Chromosomal differentiation among bisexual European species of Saga (Orthoptera: Tettigoniidae: Saginae) detected by both classical and molecular methods

European Journal of Entomology, 2009
by BEATA GRZYWACZ, ELŻBIETA WARCHAŁOWSKA-ŚLIWA, ANNA MARYAŃSKA-NADACHOWSKA, TATJANA V. KARAMYSHEVA, NIKOLAI B. RUBTSOV, DRAGAN P. CHOBANOV

INTRODUCTION

The subfamily Saginae is an ancient, highly specialized group of carnivorous bush-crickets, the ancestors of which can be traced back to the mid-Jurassic and gave rise to several groups at present occupying arid regions of the Old World, Australasia, and Central America (partim. Kaltenbach in Harz, 1969; Gorochov, 1995; etc.). The subfamily itself (sensu Eades et al., 2007; OSF online) includes 4 genera, distributed over two disjunct regions ? southern and southeastern Sub-Saharan Africa (3 genera) and part of the Western Palearctic (genus Saga). The genus Saga Charpentier, 1825, comprised of 13 species, probably originated and underwent an early radiation in the Miocene of the Aegean, associated with the aridization and isolation of the area (for similar his- toric distributional patterns of other genera see e.g. La Greca, 1999; Ciplak, 2004). The centre of biodiversity includes Asia Minor and the Southern Balkans with most taxa having relatively restricted ranges. Nevertheless, the parthenogenetic species Saga pedo (Pallas) occurs over a territory several times larger than the total area of the ranges of all other species. In continental Europe, five species and two subspecies have been recorded so far (Kaltenbach, 1967). S. rhodiensis Salfi, although occur- ring on a Greek island and here also considered as "Euro- pean", actually represents an element of the Mediterra- nean fauna of Asia Minor. Most of the Palearctic tettigonnids of the family Tetti- goniidae have a karyotype consisting of 2n = 31 acrocen- tric chromosomes in the male with an X0-XX sex chromosome mechanism. This karyotype was suggested as ancestral for most species of this family (e.g. Hewitt, 1979; Warcha owska- liwa, 1998; Warcha owska- liwa et al., 2005). The genus Saga is characterized by extreme karyotypic diversification. A previous study carried out on this genus by Matthey (1946) revealed that S. cappa- docica, S. ephippigera, and S. ornata have the ancestral karyotype (2n = 31 %). The tetraploid, parthenogenetic species S. pedo is characterized by the karyotype 4n = 68 (Matthey, 1939, 1941, 1948; Goldschmidt, 1946). How- ever, the chromosome complement of European species of Saga, based on an earlier study of S. hellenica, S. nato- liae, and S. rhodiensis, is 2n = 29 %/30&. Robertsonian centric fusion and tandem translocation are thought to be the main mechanisms responsible for the karyotypic variation observed within this group (Warcha owska- liwa, 1998; Warcha owska- liwa et al., 2007). In a pre- vious paper, we investigated heterochromatin in three species using different staining methods, preliminary C-banding, silver impregnation (Ag-NORs), chromo- mycin A3 (CMA3) and 4-6-diamidino-2-phenylindole (DAPI). CMA3 and DAPI are useful for detecting CG- and AT-enriched chromosome regions, respectively. Some similarities and differences within the C-positive constitutive heterochromatin regions were found, revealing that taxonomically closely related species with the same chromosome number show different patterns of C-bands. Cytogenetic differences between these species suggest that chromosomal divergence occurred during their speciation (Warcha owska- liwa et al., 2007). Eur. J. Entomol. 106: 1?9, 2009 http://www.eje.cz/scripts/viewabstract.php?abstract=1419 ISSN 1210-5759 (print), 1802-8829 (online) Chromosomal differentiation among bisexual European species of Saga (Orthoptera: Tettigoniidae: Saginae) detected by both classical and molecular methods EL BIETA WARCHA OWSKA- LIWA1, BEATA GRZYWACZ1, ANNA MARYA SKA-NADACHOWSKA1, TATJANA V. KARAMYSHEVA2, NIKOLAI B. RUBTSOV2 and DRAGAN P. CHOBANOV3 1Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Krak?w, Poland; e-mail: warchalowska@isez.pan.krakow.pl 2Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia 3Institute of Zoology, Bulgarian Academy of Sciences, Tsar Osvoboditel 1, 1000 Sofia, Bulgaria Key words. Orthoptera, Saginae, Saga, karyotype, Ag-NOR, FISH, chromosome, rDNA, telomeric repeats Abstract. We report the karyotype characteristics including chromosome numbers of Saga campbelli campbelli, S. c. gracilis, and S. rammei using the following classical cytogenetic methods: C-banding, silver staining, and fluorochrome staining DAPI and CMA3. We also present FISH data showing the distribution of telomeric repeats and 18S rDNA on the chromosomes of these species and the results of similar studies cited in the literature on S. hellenica, S. natoliae, and S. rhodiensis. The five European Saga species exhibit a high rate of karyotype evolution. In addition to changes in chromosome number and morphology (by chromosomal inversion and/or chromosome fusion), interspecific autosomal differentiation involved changes in the distribution and quantity of constitutive heterochromatin and GC-rich regions, as well