Chromosome diversity in Buthidae and Chactidae scorpions from Brazilian fauna: Diploid number and distribution of repetitive DNA sequences.

In this work, we analyzed cytogenetically eight Chactidae and Buthidae, including the localization of repetitive DNA sequences. The chactids possess monocentric chromosomes and the highest diploid numbers (2n=50 in Brotheas amazonicus, 2n=36 in Chactopsis amazonica, 2n=30 in Neochactas sp.) when compared with buthids (2n=10 in Tityus bahiensis, 2n=14 in Tityus apiacas and Tityus metuendus, 2n=18 in Tityus aba, 2n=26 in Ischnotelson peruassu). The localization of rDNA genes and (TTAGG)n sequences exhibited a conserved pattern of two terminal/subterminal ribosomal cistrons and terminal telomere signals. However, the comparison between the data of C-banding, DAPI after FISH and Cot-DNA fraction indicated a variable quantity and distribution of these regions, as follow: (i) positive heterochromatin and Cot-DNA signals (B. amazonicus and I. peruassu), (ii) small blocks of heterochromatin with large Cot-DNA signals (T. metuendus), (iii) positive heterochromatic regions and absence of Cot-DNA signals (T. aba and T. apiacas), and (iv) negative heterochromatin and Cot-DNA signals (T. bahiensis). Therefore, our results revealed that there still is not a clear relation between quantity of heterochromatin and presence of monocentric or holocentric chromosomes and occurrence of chromosomal rearrangements, indicating that repetitive regions in scorpions must be analyzed using different cytogenetic approaches.

Venomous Loxosceles Species (Araneae, Haplogynae, Sicariidae) from Brazil: 2n♂ = 23 and X1X2Y Sex Chromosome System as Shared Characteristics.

The family Sicariidae comprises the genera Hexophthalma, Sicarius and Loxosceles. This latter is subdivided in eight monophyletic groups based on genitalia morphology and molecular analyses: amazonica, gaucho, laeta, and spadicea (South America); reclusa (North America); rufescens (Mediterranean); spinulosa and vonwredei (Africa). In Brazil, the genus Loxosceles is represented by 50 species. The mitotic and meiotic characteristics of eight Loxosceles species were analyzed in order to discuss the chromosome evolution, as well as the correspondence between cytogenetic data and morphological/molecular data for the delimitation of the South American groups of species belonging to this genus. All species studied in this work showed 2n♂ = 23, including a X1X2Y sex chromosome system (SCS). Despite the similarity of diploid number and SCS, the species studied here differed regarding the chromosome morphology of some autosomal pairs, presence of secondary constrictions, size of X chromosomes and localization of Ag-NOR/rDNA sites. Based on all these chromosomal data, we verified a close relationship between Loxosceles species belonging to the amazonica and gaucho groups. Transmission electron microscopy (TEM) analysis of spread pachytene cells of L. gaucho showed regular synapsis between homologous autosomal chromosomes, but asynaptic behavior of the sex chromosomes. The axial elements of the sex chromosomes undergo conspicuous morphological modifications resulting in shortening of their length.

Venomous Loxosceles Species (Araneae, Haplogynae, Sicariidae) from Brazil: 2nmarte= 23 and X1X2Y sex chromosome system as shared characteristics

The family Sicariidae comprises the genera Hexophthalma, Sicarius and Loxosceles. This latter is subdivided in eight monophyletic groups based on genitalia morphology and molecular analyses: amazonica, gaucho, laeta, and spadicea (South America); reclusa (North America); rufescens (Mediterranean); spinulosa and vonwredei (Africa). In Brazil, the genus Loxosceles is represented by 50 species. The mitotic and meiotic characteristics of eight Loxosceles species were analyzed in order to discuss the chromosome evolution, as well as the correspondence between cytogenetic data and morphological/molecular data for the delimitation of the South American groups of species belonging to this genus. All species studied in this work showed 2nmarte = 23, including a X1X2Y sex chromosome system (SCS). Despite the similarity of diploid number and SCS, the species studied here differed regarding the chromosome morphology of some autosomal pairs, presence of secondary constrictions, size of X chromosomes and localization of Ag-NOR/rDNA sites. Based on all these chromosomal data, we verified a close relationship between Loxosceles species belonging to the amazonica and gaucho groups. Transmission electron microscopy (TEM) analysis of spread pachytene cells of L. gaucho showed regular synapsis between homologous autosomal chromosomes, but asynaptic behavior of the sex chromosomes. The axial elements of the sex chromosomes undergo conspicuous morphological modifications resulting in shortening of their length.

