Research Article |
Corresponding author: Elżbieta Warchałowska-Śliwa ( warchalowska@isez.pan.krakow.pl ) Academic editor: Diogo Cabral-de-Mello
© 2017 Elżbieta Warchałowska-Śliwa, Beata Grzywacz, Klaus-Gerhard Heller, Dragan P. Chobanov.
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:
Warchałowska-Śliwa E, Grzywacz B, Heller K-G, Chobanov DP (2017) Comparative analysis of chromosomes in the Palaearctic bush-crickets of tribe Pholidopterini (Orthoptera, Tettigoniinae). Comparative Cytogenetics 11(2): 309-324. https://doi.org/10.3897/CompCytogen.v11i2.12070
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The present study focused on the evolution of the karyotype in four genera of the tribe Pholidopterini: Eupholidoptera Mařan, 1953, Parapholidoptera Mařan, 1953, Pholidoptera Wesmaël, 1838, Uvarovistia Mařan, 1953. Chromosomes were analyzed using fluorescence in situ hybridization (FISH) with 18S rDNA and (TTAGG)n telomeric probes, and classical techniques, such as C-banding, silver impregnation and fluorochrome DAPI/CMA3 staining. Most species retained the ancestral diploid chromosome number 2n = 31 (male) or 32 (female), while some of the taxa, especially a group of species within genus Pholidoptera, evolved a reduced chromosome number 2n = 29. All species show the same sex determination system X0/XX. In some taxa, a pericentric inversion has changed the morphology of the ancestral acrocentric X chromosome to the biarmed X. The rDNA loci coincided with active NORs and C-band/CG-rich segments. A comparison of the location of the single rDNA/NOR in the genus Pholidoptera suggests that reduced chromosome number results from Robertsonian translocation between two pairs of autosomes, one carrying the rDNA/NOR. The results constitute a step towards better understanding of the chromosomal reorganization and evolution within the tribe Phaneropterini and the whole subfamily Tettigoniinae.
Orthoptera , Pholidopterini , karyotype, FISH, 18S rDNA, telomeric DNA, NOR, C-banding, fluorochrome staining
Tettigoniinae Krauss, 1902 is amongst the largest groups of Tettigoniidae (also known as the bush-crickets or the katydids) with over 500 species occurring over five continents with the exception of the equatorial and subequatorial climatic zones (
The diploid chromosome number (2n), chromosome morphology (FN), and type of sex determination systems of Tettigoniinae have been described for more than 100 species out of 35 genera. Most of the Old World species have 31 acrocentric chromosomes, but the few New World genera diverge from this standard karyotype with one or more pairs of metacentric autosomes and often metacentric X chromosome. Hence, the karyotypes in this group range from 23 to 33 in male (reviewed in
Molecular cytogenetic methods through mapping molecular markers such as repetitive or unique sequences on chromosomes have been successfully applied for interspecific comparative karyotype structure and evolution in insects, especially coleopterans (e.g.
Until now, the karyotypes of 16 species/subspecies of Pholidopterini, occasionally examined using C-banding and NOR staining have been studied (
A total of 74 specimens of the genera Eupholidoptera, Parapholidoptera, Pholidoptera and Uvarovistia (Pholidopterini), belonging to 38 species/subspecies were studied cytologically. Table
The best chromosome preparations were used for fluorescence in situ hybridization (FISH) with 18S ribosomal DNA (rDNA) and telomeric DNA (TTAGG)n. FISH was carried out as described earlier in
Pholidopterini taxa: collection sites, comparison of the number and morphology of chromosomes, distribution of rDNA cluster and NOR.