as the number and location of NORs. In the present study we focused on testing a hypothetical model of karyotype evolution in Saga, with particular reference to the cytogenetic mapping of rDNA and telomeric sequences. Variation in the distribution of rDNA and location of Ag-NORs are novel phylogenetic markers for the genus Saga. 1 À; Lemonnier-Darcemont et al. (2008) reached the same conclusion after studying of S. campbelli and S. rammei, which both have a chromosome number of 2n = 27 %. In this study, five species (and one subspecies) from Europe were subjected to molecular cytogenetic analyses in order to clarify the mechanism of karyotype evolution within the genus Saga. This analysis included fluores- cence in situ hybridization (FISH), which revealed pres- ence of specific DNA within chromosomes (Nath & Johnson, 2000; Schwarzacher, 2003). Changes in the number and distribution of repetitive sequences within chromosomes provide excellent markers for chromosome evolution in many species. Ribosomal DNA (rDNA) genes are useful chromosome markers for interspecific comparative karyotyping in insects at the level of genera (Galli?n et al., 1999; Martinez-Navarro et al., 2004; Zacaro et al., 2004) and population (Martinez-Navarro et al., 2004). FISH using rDNA of grasshoppers can be used on a wider range of taxa within an order (e.g. Bridle et al., 2002; Cabrero et al., 2003; Souza et al., 2003; Martinez- Navarro at al., 2004; Loreto et al., 2008). Other repetitive sequences, so called telomeric DNA, are located mainly at the chromosome termini. Telomeres play an important role in maintenance of chromosomal stability and pre- serve genome integrity. Telomeres were used as markers for identification of chromosome ends. In the majority of insect orders, including Orthoptera, the telomeres are composed of mutliple copies of short, tandemly arranged TTAGG sequences (Okazaki et al., 1993; Sahara et al., 1999; Frydrychov? & Marec, 2002; Frydrychov? et al., 2004; V?tkov? et al., 2005). The clusters of telomeric repeats and telomeric-like sequences were deemed useful in the identification of chromosomal rearrangements related to changes in chromosome number and evolution in insects (e.g., L?pez-Fern?ndez et al., 2004). The present study reports the results of a cytogenetic analysis of S. campbelli campbelli, S. campbelli gracilis, and S. rammei using classical methods (C-banding, silver, DAPI, and CMA3 staining). In addition, FISH with 18S rDNA and (TTAGG)n-specific telomeric DNA (tDNA) probes for mapping these repeats within the chromosomes were applied to these species and the results compared with those obtained previously for S. hellenica, S. nato- liae and S. rhodiensis (Warcha owska- liwa et al., 2007). Chromosomal localization of the clusters of these repeats, along with results of routine cytogenetic techniques, gave us an insight into chromosomal evolution in the bisexual European species of the genus Saga. MATERIAL AND METHODS A cytogenetic analysis of the following nymphs, adult males and female bush-crickets, collected in Bulgaria and Macedonia, was undertaken: Saga campbelli campbelli Uvarov, 1921, 3 male and one female nymph and imago, Bulgaria: Maleshevska Mt., July 2006 (41?43N, 23?06E) leg. Chobanov D; Saga camp- belli gracilis Kis, 1962, 5 male nymphs, Bulgaria: E Rodopi Mts, north of Plevoun Vill., vi.2006 (41?27N, 26?01E), leg. Chobanov D., Warcha owska- liwa E.; Saga rammei Kalten- bach, 1965, 1 male, Macedonia, Bogoslovec Vill., July 2006 (41?46N, 22?01E), leg. Chobanov D. Specimens are deposited in the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences (Krak?w) and in the Institute of Zoology, Bulgarian Academy of Sciences (Sofia). The testes and ovarioles were excised, incubated in a hypo- tonic solution (0.9% sodium citrate), and then fixed in ethanol : acetic acid (3 : 1). The fixed material was squashed in 45% acetic acid. Cover slips were removed by the dry ice procedure and then the slides were air dried. The C-banding was carried out according to Sumner (1972) with a slight modification. The silver staining method for nucleolar organizer regions (NORs) was performed as previously reported (Warcha owska- liwa & Marya ska-Nadachowska, 1992). In order to reveal the molecular composition of C-heterochromatin, some slides were stained with CMA3 to reveal GC enriched regions and DAPI to reveal AT enriched regions (Schweizer, 1976). Chromosomes were classified on the basis of the criteria proposed by Levan et al. (1964). DNA probe preparation Three different 18S rDNA probes were used for the detection of rDNA sequences on chromosomes using FISH: (1) a 3.2 kb fragment of human 18S rDNA cloned in pHr13 (rDNA-probe) (Malygin et al., 1992), (2) and (3) about 1.8 kb 18S rDNA frag- ment amplified from genomic DNAs of Isophya rammei (Orthoptera) and Philenus spumarius (Homoptera), respectively, using polymerase chain reaction (PCR) and primers 18Sai for- ward (5'-CCT GAG AAA CGG CTA CCA CAT C-3') and 18Sbi reverse (5'-GAG TCT CGT TCG TTA TCG GA-3') (Whiting et al., 1997). The PCR reactions were carried out in 25 ?l reaction volumes containing 1.5 mM MgCl2