Venomous Loxosceles Species (Araneae, Haplogynae, Sicariidae) from Brazil: 2nmarte= 23 and X1X2Y sex chromosome system as shared characteristics

The family Sicariidae comprises the genera Hexophthalma, Sicarius and Loxosceles. This latter is subdivided in eight monophyletic groups based on genitalia morphology and molecular analyses: amazonica, gaucho, laeta, and spadicea (South America); reclusa (North America); rufescens (Mediterranean); spinulosa and vonwredei (Africa). In Brazil, the genus Loxosceles is represented by 50 species. The mitotic and meiotic characteristics of eight Loxosceles species were analyzed in order to discuss the chromosome evolution, as well as the correspondence between cytogenetic data and morphological/molecular data for the delimitation of the South American groups of species belonging to this genus. All species studied in this work showed 2nmarte = 23, including a X1X2Y sex chromosome system (SCS). Despite the similarity of diploid number and SCS, the species studied here differed regarding the chromosome morphology of some autosomal pairs, presence of secondary constrictions, size of X chromosomes and localization of Ag-NOR/rDNA sites. Based on all these chromosomal data, we verified a close relationship between Loxosceles species belonging to the amazonica and gaucho groups. Transmission electron microscopy (TEM) analysis of spread pachytene cells of L. gaucho showed regular synapsis between homologous autosomal chromosomes, but asynaptic behavior of the sex chromosomes. The axial elements of the sex chromosomes undergo conspicuous morphological modifications resulting in shortening of their length.

Mechanisms of karyotype evolution in the Brazilian scorpions of the subfamily Centruroidinae (Buthidae).

The recently-revised subfamily Centruroidinae is part of the New World clade of buthid scorpions. In this study, we analyzed the cytogenetic characteristics of nine of the 10 Brazilian centruroidines, and one undescribed species of the genus Ischnotelson, using a phylogenetic approach to determine the chromosomal rearrangements responsible for the differentiation of karyotypes among the species. The cytogenetic data recorded in the present study supported the new taxonomic arrangement of the Centruroidinae, with all the species of the same genus sharing the same or similar diploid numbers, i.e., 2n = 20 or 22 in Troglorhopalurus lacrau and T. translucidus, 2n = 25 or 26 in Ischnotelson sp., I. guanambiensis and I. peruassu, and 2n = 28 in Jaguajir agamemnon, J. pintoi and J. rochae. The karyotype modelling in the ChromEvol software indicated 2n = 18 as the ancestral diploid number of the Centruroidinae. The differentiation of karyotypes among the centruroidine genera was based on increasing chromosome numbers resulting from progressive fission events. These changes probably occurred prior to the diversification of the genera Ischnotelson, Jaguajir, Physoctonus and Rhopalurus, and appear to have played a more important role in karyotype evolution at the intergeneric level than the interspecific one. However, the observed increase in diploid numbers was not accompanied by changes in the number or location of ribosomal genes or telomeric sequences. The identification of meiotic cells in female specimens also allowed us to discuss the mechanisms of achiasmatic meiosis in scorpions.

Insights into the origin of the high variability of multivalent-meiotic associations in holocentric chromosomes of Tityus (Archaeotityus) scorpions.