Species | Collection sites | Geographical coordinates | No. | 2n, FN | References | rDNA-NOR |
---|---|---|---|---|---|---|
Eupholidopteraanatolica (Ramme, 1930) | TR: Antalya, Termessos | 36°58'N, 30°30'E | 2m | 31,31 |
|
no |
Eupholidoptera annulipes (Brunner von Wattenwyl, 1882) | TR: Mersin, below Güzeloluk | 36°44'N, 34°9'E | 2m | 31,31 |
|
no |
Eupholidoptera astyla (Ramme, 1927) | GR: 1) Crete, Rethimni, Mt. Ida, spring of Skaronero, above Kamares | 1) 35°10'N, 24°48'E | 1m | 31,31 | this study | 3/4i-3/4i |
2) Crete, Rethimini, Skaleta | 2) 35°24'N, 24°37'E | 1m | ||||
Eupholidoptera epirotica (Ramme, 1927) | GR: Aitolia-Akarnania (Central Greece), Mt. Karnania above Thirion | 38°48'N, 20°58'E | 6m, 1f | 31,31 |
|
no-3/4i |
Eupholidoptera chabrieri garganica (La Greca, 1959) | GR: Kerkyra, near Agios Spiridon | 39°48'N, 19°50'E | 3m | 31,31 |
|
no |
Eupholidoptera giuliae Massa, 1999 |
GR: Crete, Chania, Chora Sfakion | 35°12'N, 24°8'E | 3m | 31,31 | this study | no |
Eupholidoptera icariensis Willemse, 1980 | GR: Samos, Ikaria | 37°36'N, 26°9'E | 1m | 31,31 |
|
no |
Eupholidoptera karabagi Salman, 1983 | GR: Antalya, Termessos | 36°58'N, 30°30'E | 3m | 31,31 |
|
no-3/4i |
Eupholidoptera krueperi (Ramme, 1930) | TR: Antalya, Kozdere Köprü | 36°36'N, 30°31'E | 1m | 31,31 | this study | 3/4i-3/4i |
Eupholidoptera latens Willemse & Kruseman, 1976) | GR: Crete, Chania, Rodopos | 35°33'N, 23°45'E | 2m | 31,31 | this study | no |
Eupholidoptera megastyla (Ramme, 1939) | GR: 1) Myrsini, Pd Peloponez | 39°25'N, 21°10'E | 4 m | 31,31 | this study | 3/4i-3/4i |
2) Arta, Tsoumerka Mt. | 2m |
|
no | |||
Eupholidoptera mersinensis Salman, 1983 | TR: 1) Mersin, Fýndýkpýnarý | 1) 36°50'N, 34°20'E | 1m | 31,31 |
|
no 3/4i |
2) Mersin, below Güzeloluk (near Köserlý) | 2) 36°45'N, 34°7'E | 1m | ||||
Eupholidoptera prasina (Brunner von Wattenwyl, 1882) | TR: Mersin, Kasyayla (above Anamur) | 36°15'N, 32°54'E | 5m | 31,31 |
|
no |
Eupholidoptera schmidti (Fieber, 1861) | MK: 1) Gorna Belica | 1) 41°13'N, 20°33'E | 2m | 31,31 | this study | 3/4i-3/4i |
2) Korab Mt. | 2) 41°41'N, 20°39'E | 1m | ||||
Eupholidoptera smyrnensis (Brunner von Wattenwyl, 1882) | 1) TR: N Sindrigi | 1) 39°18'N, 28°12'E | 1m | 31,31 | this study | 3/4i-3/4i |
2) BG: Melnik | 2) 41°31'N, 23°23'E | 2m | ||||
Eupholidoptera tauricola (Ramme, 1930) | TR: Mersin, below Güzeloluk | 36°44'N, 34°9'E | 2m | 31,31 |
|
no |
Eupholidoptera tahtalica (Uvarov, 1949) | TR: Aspendos close Antalya | 36°56'N, 31°10'E | 1m | 31,31 | this study | 3/4i-3/4i |
Parapholidoptera castaneoviridis (Brunner von Wattenwyl, 1882) |
BG: 1) Stara Planina Mts, Karandila site | 1) 42°43'N, 26°22'E | 2m | 31,32 | this study | 3/4i-3/4i |
2) Strandzha Mts, Kovach place | 2) 42°05'N, 27°25'E | 1m | ||||
Parapholidoptera cf. belen Ünal, 2006 | TR: Yiglica | 40°58'N, 31°34'E | 1m | 31,31 | this study | 3/4i-3/4i |
Parapholidoptera distincta (Uvarov, 1921) | GE: E Nailevi | 41°38'N, 42°35'E | 2m | 31,31 +B |
this study | 3/4i-3/4i |
Parapholidoptera noxia (Ramme, 1930) | GE: 1) Gombori range | 1) 41°52'N, 45°18'E | 1m | 31,31 | this study | 3/4i-3/4i |
2) SW of Gora vill. | 2) 41°14'N, 44°17'E | 2m | ||||
