n consisted exclusively of biarmed elements. These observations suggest the major role of chromosome fusions or fissions in the karyotype evolution of charinids. To infer the direction of this process and, hence, reconstruct the ancestral charinid karyotype, the obtained karyotype patterns were plotted onto the recent charinid phylogenetic hypothesis [10], as seen in Figure 6. neocaledonicus, the NOR distribution has been assessed by both silver staining and rDNA FISH techniques.On the contrary, in two remaining species, only rDNA FISH has been carried out. We did not include the karyotype data of Millot and Tuzet [16] on Sarax sarawakensis. The authors indicated 2n = 25 for males, including a single X chromosome (the sex chromosome system ♂X0/♀XX). The graphical documentation of results presented by these authors is not sufficient to support their statements.
Given the considerable karyotype diversity in both analyzed genera, we are currently unable to propose a coherent scenario regarding the main trajectory of the karyotype re- Figure 6. Charinidae, karyotype features, including number and position of NOR loci. Mapped on a cladogram constructed based on the recent molecular phylogeny of Miranda et al. [10]. Abbreviations: a = acrocentric chromosome, FN = fundamental number, m = metacentric chromosome, N/A = not analyzed, I = interstitial position, NORs = distribution of nucleolar organizer regions, sm = submetacentric chromosome, st = subtelocentric chromosome, t = terminal position. * In C. neocaledonicus, the NOR distribution has been assessed by both silver staining and rDNA FISH techniques. On the contrary, in two remaining species, only rDNA FISH has been carried out. We did not include the karyotype data of Millot and Tuzet [16] on Sarax sarawakensis. The authors indicated 2n = 25 for males, including a single X chromosome (the sex chromosome system ♂X0/♀XX). The graphical documentation of results presented by these authors is not sufficient to support their statements.
In Phrynidae, the main underlying mechanism for karyotype change was proposed to be centric fusion [7]. Among our surveyed charinids, we found a putative footprint of a fusion event in the large chromosome pair of C. neocaledonicus. This pair contains the two large adjacent segments of constitutive heterochromatin, which differ considerably by their sequence composition (one was AT-rich, while the other one was GC-rich). Differential enrichment in AT vs. GC base pairs in these segments suggests that they belonged originally to two separate chromosomes, which fused during evolution. A similar situation was observed in animal taxa known for their high rate of chromosome fusions (e.g., [43]). In C. neocaledonicus, centromere-to-telomere fusion (i.e., tandem fusion) of two smaller monoarmed chromosomes is likely, as (i) the AT-and GC-rich heterochromatin usually occupy the opposite ends of the chromosomes in this species, and (ii) the pair containing two tandemly arranged blocks is acrocentric. There are no data from other amblypygids concerning the distribution of AT-and GC-rich heterochromatin. Among other arachnids, diverse patterns of GC-rich heterochromatin have been recorded in spiders and scorpions, from a scattered distribution of many GC-rich bands throughout the chromosome complement to terminal bands localized on several chromosomes (e.g., [44][45][46]). Another indication of the action of fusions in the charinid karyotype evolution is the presence of an internal telomeric site (see below).
Comparably to phrynids [7], the elevated number of biarmed chromosomes in charinids with lower 2n cannot be fully explained by only a series of fusions. If this were the case, FN would be maintained roughly the same among the species and the number of biarmed elements would increase proportionally to the decrease in 2n [26]. The conside-rable deviation in FN found in charinids suggests that their karyotypes were also shaped by rearrange-ments, which changed the chromosome morphology without affecting the 2n. For example, the karyotypes of C. neocaledonicus and C. pescotti are composed of the same number of chromosomes, but these species display different proportions of monoarmed and biarmed chromosomes, and hence different FN (96 for C. neocaledonicus and 102 for C. pescotti). Our rDNA data (see below) strongly favor the action of inversions, although the involvement of other mechanisms leading to centromeric shift (e.g., [47]) cannot be excluded.
