Research Article |
Corresponding author: Maciej Kociński ( kocinski@isez.pan.krakow.pl ) Academic editor: Diogo Cabral-de-Mello
© 2018 Maciej Kociński, Beata Grzywacz, Dragan Chobanov, Elżbieta Warchałowska-Śliwa.
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:
Kociński M, Grzywacz B, Chobanov D, Warchałowska-Śliwa E (2018) New insights into the karyotype evolution of the genus Gampsocleis (Orthoptera, Tettigoniinae, Gampsocleidini). Comparative Cytogenetics 12(4): 529-538. https://doi.org/10.3897/CompCytogen.v12i4.29574
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Five species belonging to the genus Gampsocleis Fieber, 1852 were analyzed using fluorescence in situ hybridization (FISH) with 18S rDNA and telomeric probes, as well as C-banding, DAPI/CMA3 staining and silver impregnation. The studied species showed two distinct karyotypes, with 2n = 31 (male) and 2n = 23 (male) chromosomes. The drastic reduction in chromosome number observed in the latter case suggests multiple translocations and fusions as the main responsible that occurred during chromosome evolution. Two groups of rDNA distribution were found in Gampsocleis representatives analyzed. Group 1, with a single large rDNA cluster on the medium-sized autosome found in four species, carried in the haploid karyotype. Group 2, represented only by G. abbreviata, was characterized by the presence of two rDNA signals. TTAGG telomeric repeats were found at the ends of chromosome arms as expected. The rDNA clusters coincided with active NORs and GC-rich segments.
Orthoptera , Gampsocleis , chromosome evolution, FISH, 18S rDNA, telomeric repeats, Ag-NOR, fluorochrome staining
The Gampsocleis Fieber, 1852 belongs to Gampsocleidini Brunner von Wattenwyl, 1893, a relatively small tribe of Tettigoniinae Krauss, 1902, which includes 17 currently recognized species and 7 subspecies mainly distributed in the Palearctic region (
Changes in chromosome number (karyotype variability) or structure can contribute to speciation (e.g.
The chromosome number (2n) and fundamental number (FN = numbers of chromosome arms) have been described for more than 110 species from 37 genera of Tettigoniinae. Most Palaearctic species have a diploid number of 31 (male) and 32 (female) acrocentric chromosomes with an X0/XX sex chromosome determination system. This karyotype has been suggested to be ancestral/modal for most tettigoniids (
The present study reports the chromosomal characters of five Gampsocleis species using both molecular fluorescent in situ hybridization (FISH), and conventional methods. These data are an initial step towards better understanding of the evolutionary relationships within this genus.
A total of 18 specimens (adults and nymphs) belonging to five Gampsocleis species collected over several years (1990–2016) were selected for the study (Table
Localities of taxa, comparison of chromosome number and chromosomal localization of rDNA clusters, all forming active NOR.
Species | Collection sites and data | Geographical coordinates | No. | 2n male | rDNA-FISH/ NOR |
---|---|---|---|---|---|
Gampsocleis gratiosa Brunner von Wattenwyl, 1862 | China: Beijing area; 1995 | no data | 2 | 31 | 6 |
Gampsocleis sedakovii sedakovii (Fischer von Waldheim, 1846) | Russia: Altai Mts, valley of Edigan River; 1998 | 51.1235N, 86.5149E | 3 | 31 | 6 |
Gampsocleis ussurensis Adelung, 1910 | Korea: near Hamgyong Province, near Chongjin; 1990 | 41.79556N, 129.77583E | 2 | 31 | 6 |
Gampsocleis abbreviata ebneri Uvarov, 1921 | (FYR) Macedonia: Sveti Nikola municipality, Bogoslovec ridge; 2008 | 41.78663N, 22.01893E | 2 | 23 | 5, 8/9 |
Gampsocleis abbreviata renei Miksic, 1973 | Albania: Galichitsa Mts., above Pikina Voda place, above 1600 m; 2013 | 40.91136N, 20.85197E | 1 | ||
Gampsocleis abbreviata ssp. | Greece: Central Greece, Phthiotis, Palaiochori; 2015 | 38.70813N, 22.45736E | 2 | ||
Gampsocleis glabra Herbst, 1786 | Bulgaria: Dobrich, Dobrich; 2006 | 43.60573N, 27.83478E | 2 | 23 | 5 |
Kazakhstan: (1) Aktobe, Safonowka, (2) Shimkent, Gavrilovka | 42.20608N, 70.21833E | 3 | |||
(3) Almaty, Uzunbylack; 2016 | 43.20317N, 78.98846E | 1 |