Parapholidoptera grandis (Karabag, 1952) | TR: E Ibradi | 37°03'N, 31°44'E | 4m | 31,32 | this study | 3/4i-3/4i* |
Parapholidoptera signata (Brunner von Wattenwyl, 1861) | TR: Mersin, below Güzeloluk | 36°44'N, 34°9'E | 1m | 31,31 |
|
no |
Parapholidoptera cf. signata | TR: above Zara | 39°37'N, 37°56'E | 1m | 31,31 | this study | no |
Parapholidoptera aff. syriaca | TR: Demre | 36°27'N, 30°26'E | 3m | 29,31 | this study | 3/4i-3/4i* |
Pholidoptera dalmatica maritima/ebneri | MO: Cetinje, Skader lake | 42°23'N, 55°45'E | 2m | 31,31 | this study | 3/4i-3/4i |
Pholidoptera fallax (Fischer, 1853) | 1) BG: Veliko Tarnovo 2) MO: Cetinje, Skader lake |
1) 43°05'N, 25°39'E 2) 42°23'N, 55°45'E |
2m 2m |
31,31 | this study | 3/4i-3/4i |
Pholidoptera frivaldszkyi (Herman, 1871) | 1) BG: Batak | 1) 43°05'N, 25°39'E | 3m | this study | 3/4i- 3/4i | |
2) BG: Iskar | 2) 41°57'N, 24°12'E | 1m | 31,31 |
|
no | |
3) GR: Drama, ca. 5 km north of Elatia | 3) 41°30'N, 24°18'E | 1m | ||||
Pholidoptera griseoaptera Mařan, 1953 | 1) CR: Chetyr Dag | 1) 44°44'N, 34°19'E | 1m | 31,31 | this study | 3/4i-3/4i |
2) PL: Ojców National Park, Sąspowska valley | 2) 50°15'N, 19°50'E | 6m |
|
no-3/4i | ||
Pholidoptera littoralis (Fieber, 1853) | BG: Belogradchik | 43°38'N, 22°41'E | 1m, 1f | 31,31 | this study | 3/4i 3/4i |
Pholidoptera pustulipes (Motschulsky, 1846) | CR: 1) Chetyr Dag | 1) 44°44'N, 34°19'E | 1m | 31,31 | this study | 3/4i-3/4i* |
2) Karadag Reserve | 2) 44°55'N, 35°12'E | 1m, 1f | ||||
Pholidoptera aptera aptera (Fabricius, 1793) | PL: Pieniny Mts, polana Walusiówka | 49°25'N, 20°28'E | 4m | 29,31 |
|
no-1p |
Pholidoptera aptera bulgarica Mařan, 1953 | BG: E Rhodopi Mts, Shturets vill | 41°37'N, 25°32'E | 1m | 29,31 | this study | 1p – 1p |
Pholidoptera cf. apterabulgarica | MK: Strumica, Cham Chiflik | 41°25'N, 22°38'E | 1f | 30,32 | this study | 1p-1p |
Pholidoptera aptera karnyi Ebner, 1908 | BG: 1) Stara Planina Mts, Uzana place | 1) 42°46'N, 25°14'E | 2m 1m | 29,31 | this study | 1p-1p |
2) Lyulin Mt. | 2) 42°39'N, 23°12'E |
|
no | |||
Pholidoptera brevipes Ramme, 1939 | BG: 1) Sakar Mt., Matochina vill; | 1) 41°51'N, 26°33'E | 1m | 29,32 | this study | 3/4i-3/4i |
2) Gorska Polyana vill. | 2) 42°06'N, 26°58'E | 2m | ||||
Pholidoptera aff. brevipes | TR: Coroglu | 40°53'N, 32°57'E | 1m | 29,32 | this study | 3/4i-3/4i |
Pholidoptera macedonica Ramme, 1928 | 1) AL: Galichica Mt., Pilcina | 1) 40°54'N, 20°52'E | 2m | this study | 1p-1p | |
2) MK: Nidzhe Mt. | 2) 41°00'N, 21°41'E | 2f | 29,31 | |||
3) MK: Gorna Belica | 3) 41°13'N, 20°33'E | 1m | ||||
4) GR: Drama, near Elatia | 4) 41°30'N, 24°18'E | 2m |
|
no | ||
Pholidoptera rhodopensis Maran, 1953 | BG: 1) near Goce Delcev | 1) 41°47'N, 23°33'E | 2m | 29,31 | this study | 1p-1p |
2) Pirin Mt. above Bansko | 2) 41°46'N, 23°27'E | 1m | ||||
Pholidoptera stankoi Karaman, 1960 | MK: Ribnicka Riv | 41°42'N, 20°39'E | 3m | 29,31 | this study | 1p-1p |
Uvarovistia satunini (Uvarov, 1934) | TR: 1) Tunceli-Mazgirt | 1) 39°04'N, 39°34'E | 2m | 31,31 | this study | 4/5i 4/5i |
2) Yanıkcay | 2) 38°15'N, 42°54'E | 1m |
Table
Cytogenetic maps of 18S rDNA were obtained for six Eupholidoptera, six Parapholidoptera, 15 Pholidoptera taxa and one Uvarovistia species. In each of the species, FISH revealed a single cluster of rDNA (per haploid genome), located on one autosome pair (Table
