Research Article |
Corresponding author: Renata da Rosa ( renata-darosa@uel.br ) Academic editor: Vladimir Gokhman
© 2015 Marília de França Rocha, Mariana Bozina Pine, Elizabeth Felipe Alves dos Santos Oliveira, Vilma Loreto, Raquel Bozini Gallo, Carlos Roberto Maximiano da Silva, Fernando Campos de Domenico, Renata da Rosa.
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.
Citation:
Rocha MF, Pine MB, dos Santos Oliveira EFA, Loreto V, Gallo RB, da Silva CRM, de Domenico FC, da Rosa R (2015) Spreading of heterochromatin and karyotype differentiation in two Tropidacris Scudder, 1869 species (Orthoptera, Romaleidae). Comparative Cytogenetics 9(3): 435-450. https://doi.org/10.3897/CompCytogen.v9i3.5160
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Tropidacris Scudder, 1869 is a genus widely distributed throughout the Neotropical region where speciation was probably promoted by forest reduction during the glacial and interglacial periods. There are no cytogenetic studies of Tropidacris, and information allowing inference or confirmation of the evolutionary events involved in speciation within the group is insufficient. In this paper, we used cytogenetic markers in two species, T. collaris (Stoll, 1813) and T. cristata grandis (Thunberg, 1824), collected in different Brazilian biomes. Both species exhibited 2n=24,XX for females and 2n=23,X0 for males. All chromosomes were acrocentric. There were some differences in the karyotype macrostructure, e.g. in the chromosome size. A wide interspecific variation in the chromosome banding (C-banding and CMA3/DAPI staining) indicated strong differences in the distribution of repetitive DNA sequences. Specifically, T. cristata grandis had a higher number of bands in relation to T. collaris. FISH with 18S rDNA revealed two markings coinciding with the NORs in both species. However, two analyzed samples of T. collaris revealed a heterozygous condition for the rDNA site of S10 pair. In T. collaris, the histone H3 genes were distributed on three chromosome pairs, whereas in T. cristata grandis, these genes were observed on 14 autosomes and on the X chromosome, always in terminal regions. Our results demonstrate that, although the chromosome number and morphology are conserved in the genus, T. cristata grandis substantially differs from T. collaris in terms of the distribution of repetitive sequences. The devastation and fragmentation of the Brazilian rainforest may have led to isolation between these species, and the spreading of these repetitive sequences could contribute to speciation within the genus.
Chromosome banding, repetitive DNA, speciation, histone H3 gene, 18S rDNA
The genus Tropidacris Scudder, 1869 comprises the largest grasshoppers of the order Orthoptera, reaching up to 14 centimeters in length (
Tropidacris is widely spread throughout the Neotropical region. Its natural habitats extensively vary from dense rainforests to very open areas with a dry climate (
Considering the geographical distribution of the above-mentioned taxa,
In different organisms, speciation is related to important chromosomal rearrangements. Translocations, inversions, duplications and deletions can lead to chromosome segregation problems, causing different degrees of sterility. Karyotype changes, such as amplification or dispersion of repetitive DNA sequences may also have an important role in this process. These structural rearrangements lead to reproductive barriers and thus to the formation of new biological species (
There are no specific cytogenetic studies of Tropidacris, and information allowing inference or confirmation of the evolutionary events involved in speciation in the group is insufficient. Thus, we used cytogenetic markers to analyze T. collaris and T. cristata grandis, two members of the genus collected in different biomes of northeastern and southern Brazil. We intend to propose a mechanism that explains both the chromosome evolution and reproductive isolation between these taxa.
The specimens of T. collaris and T. cristata grandis were collected from two regions of Brazil (Table
a map of Brazil showing collection sites in northeastern and southern Brazil b Tropidacris collaris c Tropidacris cristata grandis.
