Cytogenetic analysis of three Ctenidae species (Araneae) from the Amazon

Abstract Cytogenetic characterization was performed on three wandering spiders: Ctenus amphora Mello-Leitão, 1930, C. crulsi Mello-Leitão, 1930 and C. villasboasi Mello-Leitão, 1949. The three species had similar karyotypes, with 2n = 28 (26 + X1X20) in males, with sex chromosomes exhibiting positive heteropicnosis in meiotic cells. 18S rDNA mapping revealed gene sites at the terminal region of one chromosomal pair for all species, with one C. crulsi individual, showing markings in two pairs. C. villasboasi showed markers only in the pachytene phase. The distribution pattern of constitutive heterochromatin was found to be characteristic for the genus, with markings in the centromeric region of all chromosomes, suggesting an acrocentric morphology for all chromosomes of the three analysed species. The results support the fusion of sex chromosomes as an evolutionary tendency for this spider group.

The order Araneae currently contains 48,455 described species, distributed in 120 families (World Spider Catalog, 2020). In Brazil, there are records of 3,203 species, of which 694 occur in the Amazon Region (Brescovit et al., 2011). Within this group, we focus here on the Ctenidae family, a group which has received increasing attention due to their value as bioindicators of environmental quality, and the use of their venom neurotoxins as therapeutic agents (Rego et al., 2005;Mestre and Gasnier, 2008;Pinheiro et al., 2009).
More than 868 species of spiders have already been characterized cytogenetically and, of these, 12 belong to the family Ctenidae (Araujo et al., 2020). The data for this family show a diploid number varying between 22 and 29 chromosomes for males, and X 1 X 2 0 and X 1 X 2 X 3 0 sex chromosome systems (Araujo et al., 2014). While these two systems are rare overall, they are common in spiders (Araujo et al., 2012), including the Ctenidae, making it possible, in this Family, to study the sex chromosome behavior during meiosis.
In Ctenidae, with the exception of Asthenoctenus borellii Simon, 1897, whose diploid number is 22, all species have 26 autosomal chromosomes plus two or three sex chromosomes (Araujo et al., 2020). Araujo et al. (2014) sug-gest that the conversion of the sexual chromosomal system X 1 X 2 0 to X 1 X 2 X 3 0, and vice-versa, is a relatively common event. However, available cytogenetic data are currently insufficient to allow inferences concerning evolutionary chromosomal tendencies within the group.
The aim of the current study is to increase the cytogenetic knowledge of the Ctenidae, and so contribute to the discussion concerning mechanisms of chromosome evolution in this family, especially regarding the behavior of sexual chromosomal systems X 1 X 2 0 and X 1 X 2 X 3 0, in males during meiosis.
In C. amphora (Figure 2A), C. crulsi ( Figure 2B) and C. villasboasi ( Figure 2C) 2n = 28, with 26 of the chromosomes being autosomal and two sexual. In pachytene ( Figure  3A, D, G) and dipotene ( Figure 3B, E, H) phase cells the sex chromosomes showed positive heteropicnosis, thus allowing their identification. Determination of the sex chromosomal system was performed by analyzing chromosome segrega-tion during metaphase II of meiosis ( Figure 3C, F, I), which shows nuclei with n = 13 and n = 15 (13 + X 1 X 2 ).
The FISH technique revealed 18S rDNA sites on pair 10 of the chromosomes in the spermatogonial metaphase of C. amphora and C. crulsi (Figure 2A, B). In addition, a C. crulsi individual from the Ducke Reserve was polymorphic compared to the other seven analyzed for this same locus, with four labeled chromosomes (Pairs 4 and 7) in spermatogonial metaphases ( Figure 2B -highlighted). Ctenus villasboasi showed marking on a pair of chromosomes in metaphase ( Figure 2C). However, chromosomal size visualized with this technique was insufficient to allow determination of which pair carried the 18S rDNA site.
C-banding showed the presence of constitutive heterochromatin in the centromeric region of all chromosomes, characterizing the morphology as acrocentric for all three species ( Figure 4A-C). Silver nitrate impregnation revealed NORs on pair 10 in the spermatogonial metaphases for C. amphora and C. crulsi (most individuals) (Figure 2A, B). C. villasboasi had no visible markings in the spermatogonial metaphases, but exhibited marking on one pachytene bivalent ( Figure 4D).
The karyotypic formula of males (2n=28 -26+X 1 X 2 0), found for all species, has been shown to be conserved in Ctenus (Araujo et al., 2020). Identification of sexual chromosomes in meiotic cells was facilitated by their high degree of condensation and positive heteropicnosis. The literature contains contrasting opinions regarding Ctenidae chromosomal morphology, with Araujo et al. (2014) considering them to be telocentric, while Kumar et al. (2017) and Rincão et al. (2017) consider that they are acrocentric. We considered the three species studied here to be acrocentric, based on C-banding, which showed markings in the centromeric regions of all chromosomes.
