Folia biologica (Kraków), vol. 53 (2005), No 3-4

Cytogenetic Variability of European (, ): Karyotypes, C- and Ag-NOR-banding

El¿bieta WARCHA£OWSKA-ŒLIWA, Klaus-Gerhard HELLER, and Anna MARYAÑSKA-NADACHOWSKA

Accepted September 6, 2005

WARCHA£OWSKA-ŒLIWA E., HELLER K-G., MARYAÑSKA-NADACHOWSKA A. 2005. Cytogenetic variability of European Tettigoniinae (Orthoptera, Tettigoniidae): karyotypes, C-and Ag-NOR-banding. Folia biol. (Kraków): 53: 161-171.

Karyotypes, C-banding pattern and localization of nucleolus organizer regions (NORs) in 34 European and subspecies belonging to the subfamily Tettigoniinae are described (the karyotypes of 26 species for the first time). In the males chromosome numbers vary from 2n=33 to 2n=23 and Fundamental Numbers (FN) from 36 to 27. The highest number of chromosomes for this group, 2n%=33 (FN=33), was found in Psorodonotus illyricus macedonicus. In species belonging to genera , , Anterastes, Bucephaloptera, Parapholidoptera, and , as well as in frivaldskyi and Pholidoptera griseoaptera,a karyotype of 2n=30+X0 (FN=31) was found. Ph. macedonica and Ph. aptera aptera are characterized by 2n=28+X0 (FN=31). In species from genera Drymadusa (D. dorsalis limbata) 2n=26+X0: FN=30 and (G. abbreviata), karyotypes of 2n=22+X0 (FN=36) were found. Ph. ma- cedonica and Ph. aptera aptera as well as G. abbreviata differ in karyotypes from other representatives of these genera. New data confirmed that Robertsonian fusions and tandem fusions played the most important role in the evolution of the chromosome set in Tettigoniinae. Cytogenetic similarities and differences between particular species are discussed.

Key words: Orthoptera, Tettigoniidae, katydids, karyotype, cytotaxonomy, C-banding, NOR.

El¿bieta WARCHA£OWSKA-ŒLIWA, Anna MARYAÑSKA-NADACHOWSKA, Department of Experi- mental Zoology, Institute of Systematics and Evolution of , Polish Academy of Scien- ces, S³awkowska 17, 31-016 Kraków, Poland. E-mail: [email protected] [email protected] Klaus-Gerhard HELLER, Department of Zoology II, University of Erlangen-Nûrnberg, E-mail: [email protected]

The systematics of the subfamily Tettigoniinae, amined in this study. Chromosomal information including the former Decticinae, also sometimes has been demonstrated to be valuable taxonomi- considered as the family Tettigoniidae (HELLER et cally in a large range of vertebrates (FONTANA & al. 1998), are unsatisfactory at present. This refers RUBINI 1990; SMITH 1990; VOLLETH &HELLER to the level of subgenera and genera (e.g. the large 1994; RUMPLER Y. 2000) and (BLACKMAN genera Metrioptera/ or Pterolepis/Rha- 1980; PETITPIERRE 1997; SHAPOSHNIKOV et al. cocleis), but more importantly also to higher cate- 1998; WARCHA£OWSKA-ŒLLIWA E. 1998). gories. Information concerning which morphologi- The diploid number, chromosome morphology, cal characters – if any – can be used to reliably de- and the type of sex determination system of Tetti- fine groups of related genera and tribes is lacking goniinae in the Palaearctic have been described for (e.g. RENTZ &COLLESS 1990). Of course, rela- about 50 species of 22 genera, including 30 species tionships have been established for some groups of of 11 genera from (JOHN &HEWITT 1968; genera, but for many they are missing. Careful CAMACHO et al. 1981; UESHIMA 1986; SOUTHERN morphological studies can sometimes improve the 1967; SERGEEV &BUGROV 1988; AREFJEV 1989; situation (see F. & L. WILLEMSE in preparation), WARCHA£OWSKA-ŒLIWA 1984, 1988, 1998; BUGROV but additional information would be highly wel- 1990; WARCHA£OWSKA-ŒLIWA &MICHAILOVA come. Therefore the karyotypes of 26 species of 1993; WARCHA£OWSKA-ŒLIWA &BUGROV 1996a; this group, previously undescribed, have been ex- WARCHA£OWSKA-ŒLIWA et al. 1987, 1992, 1994). 162 E. WARCHA£OWSKA-ŒLIWA et al.