Scorpions represent an intriguing group of animals characterized by a high incidence of heterozygous chromosomal rearrangements. In this work, we examined six species of Tityus (Archaeotityus) from Brazilian fauna with a particular focus on elucidating the rearrangements responsible for the intraspecific variability of diploid number and the presence of long chromosomal chains in meiosis. To access any interpopulation diversity, we also studied individuals from four species representing distinct localities. Most species demonstrated intraspecific polymorphism in diploid number (2n = 19 and 2n = 20 in T. clathratus, T. mattogrossensis, and T. pusillus, 2n = 16, 2n = 17 and 2n = 18 in T. paraguayensis, and 2n = 16 and 2n = 24 in T. silvestris) and multi-chromosomal associations during meiosis I, which differed even among individuals with the same chromosome number. In some species, the heterozygous rearrangements were not fixed, resulting such as in Tityus clathatrus, in 11 different chromosomal configurations recognized within a same population. Based on meiotic chromosome behaviour, we suggested that independent rearrangements (fusion/fission and reciprocal translocations), occurring in different combinations, originated the multi-chromosomal chains. To evaluate the effects of these chromosome chains on meiotic segregation, we applied the chi-square test in metaphase II cells. The non-significant occurrence of aneuploid nuclei indicated that non-disjunction was negligible in specimens bearing heterozygous rearrangements. Finally, based on our analysis of many chromosome characteristics, e.g., holocentricity, achiasmate meiosis, endopolyploidy, ability to segregate heterosynaptic or unsynapsed chromosomes, (TTAGG)n sequence located in terminal regions of rearranged chromosomes, we suggest that the maintenance of multi-chromosomal associations may be evolutionarily advantageous for these species.

Comparative cytogenetic analysis among filistatid spiders (Araneomorphae: Haplogynae)

The family Filistatidae is considered sister to Synspermiata or sister to Hypochilidae. Cytogenetic knowledge of this family could be useful for understanding the mechanism of chromosome evolution that has occurred within the group. In this work, two filistatid species belonging to distinct subfamilies, Kukulcania hibernalis (Hentz, 1842) (Filistatinae) and Misionella mendensis (Mello-Leitao, 1920) (Prithinae), were investigated using standard and differential chromosome staining. Analysis of mitotic and meiotic cells revealed the diploid 2n male = 25 for K. hibernalis and 2n male = 21 for M. mendensis. Both species exhibited a sex chromosome system of the X1X2Y type and metacentric/submetacentric chromosomes. In prophase I cells, the sex chromosomes were in a trivalent configuration with all elements associated without chiasma through their terminal regions. Both species revealed six nucleolar organizer regions on the terminal region of three autosomal pairs. In K. hibernalis, constitutive heterochromatin was located mainly in the terminal regions of autosomes and sex chromosomes while in M. mendensis, the heterochromatin occurred in the pericentromeric region of all chromosomes. Despite the scarcity of cytogenetic information for Filistatidae, the available results show the occurrence of high variability in the diploid number but with the maintenance of the X1X2Y sex chromosome system. Additionally, the karyotype differentiation in the species of this family seems to have involved not only the number of autosomes but also specific chromosomal sites, such as the constitutive heterochromatic regions.

Location of 45S Ribosomal Genes in Mitotic and Meiotic Chromosomes of Buthid Scorpions.

Buthid scorpions exhibit a high variability in diploid number within genera and even within species. Cytogenetically, Buthidae differs from other families of Scorpiones based on its low diploid numbers, holocentric chromosomes, and complex chromosomal chains, which form during meiosis. In this study, we analyzed the distribution of the 45S ribosomal DNA (rDNA) genes in the mitotic and meiotic chromosomes of seven buthid species belonging to the genera Rhopalurus and Tityus with the ultimate goal of elucidating the chromosome organization in these scorpions. The chromosome number ranged from 2n=6 to 2n=28. Despite the high variance in diploid number, all species examined carried their 45S rDNA sites in the terminal region of exactly two chromosomes. Analyses of meiotic cells revealed 45S rDNA clusters in the chromosomal chains of Rhopalurus agamemnon, Tityus bahiensis, Tityus confluens, and Tityus martinpaechi, or in bivalent-like configuration in Rhopalurus rochai, Tityus bahiensis, Tityus confluens, Tityus fasciolatus, and Tityus paraguayensis. In the species examined, the 45S rDNA sites colocalized with constitutive heterochromatin regions. In light of the high chromosome variability and maintenance of number and terminal position of 45S rDNA sites in buthids, the heterochromatin may act to conserve the integrity of the ribosomal genes.

