http://orcid.org/0000-0003-2189-1546
Figures
Abstract
Ctenidae represents one of the most representative spider families in the tropical forests of Brazil. Its largest genus, Ctenus, has approximately 220 species out of the more than 520 Ctenidae species described, and several authors consider it polyphyletic. Chromosomal data are only available for four species of Ctenus, representing a large gap in the cytogenetic knowledge about the group. This study provided cytogenetic data on two Ctenus species and one Guasuctenus (previously described as Ctenus). All showed 2n♂ = 28 (26+X1X20). Guasuctenus longipes presented two chromosome pairs containing 18S rDNA genes and C. medius, however C. ornatus showed only one chromosome pair with the 18S rDNA gene. Hybridization data using histone H3 probe indicated specific profiles: histone H3 genes were found in one chromosome pair in G. longipes, in three pairs in C. medius, and in four pairs in C. ornatus. Furthermore, supernumerary chromosomes were identified in C. ornatus presenting a meiotic behavior similar to that of sex chromosomes; and a trivalent was found in C. medius, formed by the association of one sex chromosome and an autosomal bivalent, indicating the importance of these events for the diversification of sex chromosomes in spiders. The C-banding pattern was similar between C. medius and C. ornatus with regard to the number and locations of heterochromatic bands, suggesting that heterochromatin amplification and dispersion, affect karyotypic evolution in the genus. Cytogenetic data showed similarity between C. medius and C. ornatus, and differentiation of G. longipes congruent with morphological data. Moreover, although more comparative analyses are needed to specify composition of the dispersed heterochromatin in Ctenus, the mapping of heterochromatic bands provided insights about the evolution of the karyotypes in this genus.
Citation: Rincão MP, Brescovit AD, Dias AL (2020) Insights on repetitive DNA behavior in two species of Ctenus Walckenaer, 1805 and Guasuctenus Polotow and Brescovit, 2019 (Araneae, Ctenidae): Evolutionary profile of H3 histone, 18S rRNA genes and heterochromatin distribution. PLoS ONE 15(4): e0231324. https://doi.org/10.1371/journal.pone.0231324
Editor: Arthur J. Lustig, Tulane University Health Sciences Center, UNITED STATES
Received: September 29, 2019; Accepted: March 20, 2020; Published: April 8, 2020
Copyright: © 2020 Rincão et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This research was supported by a grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – Finance code 001, Fundação Araucária (agreement 001/2017), Fundação Grupo Boticário (agreement 013/2018), and by the CNPq (303028/2014-9 to ADB).
Competing interests: No authors have competing interests.
Introduction
Ctenidae are one of the best-known spider families worldwide and contain species of medical interest, such as those of the genus Phoneutria, popularly known as “banana spider” or “armed spider”. This family has 48 genera and approximately 520 species [1] and is usually found in tropical forests. Based on morphological and molecular data, Ctenidae belong to the superfamily Lycosoidea, together with Oxyopidae, Thomisidae, Psechridae, Trechaleidae, Pisauridae, and Lycosidae [2, 3]; it constitutes the third largest clade of this superfamily.
The genus Ctenus, proposed by Walckenaer in 1805, contains more than 200 currently known species, corresponding to approximately 40% of the number of species in Ctenidae [1], and it is considered polyphyletic [3, 4, 5, 6, 7]. Polotow and Brescovit [7], in a study involving 32 species of Ctenus, showed that for genus to be considered monophyletic, 19 species should be transferred to other genera. Regarding these results, C. longipes was recently transferred to Guasuctenus Polotow and Brescovit [8], that presents today two Neotropical species [1].
Only four species of Ctenus have been karyotyped until date, representing less than 2% of the species described in the genus, indicating a significant gap in the cytogenetic knowledge about the group. Ctenus indicus (Gravely 1931), C. medius Kesselring 1891, C. ornatus (Kesselring 1877), and Ctenus sp. have 28 acrocentric/telocentric chromosomes (2n♂ = 26 + X1X20) [9, 10, 11]. Of these four species, three have data on nucleolar organizer regions (NORs) evidenced by either impregnation with silver nitrate (C. ornatus [9] and C. indicus [10]) or fluorescence in situ hybridization (FISH) (C. ornatus and C. medius [11]). Ctenus indicus is the only species with multiple NORs.