Table 1 General karyotypical features, C-heterochromatin patterns, and NORs in species of Tetti- goniinae (acro- acrocentric, subacro- subacrocentric, submeta- submetacentric, meta- metacentric, * intraspecific variation of C-heterochromatin)

Numbers of individuals, 2n, References other Species collection FN C-bands NOR than this paper locality All paracentromeric thin, 4 males, 33, 33 Psorodonotus M M interstitial, illyricus macedonicus Greece all acro 2, 3 M2,M3, most of small bivalents telomeric All paracentromeric thin Decticus 2 males, 31,31 present paper & M interstitial, M4/5 WARCHA£OWSKA- verrucivorus Poland all acro 4/5 M3-M6 terminal thick -ŒLIWA 1984 1 male, 31, 31 Platycleis (Parnassiana) very small division, without C-bands tenuis Greece all acro Metrioptera 1 male, 1 female, 31, 31 All paracentromeric thin, roeselii ambitiosa Greece all acro L1, L2,M3, X telomeric thin Metrioptera 6 males, 31, 31 All paracentromeric thin, WARCHA£OWSKA- M3/4 roeselii roeselii Poland all acro L2*, M3,M4,M5*, X telomeric, -ŒLIWA 1984 Metrioptera 2 males, 31, 31 All paracentromeric thin, oblongicollis Greece all acro L1/2 telomeric All paracentromeric thin, present paper & Metrioptera 5 males, 31, 31 L2,M3,M4 *interstitial, brachyptera WARCHA£OWSKA- Slovakia all acro L2,(thin), M3,M4 (thick) telomeric, -ŒLIWA 1984 X near centromeric and telomeric

Metrioptera 2 males, 31, 31 All paracentromeric thin, present paper & bicolor L *, L ,M,M M telomeric WARCHA£OWSKA- Poland all acro 1 2 3 4, 5 -ŒLIWA 1984 3 males, 1 Anterastes female, 31, 31 All paracentromeric thin, serbicus Greece all acro X thick centromeric and thin telomeric 1 male, 31, 31 Bucephaloptera All paracentromeric thin, bucephala Turkey all acro Parapholidoptera 1 male, 31, 31 Two medium size with thick signata Turkey all acro paracentromeric C-band, the rest thin 6 males, 1 All paracentromeric thin, Eupholidoptera 31, 31 female, M interstitial (subtelomeric) M3/4 epirotica all acro 3/4 Greece L1 telomeric heterozygous (two males) All paracentromeric thin, 2 males, 31, 31 Eupholidoptera M interstitial , tauricola Turkey all acro 3/4 L1* telomeric (both males) All paracentromeric thin, 5 males, 1 31, 31 Eupholidoptera female, M interstitial, smyrnensis all acro 3/4 Bulgaria L1 *telomeric (two males) All paracentromeric thin, 3 males, 31, 31 Eupholidoptera M interstitial chabrieri garganica Greece all acro 3/4 L1*telomeric (one males 20/04) Eupholidoptera 4 males, 31, 31 All paracentromeric thin, megastyla Greece all acro M3/4* interstitial (in one male (25 /03) Eupholidoptera 2 males, 31, 31 All paracentromeric thin, annulipes Turkey all acro M3/4 interstitial All paracentromeric thin, 2 males, 2 31, 31 Eupholidoptera sp. females M interstitial (subtelomeric) (ex Andros) all acro 3/4 Greece L1,L2 telomeric All paracentromeric thin, Eupholidoptera 5 males 31, 31 M3/4,M5 interstitial prasina Turkey all acro L1,L2,M3/4,M5 telomeric, X interstitial and telomeric Cytogenetic of European Tettigoniinae 163

Table 1 cont.