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, 2.5 mM dNTP, 10 ?M of each of the two primers, 100 ng template DNA and 5 U Taq DNA polymerase (Qiagen, Hilden, Germany). An initial period of 3 min at 94?C was followed by 30 cycles of 60 s at 94?C, 60 s at 51?C, and 1.5 min at 72?C, and concluded by a final extension step of 10 min at 72?C. Probes were labelled with biotin-11-dUTP by nick translation according to the manu- facturer's instructions (Invitrogen, Tokyo, Japan). A (TTAGG)n probe, used to visualize clusters of telomeric repeats, was generated by a non-template PCR using a modified version of L?pez-Fern?ndez et al. (2004) technique. Briefly, PCR was carried out in a 50 ?l reaction mixture containing 1.5 mM MgCl2

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, 0.2 mM each dNTP, 0.5 ?M of each of the two primers (5'-GGTTA-GGTTA-GGTTA-GGTTA-GG-3' and 5'- TAACC-TAACC-TAACC-TAACC-TAA-3') and 2 U Taq DNA polymerase. The non-template PCR was performed with an initial cycle of 90 s at 94?C, followed by 30 cycles of 45 s at 94?C, 30 s at 40?C and 60 s at 72?C, and a final extension step of 10 min at 72?C. The PCR product was then labelled with digoxigenin-11-dUTP in additional PCR cycles to produce the (TTAGG)n telomeric probe. Fluorescent in situ hybridisation FISH of chromosomes using the rDNA and telomeric probes was performed according to a standard protocol (Lichter et al., 1988) with salmon sperm DNA as the carrier DNA. Slides were treated with RNase A for 1 h at 37?C (100 ?g/ml 2 ? SSC), rinsed in 2 ? SSC, dehydrated in an ethanol series (70%, 85%, 100%) and air dried. For removal of cytoplasm, the slides were incubated in pepsin (1 mg/ml in 0.01 N% HCL) at 37?C for 20 min, fixed in 1% formaldehyde in phosphate buffered saline (PBS), 50 mM MgCl2