Analysis of heterochromatin distribution was also important for characterization of Ctenus species, since only C. ornatus presented interstitial heterochromatic blocks [11], while the other species of the genus presented centromeric/pericentromeric regions throughout the chromosome complement, with terminal blocks on the long arm of some chromosomes. The latter profile was observed by Rincão et al. [11] in other species of the family, including Enoploctenus cyclothorax (Bertkau 1880), Viracucha andicola (Simon, 1906) and Phoneutria nigriventer (Keyserling, 1891).
The scarcity of chromosomal data in other species of the genus limits establishing evolutionary relationships between karyotypes. The objective of this study was to provide for the first time the cytogenetic description of G. longipes and new populations of C. medius and C. ornatus to investigate, using chromosomal markers, the events that may have occurred during karyotype differentiation in these species and to correlate the results with cytogenetic and phylogenetic data available for this group.
Material and methods
Sample collection
Males and females, adults and juveniles, of three ctenid species, G. longipes, C. medius and C. ornatus, were collected in two conservation units, Parque Estadual Mata dos Godoy (PEMG) (W 51° 14' 31,86", S 23° 26' 29,37") and Parque Nacional do Superagui (PNS) (W 48° 13' 51,9", S 25° 27' 14,40"), state of Paraná, Brazil, as showed in Table 1. Sampling was performed with the permission of Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA/SISBIO) license number 42728–5. The specimens were stored in ethanol 70% and deposited in the arachnological collection of the Laboratório Especial de Coleções Zoológicas, Intituto Butantan (IBSP, curator AD Brescovit), São Paulo/SP, Brazil (S1 Table).
Cytogenetic analyses
Chromosomal preparations followed the protocol described by Araujo et al. [12], using testicles, ovaries and intestinal ducts. To assembly of the slides the tissue was treated and macerated in 60% acetic acid, then the slide was heated in a metal plate at 40° C. The diploid number was determined by counting 30 mitotic or meiotic cells. Chromosomes was classified according to Levan et al. [13]. C-banding followed Sumner [14], with the modifications proposed by Lui et al [15]: slides were stained with propidium iodide and photographed in gray scale. Staining with base-specific fluorochromes Chromomycin A3 (CMA3) and 4’, 6-diamidino-2-phenilindole (DAPI) followed the protocol described by Schweizer [16].
Chromosomal measurements for assembling karyotypes were performed in around 10 mitotic metaphases, using the MicroMeasure software, version 3.3 [17]. Sex chromosome systems were defined by comparing the mitotic metaphase measures and the X1:X2 size ratio in meiosis. For that were measured 30 meiotic cells. To determine the frequency of supernumerary chromosomes were analyzed 30 meiotic and mitotic cells.
Fluorescence In Situ Hybridization (FISH)
FISH was performed according to Schwarzacher and Heslop-Harrison [18], 18S rDNA probes were obtained from Ctenus ornatus by Rincão et al. [11], amplified from the plasmid DNA by PCR with the M13 primer pair, labeled with the Biotin-Nick Translation Mix (Roche) kit and detected with Avidin-FITC (Invitrogen). Histone H3 probes were obtained by amplifying the genomic DNA of C. ornatus, with the aid of primers described by Colgan et al. [19]. The obtained fragments were cloned using the TOPO TA Cloning Kit (Invitrogen), in competent Escherichia coli strain (TOP 10). The insert was amplified by M13 primer pair for sequencing in ABIprism 3500 Genetic Analyzer (Aplied Biosystems), sequences were analyzed using BioEdit version 7.2.5 software [20]. A 370 bp consensus sequence was obtained, which was aligned on GenBank to confirm homology to other previously described histone H3 sequences for spiders. H3 probes were then labeled by PCR, using the M13 primer pair, with the Cy3 dUTP (GE Healthcare).
