Comparative cytogenetics among populations of two Bothriurus species (Scorpiones, Bothriuridae)

Comparative cytogenetics among populations of two Bothriurus species (Scorpiones, Bothriuridae) Juliana F. de Lima1, Marielle C. Schneider2, Leonardo S. Carvalho3, Ricardo Pinto-da-Rocha1 1Department of Zoology, Universidade de São Paulo (USP), São Paulo (SP), Brazil 2Department of Biology and Zoology, Universidade Federal de Mato Grosso (UFMT), Cuiabá (MT), Brazil 3Universidade Federal do Piauí, Campus Almícar Ferreira Sobral, Floriano (PI), Brazil Corresponding author: Juliana F. de Lima (figueiredo[email protected]) Academic editor: Snejana Grozeva|Received 14 July 2025|Accepted 23 September 2025|Published 13 November 2025 https://zoobank.org/9CC68C00-621C-4B1C-A00B-1815832B71EF Citation: Lima JF, Schneider MC, Carvalho LS, Pinto-da-Rocha R (2025) Comparative cytogenetics among populations of two Bothriurus species (Scorpiones, Bothriuridae). Comparative Cytogenetics 19: 189–208. https://doi. org/10.3897/compcytogen.19.165160 Abstract Bothriurus Peters, 1861 is one of the most diverse genera within the family Bothriuridae. However, to date, only five species have been analyzed using a cytogenetic approach. In this study, for the first time, two populations of Bothriurus asper Pocock, 1893 and nine populations of Bothriurus rochai Mello-Leitão, 1932, two species from northeastern Brazil, were analyzed with respect to diploid number, chromosomal behavior during meiosis, and the localization of heterochromatin and nucleolus organizer regions (NORs). For B. asper, a diploid number of 2n = 30 was recorded in geographically distant populations, whereas B. rochai exhibited intraspecific variation in diploid number (2n = 16 and 2n = 18), representing the lowest diploid numbers ever reported for the family Bothriuridae. Despite the variability in diploid number, the number and localization of NORs remained stable among the populations of B. rochai. When comparing heterochromatin patterns between the two species, larger blocks of constitutive heterochromatin were observed in B. asper than in B. rochai. Variation in the amount of heterochromatin among populations of B. rochai was also observed; in this case, the population with the lowest amount of heterochromatin also exhibited the greatest variation in post-pachytene cell configurations. This is the first study to cytogenetically analyze multiple populations of species within the genus Bothriurus, and it significantly expands the karyotypic information available for scorpions with monocentric chromosomes. Keywords Chromosomal rearrangements, chromosome, fluorochrome, heterochromatin, meiosis, silver impregnation CompCytogen 19: 189–208 (2025) doi: 10.3897/compcytogen.19.165160 https://compcytogen.pensoft.net Copyright Juliana F. de Lima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE COMPARATIVE Cytogenetics International Journal of Plant & Animal Cytogenetics, Karyosystematics, and Molecular Systematics A peer-reviewed open-access journal Juliana F. de Lima et al. / Comparative Cytogenetics 19: 189–208 (2025) 190 Introduction Scorpions represent the third most extensively well-studied order of arachnids from a cytogenetic perspective (Šťáhlavský et al. 2018). They have a synaptic and achiasmatic male meiosis, whose correct segregation of chromosomes is guaranteed by the permanence of the synaptonemal complex until later phases of meiosis I (Schneider et al. 2009a, 2009b; Almeida et al. 2019) and different types of chromosomes. With the exception of Buthidae, the only scorpion family that presents holocentric chromosomes, the other ten scorpion families that have been cytogenetically characterized (Bothriuridae, Chactidae, Chaerilidae, Euscorpiidae, Iuridae, Liochelidae, Scorpionidae, Scorpiopidae, Urodacidae, Vaejovidae) possess monocentric chromosomes (Melters et al. 2012; Schneider et al. 2009a, 2024). However, most of these families have at most 10 species that have been studied (Almeida et al. 2023; Schneider et al. 2024), and together, these families account for less than 40% of the cytogenetic data available for scorpions. Chromosomal rearrangements are the main drivers of karyotypic differences (Mezzasalma et al. 2024) and can generate polymorphism in natural populations (White 1973; Joron et al. 2011). Fissions/fusions rearrangements and translocations