Comparative cytogenetics of seven Ctenidae species (Araneae).

The present study elevates the number of cytogenetically analyzed ctenid species and genera from two to eight and six, respectively, presenting comparisons between chromosomal data obtained and the phylogenetic hypothesis proposed in the literature. Six ctenid species presented 13 autosomal pairs, exhibiting either X1X2O (Ctenus ornatus, Ctenus sp., Parabatinga brevipes and Phoneutria nigriventer) or X1X2X3O sex chromosome systems (Nothroctenus sp. and Viracucha andicola). Asthenoctenus borellii showed 2n ♂ = 20 + X1X2O. In all species, the chromosomes were telocentric. Some cells of one C. ornatus specimen exhibited one extra chromosome that, considering the behavioral similarities between the two chromosomes, can be considered to be supernumerary, derived from or giving rise to a sex chromosome. Silver impregnation revealed nucleolar organizer regions on one autosomal pair of C. ornatus and P. nigriventer (Cteninae) and two pairs of V. andicola (Acanthocteninae). Chromosomal data suggests that the X1X2X3O system arose several times in the evolution of entelegyne spiders, and that conversion of an X1X2O system into an X1X2X3O system and vice-versa has been a relatively common event in spiders. All the chromosomal data corroborate the close relationship between Ctenus and Phoneutria, the placement of P. brevipes within Cteninae, the placement of Anahita in a separated branch within Cteninae, and the inclusion of A. borellii in a distinct group within the ctenids (Viridasiinae), all of which are as proposed by phylogenetic hypotheses available in the literature.

High chromosome variability and the presence of multivalent associations in buthid scorpions.

In this study, we investigated the mitotic and meiotic chromosomes of 11 Buthidae scorpion species, belonging to three genera (Ananteris, Rhopalurus and Tityus), to obtain detailed knowledge regarding the mechanisms underlying the intraspecific and/or interspecific diversity of chromosome number and the origin of the complex chromosome associations observed during meiosis. The chromosomes of all species did not exhibit a localised centromere region and presented synaptic and achiasmatic behaviour during meiosis I. Spermatogonial and/or oogonial metaphase cells of these buthids showed diploid numbers range from 2n = 6 to 2n = 28. In most species, multivalent chromosome associations were observed in pachytene and postpachytene nuclei. Moreover, intraspecific variability associated with the presence or absence of chromosome chains and the number of chromosomes in the complex meiotic configurations was observed in some species of these three genera. Silver-impregnated cells revealed that the number and location of nucleolar organiser regions (NORs) remained unchanged despite extensive chromosome variation; notably, two NORs located on the terminal or subterminal chromosome regions were commonly observed for all species. C-banded and fluorochrome-stained cells showed that species with conspicuous blocks of heterochromatin exhibited the lowest rate of chromosomal rearrangement. Based on the investigation of mitotic and meiotic cells, we determined that the intraspecific variability occurred as a consequence of fission/fusion-type chromosomal rearrangements in Ananteris and Tityus species and reciprocal translocation in Rhopalurus species. Furthermore, we verified that individuals presenting the same diploid number differ in structural chromosome organisation, giving rise to intraspecific differences of chromosome association in meiotic cells (bivalent-like elements or chromosome chains).

Chromosomes of Theridiidae spiders (Entelegynae): Interspecific karyotype diversity in Argyrodes and diploid number intraspecific variability in Nesticodes rufipes.