All paracentromeric thin, Eupholidoptera 2 males, 31, 31 M3/4,M5 interstitial anatolica Turkey all acro L1,L2,M3/4,M5 telomeric X interstitial? All paracentromeric thin, Eupholidoptera 2 males, 31, 31 M interstitial, M3/4 mersinensis Turkey all acro 3/4 L1 –M7/8 telomeric All paracentromeric thin, Eupholidoptera 3 males, 31, 31 M4/5 interstitial M3/4 karabagi Turkey all acro L1,L2,M3, M4,M6/7 telomeric X interstitial 1 male, 31, 31 Eupholidoptera very small division, without C bands icariensis Greece all acro present paper & Pholidoptera 1 male, 31, 31 All paracentromeric thin, WARCHA£OWSKA- M3 frivaldskyi Greece all acro M3 interstitial thin -ŒLIWA,MICHAILOVA 1993

All paracentromeric thin, present paper & Pholidoptera 6 males, 31, 31 M giseoaptera M interstitial thin 3 WARCHA£OWSKA- Poland all acro 3 -ŒLIWA 1984

29, 31 All paracentromeric thin, Pholidoptera 2 males, L1 submeta; L1 telomeric macedonica Greece L2-S14 , X acro 29, 31 present paper & Pholidptera 4 males, L aptera aptera L1 submeta; All paracentromeric thin 1 WARCHA£OWSKA- Poland all acro -ŒLIWA 1988 27, 30 All paracentromeric thin, L1 meta/ Drymadusa 1 male, /submeta; M2 interstitial near centromeric dorsalis limbata Turkey L2-S13 acro; L1, M3-S13 telomeric (M3,4,5 thick) X submeta/ X telomeric in longer arm /subacro 25, 27 L1 meta; All paracentromeric medium size, 1 male, Pterolepis M2-S12, L interstitial + telomeric (in both arms) ferdinandi Greece X acro 1 B chromo- some 25, 27 Pterolepis 2 males, L1 meta; All paracentromeric medium size, germanica Greece M2-S12, L1 interstitial + telomeric (in both arms) X acro 25, 27 All paracentromeric medium size. Pterolepis 3 males, L1 meta; L1 interstitial+ telomeric (in both arms); insularis Greece M2-S12, M2,M3,M4 interstitial, X acro all telomeric thin 25, 27 Pterolepis 2 males, 1 female, L1 meta; All paracentromeric medium size, sp. nova Greece M2-S12, M2,M3,M4 interstitial and telomeric X acro

25, 27 All paracentromeric medium size, Pterolepis 3 males, L1 meta; L1 interstitial + interstitial (both arms), edentata Greece M2-S12, M2/3 interstitial, X acro M4/5 thick telomeric, X thin telomeric 23, 36 L1 submeta; M2,M6, All paracentromeric thin (excluding pair S ) Gampsocleis 3 males 7/8 M WARCHA£OWSKA- abbreviata M7,S9,S11 X,S telomeric thin 6 -ŒLIWA 1998 Greece X meta; 9 M3-M5,S8, S10 acro 164 E. WARCHA£OWSKA-ŒLIWA et al.