Fluorescence in situ hybridization (FISH) was performed as described by
The distribution of heterochromatin was revealed by C-banding techniques, as described by
We observed two different karyotypes with the sex determination system X0 in males of five species of the genus Gampsocleis (Table
Examples of Gampsocleis species with 2n = 31 chromosomes (male): G. s. sedakovii (a, c–e) and G. ussuriensis (b) studied using different techniques: FISH with both 18S rDNA (green) and telomeric TTAGG (red) probes (a) in diakinesis revealed a single rDNA locus located distally on the 6th bivalent (white arrow) and one active NOR visualized by AgNO3 staining (b) in diplotene (black arrow). C-banding (c) as well as fluorochrome staining of heterochromatin with DAPI (blue) and CMA3 (green) bands in diakinesis (d and e, respectively); black arrows indicate a C-band, and white arrows indicate very weak DAPI+ and bright CMA3+ signals located in a distal region on the 6th bivalent. C (a–c), centromere; X (c–e), sex chromosome. Scale bar: 10 µm.
Gampsocleis species with 2n = 23 chromosomes (male): G. glabra (a–e) and G. abbreviata (f–j) studied using different techniques: FISH using 18S rDNA (green) and telomeric TTAGG (red) probes (a, f) and silver staining in diakinesis (b,g), C-banding of spermatogonial metaphase (c) and diplotene (h), and fluorochrome staining of heterochromatin with DAPI (blue) and CMA3 (green) (d, i, e, j). Arrows indicate rDNA clusters located near the telomeric region on the 5th bivalent (a, f) and in a telomeric position on the short bivalent (f); active NORs co-localized with rDNA (b, g, black arrows); thin C-bands (c, h, black arrows) and weak DAPI+ (d, i, white arrows) and bright CMA3+ signals located near the telomeric region on the medium-sized bivalent (e, j, white arrows) as well DAPI-/CMA3+ signals on the telomeric region of the short bivalent (i,j, white arrows). Bi-armed chromosomes are marked by asterisks (b). X, sex chromosome. Scale bar: 10 µm.
The localization of 18S rDNA in Gampsocleis was revealed by FISH and its activity analyzed by silver impregnation is summarized in Table
After both C-banding and DAPI/CMA3 double staining, chromosome regions in the analyzed species showed discrete quantitative and qualitative variation in their constitutive heterochromatin. In G. s. sedakovii, G. s. obscura, G. glabra, and G. ussuriensis paracentromeric C-bands was uniformly present in long and medium-sized chromosomes, distal and interstitial bands are found to vary in size between these species, as described previously (
Our results are in line with previous studies (for a review see
In cytogenetic studies, the application of a variety of staining methods (classical and molecular) generally enables a better characterization of tettigoniid karyotypes and identification of genus/species-specific patterns (
Discrete quantitative and qualitative differences in constitutive heterochromatin were discovered in the chromosomes of the analyzed species after both C-banding and DAPI/CMA3 double staining. The constitutive heterochromatin of all species analyzed was located in the paracentromeric and distal regions in some chromosomes and differed in size between species; similar observations were reported in previous studies of Gampsocleidini (
Previous data (
Species of Gampsocleis can be assigned into two groups distinguished by both the chromosome number and geographic range, in accordance with previous studies (
In conclusion, the present study offers new insights into the karyotype characteristics of bushcrickets that may be useful for interpret or understand relationships within the genus Gampsocleis as well as the subfamily Tettigoniinae. Changes observed in karyotypes may probably also play an important role in speciation. Additional species and methods (morphological and genetic characters) should be examined in order to further elucidate the relationships within the genus Gampsocleis and the tribe Gampsocleidini.
This work was supported by the Ministry of Science and Higher Education carried via the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences “Grant to Young Researchers”; decision number 6085/E-47/M/2017.