Theridiidae is a derived family within the Araneoidea clade. In contrast to closely related groups, the 2n(male) = 20+X(1) X (2) with acro/telocentric chromosomes is the most widespread karyotype among the theridiid spiders. In this work, the cytogenetic analysis of Argyrodes elevatus revealed original chromosome features different from those previously registered for Theridiidae, including the presence of 2n(male) = 20+X with meta/submetacentric chromosomes. Most individuals of Nesticodes rufipes showed family conserved karyotype characteristics. However, one individual had a 2n(male) = 24 due to the presence of an extra chromosome pair, which exhibited regular behavior and reductional segregation during meiosis. After silver staining, mitotic cells exhibited NORs localized on the terminal regions of the short arms of pairs 2, 3, and 4 of A. elevatus and on the terminal regions of long arms of pair 4 of N. rufipes. The comparative analysis with data from phylogenetically related species allowed the clarification of the origin of the interspecific and intraspecific chromosome variability observed in Argyrodes and in N. rufipes, respectively.

Chromosomes of Theridiidae spiders (Entelegynae): interspecific karyotype diversity in Argyrodes and diploid number intraspecific variability in Nesticodes rufipes

Theridiidae is a derived family within the Araneoidea clade. In contrast to closely related groups, the 2n(male) = 20+X1X2 with acro/telocentric chromosomes is the most widespread karyotype among the theridiid spiders. In this work, the cytogenetic analysis of Argyrodes elevatus revealed original chromosome features different from those previously registered for Theridiidae, including the presence of 2n(male) = 20+X with meta/submetacentric chromosomes. Most individuals of Nesticodes rufipes showed family conserved karyotype characteristics. However, one individual had a 2n(male) = 24 due to the presence of an extra chromosome pair, which exhibited regular behavior and reductional segregation during meiosis. After silver staining, mitotic cells exhibited NORs localized on the terminal regions of the short arms of pairs 2, 3, and 4 of A. elevatus and on the terminal regions of long arms of pair 4 of N. rufipes. The comparative analysis with data from phylogenetically related species allowed the clarification of the origin of the interspecific and intraspecific chromosome variability observed in Argyrodes and in N. rufipes, respectively.

Complex meiotic configuration of the holocentric chromosomes: the intriguing case of the scorpion Tityus bahiensis.

Mitotic and meiotic chromosomes of Tityus bahiensis were investigated using light (LM) and transmission electron microscopy (TEM) to determine the chromosomal characteristics and disclose the mechanisms responsible for intraspecific variability in chromosome number and for the presence of complex chromosome association during meiosis. This species is endemic to Brazilian fauna and belongs to the family Buthidae, which is considered phylogenetically basal within the order Scorpiones. In the sample examined, four sympatric and distinct diploid numbers were observed: 2n = 5, 2n = 6, 2n = 9, and 2 = 10. The origin of this remarkable chromosome variability was attributed to chromosome fissions and/or fusions, considering that the decrease in chromosome number was concomitant with the increase in chromosome size and vice versa. The LM and TEM analyses showed the presence of chromosomes without localised centromere, the lack of chiasmata and recombination nodules in male meiosis, and two nucleolar organiser regions carrier chromosomes. Furthermore, male prophase I cells revealed multivalent chromosome associations and/or unsynapsed or distinctly associated chromosome regions (gaps, less-condensed chromatin, or loop-like structure) that were continuous with synapsed chromosome segments. All these data permitted us to suggest that the chromosomal rearrangements of T. bahiensis occurred in a heterozygous state. A combination of various factors, such as correct disjunction and balanced segregation of the chromosomes involved in complex meiotic pairing, system of achiasmate meiosis, holocentric nature of the chromosomes, population structure, and species dispersion patterns, could have contributed to the high level of chromosome rearrangements present in T. bahiensis.

Strategies of karyotype differentiation in Elateridae (Coleoptera, Polyphaga).