Species | Number of specimens | Collection sites |
---|---|---|
Tropidacris collaris | 4♀, 8♂ 4♀, 7♂ |
Refúgio Ecológico Charles Darwin, Igarassu, Pernambuco, Brazil 08°03.00'S, 35°13.00"W (DMS) Gurjaú, Cabo de Santo Agostinho, Pernambuco, Brazil 08°10'00"S, 35°05'00"W (DMS) |
Tropidacris cristata grandis | 5♀, 10♂ | Iguaçu National Park, Foz do Iguaçu, Paraná, Brazil 25°37’40.67"S, 54°27’45.29"W (DMS) |
The samples were anesthetized and dissected before fixing their testes and gastric caeca in methanol: acetic acid 3:1. The females were injected with 0.1% colchicine 6h prior to dissection. For mitotic and meiotic analyses, air-dried chromosome preparations were made from tissues macerated in one drop of 2% lacto-acetic orcein. For banding techniques and FISH, squashed preparations with 45% acetic acid were made, and then coverslips were removed after freezing the preparations by immersion in liquid nitrogen for a few seconds.
The distribution of heterochromatin was analyzed with Giemsa C-banding after treatments with 0.2M HCl for 15 min at 25 °C, 5% Ba(OH)2 at 60 °C in a waterbath for 1 min and 2×SSC for 30 min at 60 °C (
In addition to the karyotype studies, genomic DNA from one male of each species was extracted from the muscle tissue sample. After proteinase K (20 mg/ml) digestion for three hours at 65 °C, phenol/Tris-HCl (pH 8.0) was added, followed by centrifugation and washing with phenol/Tris-HCl (pH 8.0) and chloroform-isoamyl alcohol. After an additional centrifugation, chloroform-isoamyl alcohol was added. Then, DNA was precipitated with absolute ethanol for 12 hours at –20 °C and eluted in TE 1/10 + RNAse.
Unlabeled 18S rDNA and histone H3 gene probes were generated by polymerase chain reaction (PCR) using the following primers: 18S rDNAF 5‘-CCTGA GAAACGGCTACCACATC-3’ and 18S rDNAR 5‘-GAGTCTCGTTCGTTATCGGA-3’ (
All images were acquired with a Leica DM 4500 B microscope equipped with a DFC 300FX camera and Leica IM50 4.0 software, and optimized for best contrast and brightness with iGrafx Image software.
The analysis of mitotic and meiotic chromosomes of T. collaris revealed 2n=24, XX (Fig.
Karyotypes of the studied species. a female karyotype of T. collaris, conventional staining b male karyotype of T. collaris, C-banding c female karyotype of T. cristata grandis, conventional staining d male karyotype of T. cristata grandis, C-banding. Arrows indicate interstitial bands. Bar = 10 µm.
Meiotic stages of T. collaris. a pachytene b diakinesis c C-banding d CMA3 staining e DAPI staining. Arrows indicate C+ subterminal blocks in larger pairs and in M4. Arrowhead shows the heterozygous form. Bar = 10 µm.
Heterochromatic blocks revealed by C-banding were located in the pericentromeric regions of all chromosomes and M4 showed the largest heterochromatic block. The medium-sized chromosomes carried small distal blocks, except for M8 without distal blocks, and the X chromosome with a large distal block (Fig.
The triple staining CMA3/DA/DAPI revealed CMA3+ blocks on the bivalents M4, S10 and S11, the first block being of lower intensity (Fig.
Fluorescence in situ hybridization (FISH) revealed two markings in S10 and S11 coinciding with the NORs (Fig.
All samples of T. cristata grandis exhibited 2n=23 in males and 2n=24 in females, featuring a sex chromosome system of the X0/XX type (Fig.
The analysis of meiotic cells in males revealed eleven bivalents corresponding to the autosomes at the pachytene stage; one positively heteropycnotic univalent (the X chromosome); and a structure in a particular bivalent pointing to a secondary constriction (Fig.
Meiotic stages of T. cristata grandis. a pachytene with positively heteropycnotic X chromosome b diplotene/diakinesis c two daughter cells at anaphase I with visible X chromosome in one of them d C-banding with pericentromeric and terminal blocks of heterochromatin in most chromosomes and positively heteropycnotic X chromosome e CMA3 staining f DAPI staining. Arrows and arrowheads indicate secondary constrictions and the megameric chromosome respectively. Bar = 10 µm.
Heterochromatin revealed by C-banding was observed mainly as pericentromeric bands in all chromosomes. In pairs L1, L2, M4, M8, S10 and S11, these bands were small while they were more developed in the other pairs. Terminal heterochromatic bands were observed on the long arm of pairs L1, L2 and M3. However, a heteromorphic pair was found in two specimens, where one of the L1 homologues did not carry this heterochromatic band. Furthermore, two pairs showed discrete bands on the long arm, one distal on pair L2 and another (proximal) on the X chromosome (Fig.