Two species of Parapholidoptera and one Pholidoptera exhibited heteromorphism in the rDNA-FISH signal and NOR in terms of the size and presence/absence between homologous chromosomes (indicated with an asterisk in Table
Generally, regardless of the number of chromosomes in karyotype, after both C-band staining and fluorochrome double-staining, chromosome regions showed a similar pattern in analyzed species in terms of constitutive heterochromatin. Most species had heterochromatin blocks in the paracentromeric region with thin C-bands, very weakly staining with DAPI and CMA3 (CMA3-) negative (not shown). Only in the first chromosome pair of Parapholidoptera grandis, thick C-bands in paracentromeric and distal regions were visualized with both DAPI-positive (AT-rich) and CMA3-positive (GC-rich) signals (not shown). In this case, a heterochromatin heteromorphism in respect to the pattern of C-bands (Fig.
Examples of chromosome banding in different species of the tribes Eupholidoptera (a–c) and Parapholidoptera with 2n = 31 (d–i) and 2n = 29 (j–l) studied using different techniques. 18S rDNA FISH revealed a single interstitial cluster (per haploid genome) located in the 3/4 or 4/5 bivalent (a, b, g, j) and co-localized with the active NOR visualized by AgNO3 staining (c–e, k). a E. astyla, diakinesis and b E. megastyla, spermatogonial metaphase with 18S rDNA loci (green, arrows) located close to the paracentromeric region of bivalent 3/4 and telomeric DNA probes (red) c E. schmidti, diakinesis with NOR (arrow) and selected C+, DAPI- and CMA3+ bands located interstitially on 3/4 bivalent (in the right corner) dP. cf. belen and e P. distincta, diplotene, arrows indicate NOR located in the middle of bivalent 3/4 f P. distincta, anaphase I with B chromosomes g, h, i P. grandis g spermatogonial metaphase with the rDNA cluster with different size between homologous chromosomes (arrows) h heterochromatin heteromorphism in respect to the pattern of C-bands in the first autosome pair (1, asterisks) i two metaphase II with bi-armed X chromosome j, k, lP. aff. syriacaj spermatogonial metaphase, rDNA-FISH signal present/absent in homologous chromosomes (arrows and an asterisk) correspond to k NOR l two metaphase II with bi-armed first pair of autosomes (1) and acrocentric X. Bar = 10 µm.
FISH using 18S rDNA (green) and telomeric TTAGG (red) probes on male (a, b, d, g, h) and female (e) karyotypes and silver staining (c, f, i). Diplotene of Pholidoptera (a–g) and Uvarovistia (h, i) species. White arrows point to rDNA clusters on the medium acrocentric pair or on the bi-armed first pair of autosomes. Black arrows indicate the active NOR co-localized with rDNA. a P. fallax and b, c P. pustulipes (2n = 31). Asterisks point to differences in size/strength of rDNA/NOR between homologous chromosomes d–f P. macedonica (2n = 29). Arrows indicate high-intensity rDNA signal and NOR located in the paracentromeric region of the bi-armed first pair of autosomes g P. brevipes (2n = 29). Arrows point 18S rDNA cluster on medium-sized bivalent h, i U. satunini (2n = 31). Bar = 10 µm.