The chromosome study of five species of the family Elateridae, belonging to the subfamilies Agrypninae and Elaterinae, and the analysis of the cytogenetic data previously recorded for this family permitted the establishment of the main strategies of karyotypic differentiation that has occurred in the elaterids. In Agrypninae, the three species studied (Conoderus fuscofasciatus, Conoderus rufidens, and Conoderus sp.) showed the male karyotype 2n=16+X0. This karyotypic uniformity detected in these Conoderus species has also been shared with other species of the same genus, differing considerably from chromosomal heterogeneity verified in the subfamily Agrypninae. The use of the C-banding technique in C. fuscofasciatus and Conoderus sp. revealed constitutive heterochromatin in the pericentromeric region of the majority of the chromosomes. In C. fuscofasciatus, additional constitutive heterochromatin were also observed in the long arm terminal region of almost all chromosomes. Among the representatives of Elaterinae, the karyotype 2n=18+Xy(p) of Pomachilius sp.2 was similar to that verified in the majority of the Coleoptera species, contrasting with the chromosomal formula 2n=18+X0 detected in Cardiorhinus rufilateris, which is most common in the species of Elaterinae. In the majority of the elaterids, the chromosomal differentiation has frequently been driven by reduction of the diploid number; but, among the four cytogenetically examined subfamilies, there are some differences in relation to the trends of karyotypic evolution.

Astylus variegatus (Coleoptera, Melyridae): Cytogenetic study of a population exposed to agrochemical products

In this work we describe the cytogenetic analyses performed in specimens of Astylus variegatus (Germar, 1824) collected in two localities: one area of natural vegetation and one of agricultural crops, where agrochemical products were used. Astylus variegatus had karyotypes with 2n(male) = 16+Xy p and 2n (female) = 16+XXp, with exclusively metacentric chromosomes. Pachytene spermatocytes showed synapsed autosomal bivalents and non-associated sex chromosomes. In diplotene, the autosomal bivalents exhibited one or two terminal chiasmata and the Xy p had a typical parachute configuration. In meiotic cells of some specimens, an extra chromosome, interpreted as a B chromosome, was observed. C-banding showed constitutive heterochromatin in the pericentromeric region of all chromosomes, with the exception of the y p. Silver nitrate staining revealed one nucleolus organizer region (NOR) on the terminal region of the short arm of the second autosome pair. Silver staining of meiotic cells confirmed the NOR pattern detected in mitotic cells and revealed an argentophilous material on the Xy p. A cytogenetic comparison between the two populations of A. variegatus showed a statistically significant divergence (chi2 = 117.10; df = 1) in the number of aneuploid cells and a higher frequency of B chromosome in the population exposed to agrochemicals.

Evolutionary chromosomal differentiation among four species of Conoderus Eschscholtz, 1829 (Coleoptera, Elateridae, Agrypninae, Conoderini) detected by standard staining, C-banding, silver nitrate impregnation, and CMA3/DA/DAPI staining.

The speciose Brazilian Elateridae fauna is characterized by high karyotypic diversity, including one species (Chalcolepidius zonatus Eschscholtz, 1829) with the lowest diploid number within any Coleoptera order. Cytogenetic analysis of Conoderus dimidiatus Germar, 1839, C. scalaris (Germar, 1824,) C. ternarius Germar, 1839, and C. stigmosus Germar, 1839 by standard and differential staining was performed with the aim of establishing mechanisms of karyotypic differentiation in these species. Conoderus dimidiatus, C. scalaris, and C. ternarius have diploid numbers of 2n(male) = 17 and 2n(female) = 18, and a X0/XX sex determination system, similar to that encountered in the majority of Conoderini species. The karyotype of C. stigmosus was characterized by a diploid number of 2n = 16 and a neoXY/neoXX sex determination system that was highly differentiated from other species of the genus. Some features of the mitotic and meiotic chromosomes suggest an autosome/ancestral X chromosome fusion as the cause of the neoXY system origin in C. stigmosus. C-banding and silver impregnation techniques showed that the four Conoderus species possess similar chromosomal characteristics to those registered in most Polyphaga species, including pericentromeric C band and autosomal NORs. Triple staining techniques including CMA3/DA/DAPI also provided useful information for differentiating these Conoderus species. These techniques revealed unique GC-rich heterochromatin associated with NORs in C. scalaris and C. stigmosus and CMA3-heteromorphism in C. scalaris and C. ternarius.