have been reported in different scorpion species (e.g. Schneider et al. 2009a, 2009b; Mattos et al. 2013; Adilardi et al. 2020; Lima et al. 2023), and are the primary factors responsible for the high intraspecific karyotype variability and the diverse chromosomal configurations observed during meiosis (Mattos et al. 2018). The identification and characterization of polymorphism expand our understanding of chromosomal evolution (e.g. Jacobina et al. 2009; Mattos et al. 2013; Adilardi et al. 2014, 2016, 2020; Almeida et al. 2017; Ubinski et al. 2018; Lima et al. 2023). In general, cytogenetics is a powerful tool for the characterization of natural groups and has proven effective in characterizing different scorpion lineages (Ojanguren-Affilastro et al. 2017; Šťáhlavský et al. 2018; Štundlová et al. 2019) as well as in identifying population-level polymorphism (e.g. Mattos et al. 2013, 2018; Ubinski et al. 2018; Adilardi et al. 2020; Lima et al. 2020, 2023). Thus, different cytogenetic markers have been widely applied in scorpion studies for the characterization of species and populations. Among the most frequently used are constitutive heterochromatin regions and nucleolus organizer regions (NORs). The pattern of distribution and amount of constitutive heterochromatin is variable within Scorpiones and can assist in distinguishing species and populations (e.g. Mattos et al. 2013; Adilardi et al. 2015; Almeida et al. 2017; Ojanguren-Affilastro et al. 2017; Lima et al. 2023). Although the number and position of NORs appear to be stable for some Buthidae species (Šťáhlavský et al. 2020), as observed in representatives of the genera Ischnotelson Esposito, Yamaguti, Souza, Pinto da Rocha et Prendini, 2017, Jaguajir Esposito, Yamaguti, Souza, Pinto da Rocha et Prendini, 2017, Physoctonus Mello-Leitão, 1934, Rhopalurus Thorell, 1876, and Tityus C. L. Koch, 1836 (Adilardi et al. 2013, 2014; Mattos et al. 2013, 2014; Ubinski et al. 2018), other genera have shown variation in the number or position of these regions, for example, species of Cytogenetics of Bothriurus species 191 Compsobuthus Vachon, 1949, Hottentotta Birula, 1908, and Reddyanus Vachon, 1912 (Šťáhlavský et al. 2020). These data highlight the effectiveness of this marker in distinguishing certain scorpion taxa. The family Bothriuridae comprises 168 described species (Rein, 2025). However, only three genera (Brachistosternus Pocock, 1893, Bothriurus Peters, 1861, and Timogenes Simon, 1880) and less than 6% of the species of this family have had their chromosomes studied (Schneider et al. 2024). In Brazil, the genus Bothriurus is the most diverse, currently comprising 17 described species (Rein 2025). However, only five species of this genus have been cytogenetically characterized, and only two of them, B. araguayae Vellard, 1934 (2n = 42 and 44) (Ferreira 1968; Schneider et al. 2009b) and Bothriurus sp. (2n = 36) (Piza 1947) belong to the Brazilian fauna. Additionally, only B. araguayae and B. rochensis San Martin, 1965 (2n = 46) have been characterized in terms of chromosomal behavior during meiosis. These species exhibited distinct diploid numbers (including intraspecific variation observed among populations of B. araguayae), as well as differences in the number of NORs (4 and 6, respectively), the localization of heterochromatin, and chromosome morphology (Schneider et al. 2009b). These results reinforce the potential of cytogenetic markers in the characterization of Bothriurus species and, consequently, in biodiversity assessment. Chromosomal differences have also highlighted the utility of cytogenetic data for species-level taxonomy, as observed in representatives of Scorpiopidae (Šťáhlavský et al. 2021). Bothriurus asper Pocock, 1893 and B. rochai Mello-Leitão, 1932 are species widely distributed in northeastern Brazil (Maury 1982; Lourenço 2000; Mattoni et al. 2003; Santos-da-Silva et al. 2017). Both belong to distinct species groups named after them, which still face significant taxonomic challenges (Santos-daSilva et al. 2017). These species groups are poorly understood from taxonomic, karyological, phylogenetic, and ecological perspectives. In this context, cytogenetic analyses not only contribute to understanding the impact of chromosomal rearrangements on karyotype evolution but also enable the identification of potential diagnostic chromosomal