Up to now, cytotaxonomic studies of the Palaearctic Tettigoniinae in more than 60 species out of 22 genera, including 30 species of 11 genera from Europe showed that the most of species had 31 acrocentric chromosomes. However, in Pholidoptera macedonica and Ph. aptera, the chromosome number is reduced to 29 with one submetacentric long pair (
Representatives of four Pholidopterini genera examined in this study have two different male karyotypes including 31 or 29 chromosomes, respectively, and the same sex determination system. The diploid number 2n = 31 (male) of all species belonging to Eupholidoptera and Uvarovistia (only one species was analyzed) as well as to Parapholidoptera (excluding Parapholidoptera prope syriaca) corroborates previous studies, which revealed that most species of the Palaearctic Tettigoniinae were characterized by such basic/ancestral karyotype (e.g. for review see
The ancestral chromosome number is reduced to 2n = 29 in almost half of the analyzed Pholidoptera species and only one Parapholidoptera species (P. prope syriaca) as a result of one Robertsonian translocation (the first autosome pair in the set becomes submetacentric). This reduction appears to be more frequent in the chromosomal evolutionary history of the subfamilies Tettigoniinae and Bradyporinae (e.g.
B chromosomes are frequent in orthopterans, especially in superfamilies Pyrgomorphoidea, Grylloidea, Acridoidea, Tetrigoidea (e.g.
In the Pholidopterini chromosomes described in this paper, one 18S rDNA FISH locus (per haploid genome) coincides with a single active NOR detected by Ag-NO3 staining and with a C-band/CG-rich segment, independently from the number of chromosomes in the set. However, in analyzed species/subspecies two different patterns of the location of rDNA/NOR were observed. A single bivalent carrying 18S rDNA clusters in the interstitial region on a medium-sized chromosome (3rd/4th pair) seems to be typical feature of the representatives of Eupholidoptera, some Pholidoptera and Parapholidoptera taxa with 31 chromosomes as well as Parapholidoptera prope syriaca with chromosome number reduced to 29 in male. The 18S rDNA location on the 4/5th pair of Uvarovistia satunini might represent a derived aspect. Additionally, some structural rearrangements, e.g. a small inversion, may have been involved in changes of the rDNA location in two Parapholidoptera species. By contrast, in seven species/subspecies with 29 chromosomes, one paracentromeric rDNA site, exhibited a unique rDNA distribution pattern in the long bi-armed autosome. The distribution of rDNA loci/active NORs show that a Robertsonian translocation between the first pair and medium sized pair of chromosome-bearing rDNA cluster (probably 3rd/4th) has reduced the chromosome number from 2n = 31 (FN=31, 32) to 29 (FN=31, 32).
The presence of interstitial rDNA loci on a single bivalent (acrocentric or bi-armed) has been observed in the same subfamily in the tribe Platycleidini (
The presence of paracentromeric 18S rDNA cluster on a single bivalent was previously observed in different size chromosomes of other tettigoniids: in European (e.g.
Generally, the Pholidopterini are characterized by chromosomes with a small amount of constitutive heterochromatin in the paracentromeric region and interstitially located C-bands in a medium sized autosome. In most of species and genera belonging to European Tettigoniinae, thin paracentromeric C-bands were uniformly present in chromosomes, but the C-banding patterns and distribution of interstitial and telomeric heterochromatin are usually found to vary among genera and sometimes between species of one genus (e.g.
In conclusion, the results described in this paper demonstrate the usefulness of molecular techniques as tools for better understanding of chromosomal organization and evolutionary history in the tribe Pholidopterini. This study show that the karyotypes of the species analyzed have undergone evolution including changes in chromosome number and morphology by one Robertsonian translocation and sporadically inversion in the X chromosome. The location of the single rDNA/NOR coinciding with C-band/CG-rich segment in the genus Pholidoptera suggests that reduced chromosome number from 31 to 29 in male resulted from a Robertsonian translocation between two pairs of autosomes, one carrying the rDNA/NOR. Thus, FISH for identifying the location of 18S rDNA has proved to be a good marker for distinguishing species in this genus. On the other hand, the tendency of interstitial distribution of repeated DNA sequences may represent a cytogenetic marker for distinguishing some genera in Tettigoniinae. In contrast, interspecific autosomal differentiation has involved minor differences concerning the heterochromatin composition and distribution obtained by C-banding and fluorochrome staining. Furthermore, the additionally detected taxon-specific karyotypic differences in Pholidopterini need to be compared with phylogenetic data to get a clearer idea of their importance both for understanding the karyotype evolution and speciation within this group.
This work was supported by grant 2011/01/B/NZ8/01467 from the National Science Centre, Poland (B. Grzywacz) and project between the Bulgarian Academy of Sciences and the Polish Academy of Sciences (E. Warchałowska-Śliwa and D.P. Chobanov).