At metaphase II, fluorochrome staining showed terminal GC-rich blocks on L1 and L2 pairs and on three other chromosomes with CMA3+ blocks in the proximal regions (Fig.
Active NORs were found in one or two bivalents at pachytene (Fig.
Mitotic and meiotic cells of T. collaris (a, b, c) and T. cristata grandis (d, e, f). a, d silver nitrate impregnation b, e FISH with 18S rDNA probe c, f FISH with histone H3 gene probe. Black and white arrows, arrowheads and asterisk indicate Ag-NOR bands, rDNA sites, pericentromeric regions and chromosome pair no. 10 respectively (heterozygous condition shown in the box). Bar = 10 µm.
The observed diploid number (2n♂=23, X0) and the overall structure of the karyotype containing only acrocentric chromosomes were identical in the two species. These karyotypes are similar to those reported for most species of Romaleidae (
Although T. collaris and T. cristata grandis have the same chromosome number, they can differ in the karyotype structure. Both species have two pairs of large chromosomes. However, the karyotype of T. collaris contains six pairs of medium-sized chromosomes (M3-M8) and three small pairs (S9-S11). On the other hand, T. cristata grandis has seven medium-sized pairs (M3-M9) and two small pairs (S10-S11). Furthermore, the conventional analysis of meiocytes revealed two other differences between these species. Specifically, T. cristata grandis has a large chromosome with a secondary constriction, and a megameric chromosome. Grasshoppers of the family Romaleidae have conserved karyotypes, i.e., they reveal extensive uniformity in the chromosome number and chromosomal morphology. However, we found an extensive interspecific variability as regards chromosome banding in the species studied, which indicates a wide variation in the distribution of repetitive DNA sequences. Such variation was also observed in several other members of the group (
T. collaris and T. cristata grandis have pericentromeric C-bands in all chromosomes. However, we also noted some differences in the distribution of other heterochromatic blocks (Figs
Allopatric speciation usually occurs after the relatively long geographical isolation between populations. Consequently, these populations accumulate genetic differences that can cause reproductive incompatibilities (
Other differences were found between the two species with respect to the base content of DNA that constitutes heterochromatin. CMA3+ blocks (GC-rich) were observed in three chromosomes of T. collaris, while T. cristata grandis showed a higher number of bands. These results indicate that, in addition to the expansion of the heterochromatin, its composition has also been modified in terms of base pairs. The occurrence of DAPI+ heterochromatin (AT-rich) in T. cristata grandis also differentiates it from T. collaris, since the karyotype of the latter species does not have AT-rich regions. The same is true for the various members of this family studied to date (
Most studies of histone H3 genes in grasshoppers reveal a localization of these sequences on a single pair of chromosomes (
The wide range of variation in heterochromatin among the samples of the two species, in addition to the distribution of GC-rich blocks and co-localization with H3 histone genes in T. cristata grandis, is an exceptional feature in grasshoppers. Heterochromatin has the ability to spread to different regions and influence gene expression, leading to silencing of some genes and preventing recombination between them (
The NORs were observed in two chromosome pairs in both species, although these pairs are different. While T. collaris has NORs on two small pairs, these structures were observed on medium-sized chromosomes of T. cristata grandis.
The data presented in this study demonstrate karyotype conservation regarding the chromosome number and morphology in both species of Tropidacris when compared to other species of Romaleidae. However, they indicate that T. cristata grandis has an extremely diverse karyotype in terms of the presence and distribution of heterochromatic blocks and differentiation in the localization of histone H3 genes, showing karyotypic differences from T. collaris. While T. collaris is widely distributed in Brazil, T. cristata grandis has a restricted geographical distribution within isolated fragments of the rainforest. Originally, the Atlantic Forest extended along almost the whole east coast of Brazil, with extensive incursions into the inner parts of the country (
The authors are grateful to the Iguaçu National Park staff for their technical assistance and to Edson Mendes Francisco for his help in collecting the samples. The Instituto Brasileiro do Meio Ambiente e dos Recursos Renováveis (IBAMA), Instituto Chico Mendes de Conservação da Biodiversidade - ICMBio, protocol number 31946-2 issued authorizations for the collection. This work was supported by Universidade Estadual de Londrina.