characters and the detection of population-level polymorphism. Understanding the chromosomal organization of the genome is essential for identifying processes related to population divergence, adaptation, and, in some cases, even speciation events (Sheffer et al. 2021). Evolutionary processes may be accompanied by changes in genome organization and not solely by point mutations in the DNA sequence (Štundlová et al. 2019). The absence of information on interand intraspecific karyotypic variability in these two Bothriurus species makes them suitable models for cytogenetic characterization. Furthermore, this study addresses a broader gap in cytogenetic data available for species of the genus Bothriurus. Thus, the present work characterized different populations of B. asper and B. rochai with respect to diploid number and chromosomal behavior during meiosis, with the aim of expanding cytogenetic knowledge for the group, as well as enabling the future integration of the data obtained here into broader biodiversity research. Juliana F. de Lima et al. / Comparative Cytogenetics 19: 189–208 (2025) 192 Material and methods Sampling The number of specimens analyzed in this study and their respective collection sites in northeastern Brazil are listed in Table 1 (see Suppl. material 1: table S1, fig. S1). All specimens were deposited in the Natural History Collection of the Federal University of Piauí, Floriano, Brazil (CHNUFPI; curator: J.F. Vilela). Cytogenetic preparations Slides were prepared from the gonads of adult individuals according to the technique described by Schneider et al. (2009a). The slides were stained with 3% Giemsa solution for 12 minutes to perform the initial characterization of diploid number, chromosomal configurations in meiotic cells and chromosome morphology. The nomenclature for chromosome morphology followed Levan et al. (1964). However, only two categories were considered: meta/submetacentric for chromosomes with two arms and subtelo/acrocentric for chromosomes with only one clearly visible chromosome arm. This is because the analyses were performed in metaphase II cells, in which the chromosomes are more condensed. The diploid set length (DSL) was evaluated for B. rochai populations. At least three post-pachytene cells per population, exhibiting similar levels of chromatin condensation, were analyzed using ImageJ (Image Processing and Analysis in Java) (see Suppl. material 1: fig. S2), developed by the Research Services Branch of the U.S. National Institute of Mental Health. All cells were measured in micrometers, including all bivalents as well as the chromosomes constituting the chromosomal chain, when present. A generalized linear model (GLM) with Gaussian distribution of errors was fitted to compare the DSL among cells with different chromosomal arrangements. Model adequacy was evaluated by manually examining the dispersion parameters. A contrast analysis was conducted using the ‘coms’ function from the R package ‘RT4Bio’ (ReisTable 1. Bothriurus species analyzed in this study, including the number of individuals and their collection localities. CECeará, PBParaíba, PEPernambuco, PI-Piauí, RNRio Grande do Norte. Species Number of individuals Collection localities Bothriurus asper 4♂Teresina-PI (4°54'13.2"S, 42°47'27"W) 1♂Igarassu-PE (7°46'33"S, 34°56'54"W) Bothriurus rochai 2♂Apodi-RN (5°35' 27"S, 37°49' 39"W) 3♂Cajazeiras-PB (6°52' 37"S, 38°37'03"W) 2♂Maturéia-PB (7°16'18"S, 37°18'5.1"W) 1♂Icó-CE (6°24'46"S, 39°03'55"W) 3 ♂Quixadá-CE (4°55'57"S, 39°10'22"W) 8♂Brasileira-PI (4°06'28"S, 41°42'04"W) 2♂João Câmara-RN (5°34'02"S, 35°55'03"W) 2♀Floriano-PI (6°46 21"S, 43°03'48"W) 9♂São Raimundo Nonato-PI (8°52'45"S, 42°43'08"W) Cytogenetics of Bothriurus species 193 Júnior et al. 2015), to identify which chromosomal arrangement groups differed significantly. A figure was generated using the ‘ggplot2’ package (Wickham 2016). To detect active nucleolus organizer regions (NORs), silver ion impregnation was performed according to the method described by Howell and Black (1980) and constitutive heterochromatin regions were identified following the protocol described by Sumner (1972), with a modified incubation time of 30 seconds in 5% barium hydroxide octahydrate solution at 60 °C. Additionally, to visualize ATand GC-rich chromatin regions, some slides were stained with 4’-6-diamidino-2-phenylindole (DAPI), chromomycin A3 (CMA₃), and distamycin A, following Schweizer (1976). All chromosomal preparations were photographed using a Zeiss Axio Imager A2 light microscope with Zen 3.4 software and an Olympus BX51 fluorescence microscope with DP Controller software, using appropriate filters for fluorescent dyes. Results Bothriurus asper included two analyzed populations (Fig. 1, Table 1), with the cytogenetic description comprising specimens from the type locality, Igarassu-PE. In contrast, a total of nine populations of B. rochai were characterized, one of them from its type locality (vaguely known only as the state of Ceará; Maury, 1982) Figure 1. Distribution and collection localities in Brazil of Bothriurus asper and Bothriurus rochai analyzed in this work. Juliana F. de Lima et al. / Comparative Cytogenetics 19: 189–208 (2025) 194 (Fig. 1, Table 1). Cytogenetic analyses revealed that both B. asper and B. rochai possess monocentric chromosomes, with clearly defined primary constrictions and centromeric regions. Chromosomal characterization In the analyzed specimens of B. asper, no mitotic metaphase cells were observed. Therefore, the diploid number 2n = 30 was determined based on the analysis of post-pachytene and metaphase II cells. In the specimen from Igarassu-PE, post-pachytene nuclei exhibited two distinct configurations: 15 side-by-side bivalents (29.5% of the cells), with no evidence of chiasmata (Fig. 2A), and 13 bivalents plus one chromosomal chain composed of four elements (70.5% of the cells), arranged in a cross-shaped configuration (Fig. 2B). Metaphase II cells revealed the presence of 15 chromosomes (n = 15), including 11 meta/submetacentric and four subtelo/acrocentric chromosomes (Fig. 2C). In the four specimens from Teresina-PI, only post-pachytene cells displaying a chromosomal chain composed of four elements (13II + CIV) were observed. However, unlike the individual from Igarassu-PE, all cells showed an open quadrivalent Figure 2. Testicular cells of B. asper stained with Giemsa. Post-pachytene cell with 15 bivalents (15II) (A). Post-pachytene cells with 13 bivalents and a chain of four chromosomes (13II + CIV) (B, D, E). Meiotic metaphases with 15 chromosomes (C, F). Diagrams illustrating the chromosomal rearrangements (G, H). Arrows = chromosomal chains. Scale bars: 10 μm. Cytogenetics of Bothriurus species 195 association (Fig. 2D, E). Finally, metaphase II cells revealed a haploid number of n = 15, consisting of five meta/submetacentric and ten subtelo/acrocentric chromosomes (Fig. 2F). Schematic representations of multivalent associations, for both species, can be seen in the Fig. 2G, H. The cytogenetic characterization of B. rochai revealed intraspecific variation in diploid number (Figs 3–5) (Suppl. material 1: fig. S1). Mitotic metaphase cells from males allowed the determination of a diploid number of 2n = 18 for individuals from Brasileira-PI, Floriano-PI, São Raimundo Nonato-PI, Icó-CE, Quixadá-CE, and Maturéia-PB (Fig. 3A–F), and 2n = 16 for the populations from Apodi-RN, Cajazeiras-PB, and João Câmara-RN (Fig. 3G–I). Floriano-PI was the only population from which only female specimens were obtained; these individuals showed 18 chromosomes in mitotic metaphase cells, consistent with most of the analyzed populations (Fig. 3B). Figure 3. Mitotic metaphase cells of B. rochai stained with Giemsa. Mitotic metaphase cells showing 2n = 18 (A, B, C, D, E, F). Mitotic metaphase cells showing 2n = 16 (G, H, I). Scale bars: 10 μm. Juliana F. de Lima et al. / Comparative Cytogenetics 19: 189–208 (2025) 196 Of the eight specimens examined from Brasileira-PI, three exhibited post-pachytene nuclei composed exclusively of nine bivalents (Fig. 4A), while the remaining five showed variation in chromosomal configurations. These latter specimens presented Figure 4. Post-pachytene cells of B. rochai stained with Giemsa. Nine bivalents (A, E, F, G). Eight bivalents (J). 6II + CVI (B, H, I). 7II + CIV (C). 7II + CIV (D) (two cells shown). 