All three species showed metaphase II meiotic cells with n = 13 and n = 15 (13+X 1 X 2 ), which agrees with the information given by Araujo et al. (2014) and Rincão et al. (2017) for Ctenidae species. This feature is common for a X 1 X 2 0 sex chromosomal system, in which for males, at the end of meiosis I, two sex chromosomes migrate to the same pole cell. Araújo et al. (2012) collated proposed spider chromosomal evolution theories, which include the X0 sexual chromosomal system giving rise to the X 1 X 2 0 (White, 1940;Pätau, 1948), via part of an X chromosome fissioning and attaching to a supernumerary chromosome (Bole-Gowda, 1950), and the sexual chromosomal system X 1 X 2 X 3 0 giving rise to X 1 X 2 0 via fusion of two X chromosomes (Král, 2007).
Taking into account the cytogenetic characterization of species considered basal within the Ctenidae, such as Nothroctenus sp. and Viracucha andicola Simon, 1906 (Polotow and, and the data presented here for Ctenidae species occuping positions considered to be derived, we believe the diploid number reduction hypotheses based on chromosome fusion to be more parsimonious, since the first group has 2n = 29 and the second group has 2n = 28.  However, Rincão et al. (2020) recently found two individuals of C. ornatus showing one supernumerary chromosome and one individual with two supernumerary chromosomes. Those chromosomes showed positive heteropicnosis and behavior similar to sex chromosomes, which the author states may demonstrate conversion of sexual chromosomal system X 1 X 2 0 to X 1 X 2 X 3 X 4 0 for the first time in Ctenidae.
The FISH-obtained 18S rDNA tags for the three species in our study confirmed Ag-NOR derived data, and were similar to those described for C. ornatus and C. medius by Rincão et al. (2017). However, an individual of C. crulsi in the current study had two pairs of chromosomes with 18S rDNA labeling (Pairs 4 and 7). These additional markers were shown to be relatively minor when compared to that Cytogenetics of Ctenidae spiders 3 found in the other Ctenus species of the current study, as well as those available in the literature. Such data suggest that these alterations can be caused by chromosomal rearrangements, insertions by transposable elements or ectopic recombination, all processes that could, potentially, be involved in karyotypic differentiation and new species emergence (Stáhlavsky et al., 2018).
Considering the number of chromosomes with 18S rDNA in C. crulsi, we believe the translocation hypothesis to be the most feasible, since this mechanism of chromosome evolution depends on the interchange of segments between two non-homologous chromosomes without loss of genetic material (Gross et al., 2010). Such translocations can be simple, when only the segment of one chromosome passes to the other, or reciprocal, when two chromosomes exchange segments with each other. The translocation model for rDNA is described by Araujo et al. (2015) for the exchange of such segments between autosomal and sexual chromosomes in the genus Nephila. This shows that a possible translocation between the two types of chromosomes is possible. In the C. crulsi metaphases analyzed here, the sex chromosomes were not evident.
Silver nitrate impregnation showed NOR markers on an autosomal pair for C. amphora and C. crulsi (most individuals), and on a pachytene bivalent for C. villasboasi, a result similar to that found for C. ornatus Keyserling, 1877 (Araujo et al., 2014;Rincão et al., 2020), and C. medius (Rincão et al., 2020), but which differs from those reported for C. indicus Gravely, 1931, by Kumar et al. (2017), and for G. longipes by Rincão et al. (2020), who found NOR markings on two chromosome pairs. The NOR distribution pattern is currently known for only five species in the genus Ctenus. Therefore, we suggest that additional cytogenetic studies are still needed to establish the plesiomorphic characteristics of the Ctenidae karyotype and, thus, to be able to understand the mechanisms of chromosomal evolution that occurred in this group of spiders.
The pattern of constitutive heterochromatin distribution reported here for C. amphora, C. crulsi and C. villasboasi is similar to those found by Rincão et al. (2017) for C. medius Keyserling, 1891, E. cyclothorax Bertkau, 1880, P. nigriventer Keyserling, 1891 and V. andicola, with blocks in the centromeric regions of all the chromosomes.
According to Sumner (1972), C-banding marks centromeric and telomeric regions, possibly marking nucleolar regions and, rarely, intercalated regions of the chromosome. In the metaphases studied here, the sex chromosomes were not totally heterochromatic, thus confirming heteropycnosis of the sex chromosomes in meiosis. Heterochromatic chromosomes remain condensed throughout the cell cycle, whereas heteropycnotic chromosomes may have a higher or lower level of condensation, depending on the stage of cell division in which they are found (John, 1990).
As a result of the current study, the number of Ctenidae species with chromosomal data has been increased to 14. The data obtained for C. amphora, C. crulsi and C. villasboasi extends to seven the number of species analyzed from the genus Ctenus, and so allow a conserved karyotype to be 4 Pinto Neto et al. infered, as well as the diploid number, chromosomal formula and sex chromosome system plus mapping of the rDNA sites, constitutive of sex chromosomes in meiotic cells in pachytene and diplotene phases is a common feature for these species (Araújo et al., 2012). The results also reinforce theories of chromosome fusion as a possible evolutionary tendency in this family.