The great majority of cytotaxonomic analyses Most of the European species of Tettigoniinae have been based on the conventional staining tech- analyzed in this paper in the genera Decticus, nique. Banding techniques such as C-banding and Platycleis, Metrioptera, Anterastes, Bucephalop- Ag-NOR-banding allow for a better characteriza- tera, Parapholidoptera, Eupholidoptera, and some tion of tettigonid karyotypes and selectively reveal species of Pholidoptera,have2n%=31 (FN=31; chromosome regions consisting of constitutive only acrocentric chromosomes) (Fig. 1). How- heterochromatin and NOR-sites of the RNA ever, in Pholidoptera macedonica, the chromo- genes. New cytological markers are useful for bet- some number is reduced to 2n%=29 (FN=31) with ter insight into the pathways by which the various one submetacentric pair (L1), similar to Ph. aptera karyotypes have evolved in Tettigoniidae. Consti- (Fig. 2) described earlier (WARCHA£OWSKA-ŒLIWA tutive heterochromatin shows characteristic distri- 1988). bution patterns in karyotypes and contributes to The highest number of chromosomes for this the broad scattering of genome sizes throughout group, 2n%=33, was found in Psorodonotus illyri- biological taxa (REDI et al. 2001). cus macedonicus (Fig. 3). Data on C-banding patterns in mitotic and mei- In Drymadusa dorsalis limbata the complement otic cells, and information on the nucleolus organ- is reduced to 2n%=27, FN=30 with a meta/subme- izer region (NOR), are limited in the European tacentric L1 pair and the submeta/subacrocentric X Tettigoniinae (WARCHA£OWSKA-ŒLIWA &BUGROV chromosome (Fig. 4). 1996a; WARCHA£OWSKA-ŒLIWA et al. 1992, 1994). Here a further study on the karyotypes of 34 taxa of Five species of the genus Pterolepis are charac- % the subfamily is provided in order to obtain more terized by 2n =25. This is a chromosomal set with information on the genome organization of these one extremely long metacentric L1 (FN=27) (Fig. orthopteran species. 5a,b,c). The lowest chromosome number in this group was found in Gampsocleis abbreviata 2n%=23 (FN=36) with L1 submetacentric, five pairs metacentric, and five pairs acrocentric. The Material and Methods metacentric X chromosome is the largest element in the karyotype as in G. glabra (WARCHA£OW- Adult males and females were collected in SKA-ŒLIWA et al. 1992). Greece, Turkey, Poland, and Slovakia for the cyto- genetic study (see Appendix for taxonomic data All analyzed species show the XO% and XX& and specific localities). Specimens from Greece type of sex chromosome determination. and Turkey are deposited in Collectio Heller (CH Table 1 shows the C-banding patterns of 34 spe- + reference number), the others – in the Institute of cies, and Figures 1-13 give some examples of the Systematics and Evolution of Animals PAS results. Most of the species of Tettigoniinae have (Kraków). paracentromeric C-bands in the vicinity of the cen- Testes and ovarioles were excised, incubated in tromeric regions. In most cases, the paracentro- a hypotonic solution (0.9% sodium citrate), and meric C-bands are restricted to the centromeric then fixed in ethanol:acetic acid (3:1). The fixed region (thin C-bands), e.g. in the whole autosome material was squashed in 45% acetic acid. Cov- complement and the X chromosome of Metriop- erslips were removed by the dry ice procedure and tera roeselii (Figs 1, 6), Eupholidoptera mersinen- then preparations were air dried. The C-banding sis (Fig. 7), and (Fig. 2). In examination was carried out according to SUMNER other cases, C-bands occupy the region next to the (1972) with slight modification. The silver stain- centromere (thick C-bands) as in the two ing method for NORs was performed as previously medium-sized pairs of Parapholidoptera signata, reported (WARCHA£OWSKA-ŒLIWA &MARYAÑSKA- and in all chromosomes of Pterolepis sp.n. NADACHOWSKA 1992). The fixed material is de- In 22 species of Tettigoniinae, shown in Table 1, posited in the Institute of Systematics and Evolu- interstitial C-bands on one or more chromosomes tion of Animals PAS (Kraków). are present. Interstitial C-bands are located for in- stance near the paracentromeric region of the X chromosome in Eupholidoptera karabagi (Fig. 8), Results in the interstitial region of pairs M2,M3 in Psoro- donotus illyricus macedonicus (Fig. 3), in the pair The chromosome number (2n), morphology of M4/5 of Decticus verrucivorus (Fig. 9), in the pairs chromosomes (Fundamental Number = FN), the M3/4 and M5 of Eupholidoptera anatolica (Fig. 10), C-banding patterns, and NOR locations in the spe- in the interstitial parts of both arms in pair L1 of cies studied are reported in Table 1. The diploid Pterolepis ferdinandi as well as in pairs M2-M4 of chromosome number of 26 species representing Pterolepis insularis (Fig. 5a), and of Pterolepis sp. 10 genera was investigated for the first time. n. Only in (in one indi- Cytogenetic of European Tettigoniinae 165

X

1

X

X X 2 3 4

X X B

X

5a 5b 5c

Figs 1-5. C-banded karyotypes of males. Fig. 1. Metrioptera roeselii and Fig. 2. Pholidoptera aptera aptera – 2n=31, FN=31 and 2n=29, FN=31 respectively, metaphase I, both species with very thin paracentromeric C-bands. Fig. 3. Psorodonotus illyricus macedonicus – 2n=33, FN=33, metaphase I, arrows indicate interstitial C-bands. Fig. 4. Drymadusa dorsalis limbata – 2n=27, FN=30, metaphase I. Figs 5a, b. (a) Pterolepis insularis and (b) Pterolepis ferdinandi – 2n=25, FN=27, metaphase I with interstitial C-bands (arrows), B (supernumerary chromosome), Fig. 5c. Pterolepis edentata, mitotic metaphase with extremely long metacentric pair L1.Bar=10Fm.