FISH slides were counterstained with DAPI and analyzed on the Leica DM2000 epifluorescence microscope equipped with a Moticam Pro 282B digital camera. The images were captured using Motic Images Advanced software, version 3.2.
Results
Guasuctenus longipes
Guasuctenus longipes showed 2n = 28 (26 + X1X20) in males (Fig 1A) and 2n = 30 (26 + X1X1X2X2) in females (Fig 1B). All chromosomes were acrocentric and the autosomal complement gradually decreasing in size. The X1:X2 size ratio obtained of meiotic cells showed that X2 sex chromosome corresponds to 79% of the X1 length. Therefore, the X1 and X2 sex chromosomes corresponds, respectively, to the largest and medium-sized chromosomes of the complement (Fig 1A and 1B). FISH technique indicated chromosomal pairs 9 and 11 carrying the 18S rDNA genes and pair 4 carrying the histone H3 genes (Fig 1, boxes), all these sites were present in the terminal region of the long arm of the chromosomes.
Male and female karyotypes of G. longipes with conventional staining Giemsa (a, b, respectively); male karyotype after C banding (c), arrow points to the terminal heterochromatic blocks on the long arm of pair 4. Inset box shows chromosome pairs with 18S rDNA (green) and histone H3 (red). Scale bar = 10 μm.
C-banding technique indicated the presence of heterochromatic blocks in the centromeric region of all chromosomes and smaller blocks in the terminal region of the long arm of pair 4 (Fig 1C). Fluorochrome staining showed regions DAPI- and CMA3+ (Fig 5B), which were coincident with heterochromatic blocks.
Meiotic analyses in G. longipes indicated isopycnotic sex chromosomes in pachytene cells in females (S1A Fig). In males, sex chromosomes showed positive heteropycnosis and parallel association in the early stages of meiosis (S1B–S1D Fig). Metaphase II and anaphase II cells in males showed 13 or 15 chromosomes, confirming the X1X20 sex chromosome system (SCS) (S1E and S1F Fig). In addition, satellites were found in association at the stage of diplotene in homologous and non-homologous chromosomes, of all specimens collected in the Parque Nacional de Superagui (PNS) (S1G and S1H Fig) and coincided with 18S rDNA sequences (pairs 9 and 11) (S1G and S1H Fig -box).
Ctenus medius
Ctenus medius exhibited 2n = 28 (26 + X1X20) in males (Fig 2A) and 2n = 30 (26 + X1X1X2X2) in females (Fig 2B). All chromosomes were acrocentric and the autosomal complement gradually decreasing in size. The X1:X2 size ration obtained of meiotic cells showed that X2 sex chromosome corresponds to 90% of the X1 chromosome length. Therefore, the X1 and X2 sex chromosomes were identified as the largest chromosomes of the diploid set (Fig 2A and 2B). FISH technique indicated chromosome pair 13 carrying 18S rDNA genes and pairs 5, 7 and 9 carrying histone H3 genes (Fig 2, boxes); all sites were present in the terminal region of the long arm of the chromosomes.
Male and female karyotypes of C. medius with conventional staining Giemsa (a, b, respectively); male karyotype after C banding (c) arrowhead points the terminal heterochromatic block on the long arm of one chromosome of the pair 13; two bivalents in pachytene after C-banding, arrows show interstitial heterochromatin in one of them (d). Inset box shows chromosome pairs with 18S rDNA (green) and histone H3 (red). Scale bar = 10 μm.
C-banding analyses showed that large heterochromatic blocks were in the centromeric region of all chromosomes (Fig 2C) and in the terminal region of one chromosome of pair 13. However, cells in the pachytene indicated some bivalent had small heterochromatin dots in interstitial regions (Fig 2D). CMA3 and DAPI staining showed that heterochromatic regions are GC-rich (Fig 5C and 5D).