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, dehydrated in an ethanol series (70%, 80%, 96%) and again air dried. For each slide, 20 ng of the probe mixed with 10 ?g of soni- cated salmon sperm DNA (Invitrogen) were ethanol- -precipitated, cooled to ?20?C and resuspended in 15 ?l of hybridization mix (50% formamide, 10% dextran sulphate, 1% 2 À; 3 FN ? fundamental number of chromosome arms; * intraspecific variation of heterochromatin; 1, 2, ., the number of autosome pair; X, sex chromosome. 1 The location of NOR and CMA3 band on M6 pair of S. rhodiensis, described by Warcha owska- liwa et al. (2007), is incorrect. Warcha owska - liwa et al., 2007 S9 S9, X M8/9, X Paracentromeric: most of autosomes and X thick, M5, M6 interstitial L1, M2?M5 telomeric 29, 32 X submeta- centric, L1 metacen- tric, the remaining autosomes acrocen- tric S. rhodiensis Warcha owska - liwa et al., 2007 S9 Paracentro- meric M8/9 M8/9, X M8/9, X Paracentromeric: L1, S11?S14 thin; M2 ? S10, X thick M6 interstitial L1, M2?M8 telomeric 29, 32 X submeta- centric, L1 metacen- tric, the remaining autosomes acrocen- tric S. natoliae Warcha owska - liwa et al., 2007 M3 Paracentro- meric M3 L1, M2, M3 L1, M2, M6 Paracentromeric: L1, M4?S14, X thin; M2 and M3 thick M6 interstitial L1, M2, M3, M6 telomeric 29, 32 X submeta- centric, L1 metacen- tric, the remaining autosomes acrocen- tric S. hellenica this study S8/9 One per cell S8/9 Not analyzed Paracentromeric: L1?M8, S10, S11 thin; S9 thick* 23, 28 X, L2 subme- tacentric, L1, meta- centric, the remaining autosomes acrocentric S. rammei this study M3 Paracentro- meric M3, Subtelocen- tric S9* Not analyzed Not analyzed Paracentromeric L1, L2, M4?S13, X thin; M3 thick; M5* interstitial S8/9* subtelomeric all L, M and X telomeric 27, 32 X, L2 subme- tacentric, L1, meta- centric, the remaining autosomes acrocentric S. campbelli gracilis this study M3 Paracentro- meric M3, Subtelocen- tric S9* Paracentro- meric M3, M4, telomeric in one arm L1 Paracentro- meric: L1, L2, M3 Paracentromeric L1, L2, M4?S13, X thin; M3 thick S8/9*subtelomeric all L, M and X telomeric 27, 32 X , L2 subme- tacentric, L1, meta- centric, the remaining autosomes acrocentric S. campbelli campbelli bright CMA3 bright DAPI References rDNA -FISH signal Position of NOR Position of fluorochrome bands1 C-bands on chromosomes * intraspecific variation of C-heterochromatin (thick/thin or present/absent) 2n (male), FN and chromosome morphology Species TABLE 1. A comparison of the distribution of heterochromatin bands, NOR and rDNA on chromosomes of the European Saga spe- cies. Fig. 1. C-banding staining of male chromosome complements of Saga campbelli gracilis (a?d), and S. rammei (e, f); a) S. c. gra- cilis ? karyotype and b) spermatogonial metaphase (2n = 27), c) karyotype and d) metaphase II with 14 chromosomes, e) S. rammei ? karyotype and f) metaphase II with 12 chromosomes. The arrows indicate large blocks of heterochromatin on M3 (a?d) and an additional interstitial C-band occurring on the M5 pair (a, b) of S. c. gracilis. X, sex chromosome. Bar = 10 ?m. À; Tween 20, 2 ? SSC). Metaphase spreads were denatured simul- taneously with the DNA probe for 5 min at 75?C. Incubation was performed overnight at 37?C. After hybridization, slides were washed with 50% formamide in 2 ? SSC (3?), 2 ? SSC (2?), 0…

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