5II + CVI (K, L, M, N, O, P). Arrow = chromosomal chain. Scale bars: 10 μm. Figure 5. Metaphase II cells of B. rochai stained with Giemsa. Cells with n = 9 (A, B, C, D, E). Cells with n = 8 (F, G, H). Scale bars: 10 μm. Cytogenetics of Bothriurus species 197 cells with 9II, cells with six bivalents and one chromosomal chain composed of six chromosomes (6II + CVI) (Fig. 4B), and a few cells with seven bivalents and one quadrivalent chromosomal chain (7II + CIV) (Fig. 4C, D). For all specimens from São Raimundo Nonato-PI, Icó-CE, and Quixadá-CE, only cells with nine side-by-side bivalents were observed, with no evidence of chiasma (Fig. 4E–G). In the specimens from Maturéia-PB, post-pachytene cells also revealed the presence of six bivalents and a chromosomal chain composed of six chromosomes (6II + CVI) (Fig. 4H, I) in both individuals analyzed. However, some degree of instability was observed in the bivalent configurations within this population. While in some cells, five isolated bivalents were observed, with the ends of two pairs positioned very close to each other (Fig. 4H), other cells appeared to show a terminal association between the two bivalents (Fig. 4I). Among the populations with a diploid number of 2n = 16, only in specimens from Apodi-RN did all post-pachytene nuclei exhibit exclusively bivalents (8II) (Fig. 4J). In contrast, all cells from the João Câmara-RN and Cajazeiras-PB populations displayed five large bivalents and a chromosomal chain composed of six medium-sized chromosomes (5II + CVI) (Fig. 4K–P). However, varying degrees of association between the chromosomes forming the chain were observed. While in some cells only the terminal and subterminal regions of the chromosomes were associated (Fig. 4L), in others, an extensive portion of the chromosomes was synapsed (Fig. 4P). Metaphase II cells revealed haploid numbers of n = 9, with five meta/submetacentric and four subtelo/acrocentric chromosomes in specimens from Brasileira-PI, São Raimundo Nonato-PI, Icó-CE, Quixadá-CE, and Maturéia-PB (Fig. 5A–E), n = 8 with six metacentric and two acrocentric chromosomes in specimens from Apodi-RN and Cajazeiras-PB (Fig. 5F, H), and four meta/submetacentric and four acrocentric chromosomes in specimens from João Câmara-RN (Fig. 5G). To assess differences in chromosome size, DSL was measured in post-pachytene cells with comparable levels of chromatin condensation. The average DSL was 2.50 μm in individuals with 2n = 18 and 1.59 μm in those with 2n = 16, with specific values for cells containing only bivalents and those forming chromosomal chains, summarized in Table 2. The analysis revealed significant variation in DSL among chromosome organizations (residual deviance = 8.101, d.f. = 31; F = 9.259, p < 0.001). Post-pachytene cells of specimens with 2n = 16 and those with 2n = 18 organized as 6II + CVI exhibited smaller chromosomes, ranging from small to medium sizes. In contrast, specimens with 2n = 18 organized as 9II or 7II + CIV showed larger DSL values. Additionally, the size of the bivalents and the chromosomes involved in multivalent associations were slightly smaller in the 2n = 16 and 6II + CVI groups than in the 9II and 7II + CIV groups (Figs 4, 6). Localization of nucleolus organizer regions (NORs) For B. rochai, chromosome preparations subjected to silver ion impregnation indicated the presence of active nucleolus organizer regions at the terminal region of one chromosome pair (Fig. 7). For specimens with diploid number 2n = 18, small markings were Juliana F. de Lima et al. / Comparative Cytogenetics 19: 189–208 (2025) 204 with monocentric chromosomes. The stability of the diploid number in B. asper, in contrast to the variation observed in B. rochai, suggests distinct trajectories of karyotype evolution within the group, in which chromosomal rearrangements, especially translocations and fission/fusion events, play a central role. Additionally, the differences in the amount and distribution of heterochromatin, associated with the conservation of a single pair bearing the nucleolar organizer regions (NORs), indicate that, despite the structural differences observed, some elements of genomic organization remain conserved. Acknowledgments We thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES for the Ph.D. scholarship granted to JFL (process number 88882.333073/2019-01). We also thank CNPq (process number 306241/2022-6) and FAPESP (process number 13/50297-0) for the research grants awarded to RPR. We would like to thank two anonymous reviewers for critical reading and valuable suggestions in the manuscript. 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Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/compcytogen.19.165160.suppl1