Meiotic analyses in C. medius indicated isopycnotic sex chromosomes in pachytene cells of females (S2A Fig). In males, sex chromosomes exhibited positive heteropycnosis and parallel association in the early stages of meiosis (S2B–S2D Fig). Metaphases II showed cells with 13 or 15 chromosomes, confirming X1X20 SCS (S2E and S2F Fig). In one specimen from the population of Parque Estadual Mata dos Godoy (PEMG), we observed a trivalent formed from the association between the sex chromosome X2 and an autosomal bivalent (S2G and S2H Fig). C-banding analyses indicated the pairing of an autosomal bivalent with sex chromosome X2 in the short arm (Fig 3A). A graphical representation of the trivalent is shown in Fig 3B.
c = centromere region. (a), arrowheads show sex chromosomes and arrow show an autosomal bivalent which, in association with the X2 sex chromosome forms the trivalent; graphical representation of trivalent formation in diakinesis, showing pairing in the region of short arm between the autosomal bivalent and the X2 sex chromosome, suggesting a possible translocation (b). Scale bar = 10 μm.
Ctenus ornatus
Ctenus ornatus showed 2n = 28 (26 + X1X20) in males (Fig 4A) and 2n = 30 (26 + X1X1X2X2) in females (Fig 4B). All chromosomes were acrocentric and the autosomal complement gradually decreasing in size. The X1:X2 size ratio obtained of meiotic cells showed that X2 sex chromosome corresponds to 75% of the X1 chromosome length. However, additional analyses with C-banding, evidenced the X1 and X2 sex chromosomes as the largest and the smallest chromosomes in the complement (Fig 4A, 4B and 4C). In mitotic metaphase cells, secondary constrictions were observed in the terminal region of the long arm of pair 12 (Fig 4A). FISH technique demonstrated the presence of 18S rDNA genes in the terminal region of the long arm of pair 12 (Fig 4, box), coincident with the secondary constriction (Fig 4A) and the presence of histone H3 genes in pairs 2, 8, 11 and 13, the last two with interstitial markings (Fig 4).
Male and female karyotypes of Ctenus ornatus with conventional staining Giemsa (a, b, respectively); male karyotype after C-banding (C) and staining with fluorochrome CMA3 (d), inset boxes show the sex chromosome pair of the Parque Estadual Mata dos Godoy (c1, d1) and Parque Nacional do Superagui (c2, d2): note the difference in X1 sex chromosome banding pattern between populations. Inset box shows chromosome pairs with 18S rDNA (green) and Histone H3 (red). Scale bar = 10 μm.
C-banding technique evidenced heterochromatic blocks in the centromeric, interstitial and terminal regions of the chromosomes. However, the profile of the X1 sex chromosome differed between the two populations (Fig 4C and 4D). CMA3 and DAPI staining showed the presence of GC-rich regions, CMA3+ and DAPI-, coincident with the C-banding pattern (Figs 4D and 5E–5H).
Meiotic cells after staining with base-specific fluorochromes DAPI (a, c, e, g) and CMA3 (b, d, f, h). Arrowheads point sex chromosomes. Diplotene cells of Guasuctenus longipes (a, b); Metaphase II cells of C. medius (c, d); Pachytene cells (e, f) and diplotene cells (g, h) of C. ornatus: arrows (h) point to interstitials blocks. Scale bar = 10 μm.
Meiotic analyses in C. ornatus evidenced isopycnotic sex chromosomes in pachytene cells of females (S3A Fig). In males, sex chromosomes showed positive heteropycnosis in the early stages of meiosis (S3B and S3C Fig). Metaphases II showed cells with 13 and 15 chromosomes, confirming the X1X20 SCS (S3D Fig). In the PNS population, we observed two specimens with one supernumerary chromosome (S3E–S3G Fig), with approximately 50% of the cells in each one, and one specimen with two supernumerary chromosomes (S3H Fig), in 53% of the cells analyzed. These chromosomes showed positive heteropycnosis (S3E Fig) and behavior similar to the sex chromosomes (S3F and S3H Fig)
An idiogram of the distribution of heterochromatin, 18S rRNA, and histone H3 genes in the karyotype of G. longipes, C. medius, and C ornatus is shown in Fig 6.