We did not observe heteromorphic (i.e., morphologically differentiated) sex chromosomes in any of the studied charinids, which is in line with the observations in other ka-ryotyped amblypygids [7,18], except for charinid S. sarawakensis [16], where the authors proposed the ♂X0/♀XX sex chromosome system (2n = 25 in males). However, the pre-sence of an X0 system in S. sarawakensis is unlikely, as we did not find heteromorphic sex chromosomes in other Sarax species. The lack of heteromorphic sex chromosomes in amblypygids, nonetheless, does not rule out the possibility that homomorphic sex chromosomes (i.e., those without morphological differentiation) might be present in them. Given that mostly only males (in seven out of nine species) provided chromosome plates, one cannot exclude female heterogamety with morphologically differentiated sex chromosomes in whip spiders. Heteromorphic sex chromosomes occur in two tetrapulmonate groups, spiders [48,[67][68][69] and uropygids [70]. These orders exhibit male heterogamety. Bearing in mind that sex chromosomes of spiders exhibit a specific behavior, including a different condensation both in male and female germlines [71], we suppose that a large homomorphic chromosome pair exhibiting a distinct condensation pattern in the oogonial mitoses of S. ioanniticus females might potentially represent sex chromosomes. This hypothesis is amenable for experimental testing in further studies.

Distribution of rDNA and Telomeric Sequences
The major rDNA cluster (45S rDNA) is by far the most utilized molecular chromosomal marker across diverse eukaryotic organisms, and especially in non-model groups [72][73][74]. The major rDNA cluster visualized either by 18S or 28S rDNA probes has already been localized in some spiders [36,46,73,[75][76][77], scorpions [45,76,[78][79][80][81][82], pseudoscorpions [83], harvestmen [84][85][86][87], ticks [73,88,89], and an amblypygid [18]. Mapping of this cluster often revealed a single NOR site, which is probably an ancestral pattern in arachnids [36]. The number of NOR/rDNA loci has increased in some arachnid clades during their evolution [76,77,81,87]. In haplogyne spiders, these loci are also frequently placed on sex chromosomes [77,90]. When stable distribution patterns for rDNA loci within a certain lineage are used as markers in the phylogenetic context, they may largely contribute to the interpretation of possible karyotype rearrangements (e.g., [84]). All three charinids examined in the present study possess a single pair of NOR-bearing chromosomes, thus implying a possible ancestral condition for the whole amblypygid clade. While in two Charinus species (C. neocaledonicus and C. pescotti), the NOR occupies the terminal position on a chromosome pair, in a single surveyed Sarax species (S. seychellarum), the NOR is placed interstitially in the middle of one arm of the smallest metacentric chromosome pair. The most parsimonious explanation for the observed pattern is that a paracentric inversion shifted the NOR from the terminal location to the interstitial one in this species. Therefore, our data suggest that the major rDNA cluster might be a useful cytogenetic marker for the determination of mechanisms of karyotype repatterning in amblypygids.
According to our data, DNA molecules of charinids are terminated by the so-called insect telomeric sequence (TTAGG) n , which is ancestral for arthropods [17]. This motif has been identified in many arachnid orders [17,35,45,78,79,89,91], including a whip spider of the family Phrynichidae, Damon medius [17]. While the same telomeric motif is usually shared by taxa on higher taxonomic levels, exceptions do occur [92][93][94][95], including arachnids [17]. The presence of insect telomeric sequence, both in early-branching charinids and in a representative of phrynichids belonging to neoamblypygids, suggests that this motif is ancestral in amblypygids. This conclusion is in line with the wide distribution of this motif in arachnids, except for spiders [17]. Besides terminal (TTAGG) n sequences, we also found one ITS in S. seychellarum. This ITS could have arisen by chromosome rearrangement (e.g., [96,97]; for reviews, see [98,99]). S. seychellarum stands out from our sampling by possessing the lowest 2n (22 chromosomes), with the karyotype being composed of biarmed elements only. Therefore, the observed ITS might represent a hallmark of a previous chromosome fusion or translocation. The presence of ITS on only one of the homologues is puzzling but it might reflect the heterozygous constitution resulting from a different degree of sequence elimination/degeneration from the interstitial site. Besides chromosome rearrangement, the telomeric repeats might also be translocated to the interstitial region via transposition or during double-strand break repair processes [98,99].

Conclusions
Our cytogenetic survey in the early-branching amblypygid family Charinidae provides important clues about the pace and trajectories of karyotype differentiation in amblypygids and in tetrapulmonates in general. The data corroborate the findings from the derived lineage Phrynidae in the way that amblypygids possess highly variable 2n and chromosome morphology shaped mostly by fusions and inversions. While we suppose the general direction of karyotype change towards a reduction in 2n via fusions in amblypygids, and some of our findings support this view, larger sampling, including representatives of other amblypygid families, is needed to clarify this hypothesis.