Discussion
Karyotypic analysis
The karyotypes of G. longipes, first described, C. medius and C. ornatus showed a conserved pattern, similar to the ancestral karyotype described for the superfamily Lycosoidea [21, 22]. This karyotype with 2n♂ = 26 + X1X20, is present in all Ctenus species and varies only in the size ratio of the sex chromosomes [9, 10, 11, 23]. The divergence about X1:X2 size ratio and the definition of the X2 sex chromosome in C. ornatus, may be explained by higher condensation of the sex chromosomes in meiosis, as discussed by Dolejš et al. [21]. Therefore, the karyotypic structure is well conserved in Ctenus, indicating the need to evaluate other characteristics to differentiate the species of the genus from a cytogenetic point of view.
A common feature in spider karyotype is the presence of secondary constrictions [21, 22, 24], which had been described in Ctenidae only in Nothoctenus sp. and Ctenus sp. [9]. In C. ornatus and G. longipes, secondary constrictions were shown to be associated to 18S rDNA, and this type of association is recurrent in several animal groups, such as the grasshopper Abracris flavolineata De Geer 1773 [25] and the fish Loricariichthys platymetopon Isbrücker and Nijssen, 1979 [26].
Araujo et al. [9] also reported the occurrence of one supernumerary chromosome in a male specimen of C. ornatus with meiotic behavior of univalent similar to that in sex chromosomes. Since this system is recurrent in spiders, the authors proposed that this may be an origin event of X1X2X30 SCS [9, 27, 28, 29]. This same event may be occurring in the cells of the analyzed specimens in this study. And the presence of two supernumerary chromosomes in other specimen may demonstrate the conversion for a X1X2X3X40 SCS in Ctenidae for the first time, thus occurring systems involving neo-X3 and neo-X4 sex chromosomes.
The occurrence of supernumerary can be considered recurrent in C. ornatus because the distance from the region sampled in this study to the region sampled by Araujo et al. [9] was >300 km. However, further analyses are necessary to determine the frequency of these chromosomes in different populations of this species and elucidate the mechanisms involved in its formation.
A very striking feature of the karyotype of all analyzed Lycosoidea species is the difference in the size of the sex chromosomes, which was also observed among the three species analyzed here, in the ratio of size X1: X2. This chromosome variation between species may be the result of translocations between autosomes and sex chromosomes, as discussed earlier by Dolejš et al. [21], for Lycosidae species. Probably, the amount of heterochromatin is not necessarily involved in this polymorphism because in C. ornatus, despite the very evident heterochromatic bands, the sex chromosome X2 was identified as the smallest complement chromosome. Therefore, the presence of a trivalent in a specimen of C. medius from PEMG, involving the association of an autosomal bivalent with a sex chromosome, reinforces the hypothesis of Dolejš et al. [21] that translocations are one of the main mechanisms responsible for the size differentiation of sex chromosomes in this superfamily.
Inferences about the distribution of repetitive DNA in Cteninae
Repetitive DNA sequences in the terminal regions of the chromosomes are common in Ctenidae, including heterochromatic regions and 18S rRNA genes [10, 11]. Dolejs et al. [21] suggest that NORs in the terminal region of the long arm of autosomal chromosomes constitute a plesiomorphic condition in Araneomorphae, which appears to apply to Ctenidae, even in subfamilies such as Acantheinae and Acanthocteninae [11], which have ancestral morphological traits [7].
Guasuctenus longipes (present study), C. indicus [10] and Phoneutria nigriventer [11] were the only species in Cteninae that demonstrated to have more than one chromosomal pair carrying 18S rDNA genes. In this respect, C. medius and C. ornatus had a similar profile, in addition to having an increase in the number of histone H3 genes and heterochromatin distribution; the latter was significant in C. medius owing to the size of the heterochromatic blocks and in C. ornatus because of the number and the distribution profile of these blocks (Fig 6).
Results of a comparative analysis of the most recent cytogenetic and phylogenetic data for Cteninae indicated that the close relationship between C. medius and C. ornatus found in the present study, and the very different karyotype profile for G. longipes (previously described as C. longipes), are well supported by the morphological analysis of Polotow and Brescovit [7], who studied 32 species of Ctenus and concluded that the genus was polyphyletic. These authors indicated that C. medius was grouped in a clade (Clade F) together with C. ornatus, C. vehemens, C. fernandae and C. paubrasil, among other species, whereas G. longipes belonged to a separate clade (Clade K) and was more phylogenetically related to species of the genera Parabatinga and Isoctenus.
The presence of interstitial heterochromatin bands in C. ornatus may appear to be an apomorphic feature of the species, but it is not unique to it. C-banding analyses in pachytene cells of C. medius indicated the presence of a few interstitial heterochromatic bands in some chromosomes, but not in mitotic metaphase, probably because of their high degree of condensation. Moreover, the colocalization of histone H3 and the heterochromatic bands, in specially the interstitial bands in C. ornatus, suggest a direct relationship of evolution between histone genes and heterochromatin in Ctenus.
Therefore, although cytogenetic data are not available for other species of the clade where C. medius and C. ornatus are grouped, it can be concluded that amplification and dispersion events of heterochromatin and repetitive DNA sequences, such as those originated by transposons and ectopic exchanges [30], were significant for the evolutionary history of the karyotypes of both species. The differences in the sex chromosome X1 between two C. ornatus populations suggest that these mechanisms continue to occur. Furthermore, the presence of interstitial histone H3 sites may be evidence that paracentric inversions also played a role in karyotype differentiation in this group of spiders.
This study, besides bringing the first physical mapping data of histone H3 in spiders and the first cytogenetic description in a new genus, shows the potential of chromosomal markers in the karyotype characterization in Araneae. In addition to histone genes, heterochromatic and 18S rDNA profiles showed a similarity between C. medius and C. ornatus, and a differentiation of G. longipes congruent with the morphological data.
Supporting information
S1 Table. Specimens separated by collection sites.
PNS = Parque Nacional de Superagui; PEMG = Parque Estadual Mata dos Godoy. The numbers determine specimen voucher deposited in the arachnological collection of the Laboratório Especial de Coleções Zoológicas, Intituto Butantan (IBSP, curator AD Brescovit), São Paulo/SP, Brazil. (PDF 41 kb).
https://doi.org/10.1371/journal.pone.0231324.s001
(PDF)
S1 Fig.
Female (a) and male (b-h) meiotic cells of G. longipes with conventional staining Giemsa. Arrowheads point sex chromosomes. Pachytene cells (a, b); diplotene (c); diakinesis (d); metaphase II (e); anaphase II (f); diplotene (g, h); arrows show associated satellites between homologous (g) and non-homologous chromosomes (h) in all individuals of the PNS population coinciding with 18S rDNA sites (boxes). (PDF 211 kb).
https://doi.org/10.1371/journal.pone.0231324.s002
(PDF)
S2 Fig.
Female (a) and male (b-h) meiotic cells of C. medius with conventional staining Giemsa. Arrowheads point sex chromosomes. Pachytene cells (a, b); diakinesis cell (c); metaphase I cell (d); metaphases II cells, with 15 and 13 chromosomes (e, f, respectively); diakinesis (g) and pachytene cells (h), showing the trivalent observed in the individual of PEMG. (PDF 1312 kb).
https://doi.org/10.1371/journal.pone.0231324.s003
(PDF)
S3 Fig.
Female (a) and male (b-h) meiotic cells of C. ornatus with conventional staining Giemsa. Arrowheads point sex chromosomes. Pachytene cells (a, b); diakinesis cell (c); metaphases II cells; pachytene, diakinesis and metaphases II cells (e, f, g, respectively, showing one supernumerary chromosome (S), totally heteropicnotic (e); diakinesis cell evidencing two supernumerary chromosomes (S1 and S2) with similar behavior to sex chromosomes. (PDF 215 kb).
https://doi.org/10.1371/journal.pone.0231324.s004
(PDF)
Acknowledgments
The authors thank Robson Rockembacher (UEL) for their assistance with sample collection; the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) and Instituto Ambiental do Paraná (IAP) for the assistance in the Conservation Units sampled.
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