vidual) variation in M4 due to the presence or ab- verrucivorus). However, in some species of the sence of interstitial C-bands is observed (Fig. 11). genera Pterolepis and in Drymadusa dorsalis When telomeric C-bands are present, they are lo- these segments are larger (thick). Differences in cated in chromosomes of different sizes and in the amount of heterochromatin are clearly visible most cases they are thin (Table 1, Fig. 6). How- in the interphase nuclei and in the early prophase ever, in three pairs of autosomes (M -M )ofDecti- as e.g. in the genera Psorodonotus, Metrioptera, 3 6 Pholidoptera, Decticus, Drymadusa, and Pterole- cus verrucivorus andinM2,M3 of Metrioptera brachyptera, these bands are thick (Figs 9, 11). It is pis (Figs 13a-f). also worth mentioning that sometimes size varia- A B chromosome was detected in one male of tion of telomeric C-bands of two homologous Pterolepis ferdinandi. It was a acrocentric small chromosomes may be present, e.g. in two pairs (L2, element in the complement (Fig 5b). M5)ofMetrioptera roeselii, or in some individuals The location of chromosome NOR’s was shown in four species of Eupholidoptera (Fig. 12). in only 9 of the species studied. For other species To sum up information connected with hetero- the NOR was not revealed owing to the absence of chromatin, the C-banding technique revealed that diplotenes in the studied cells. Eight species, most of the examined species with 2n=31 in males namely Decticus verrucivorus, Metrioptera roese- show very small (thin) segments of constitutive lii, Eupholidoptera epirotica, E. mersinensis, E. heterochromatin in all autosomes and in X chro- karabagi, Pholidoptera griseoaptera,andPh. ap- mosomes (excluding terminal C-bands in Decticus tera each showed a single active NOR located on 166 E. WARCHA£OWSKA-ŒLIWA et al.

Figs 6-13. Fig. 6. Metrioptera roeselii, mitotic metaphase I, arrows indicate telomeric C-bands. Fig. 7. Eupholidoptera smyrnensis, metaphase I. Fig. 8. Eupholidoptera karabagi, meiotic prophase, the X chromosome with interstitial C-band (arrow). Fig. 9. Decticus verrucivorus, mitotic metaphase, arrows indicate telomeric C-bands. Fig. 10. Eupholidoptera anatolica, mitotic with interstitial C-bands. Fig. 11. Metrioptera brachyptera, diakinesis, _ indicates variability of interstitial heterochromatin, arrows indicate telomeric C-bands, thick in autosomes and thin in the X chromosome. Fig. 12. Eupholidoptera chabrieri garganica, metaphase I, _ indicates variability in telomeric C-band. Figs 13a-f. Nuclei with heterochromatin: (a) Metrioptera roeselii, (b) Pholidoptera aptera aptera, (c) Psorodonotus illyricus macedonicus, (d) Decticus verrucivorus, (e) Drymadusa dorsalis limbata, (f) Pterolepis insularis.Bar=10Fm. Cytogenetic of European Tettigoniinae 167

14a 14b 14c

14d 14e 14f

Fig. 14a-f. Ag-staining areas of the NORs, diplotene/diakinesis: (a) Decticus verrucivorus, (b) Metrioptera roeselii, (c) Eupholidoptera karabagi, (d) Pholidoptera griseoaptera, (e) Pholidoptera aptera aptera, (f) Gampsocleis abbreviata (arrows). Bar = 10 Fm.

the medium-sized bivalents (M3/4 and M4/5), and in SKA-ŒLIWA 1998), shows 2n=33?. One of the pos- Gampsocleis abbreviata on the M6 bivalent. Ac- sible mechanisms producing this unusually high tive NORs were detected distally in D. verrucivo- number rests in the relatively complicated chro- rus, M. roeselii, G. abbreviata, and in a near distal mosomal reorganization that could be connected position in Eupholidoptera epirotica, whereas with chromosome aneuploidy. Taxonomically this they are located interstitially in Eupholidoptera character can in the future be used to clarify the mersinensis , E. karabagi, Pholidoptera frivald- problematic relationships between Psorodonotus sky, P. griseoaptera,andGampsocleis. Pholidop- and the genus Uvarovina (Peltastes) (see STORO- tera aptera aptera showed one NOR located on L1 ZHENKO 2004). probably near a distal position. (Figs 14a-f). Nine species have chromosome numbers lower than the modal number of the subfamily. They may have their origin in independent Robertsonian Discussion fusions of acrocentric chromosomes. Interest- ingly, the simplest case, the fusion of two pairs of Most species of the Palearctic Tettigoniinae chromosomes resulting in 2n=29, was only found have been found to have 2n=31 with only acrocen- here in the closely related Pholidoptera aptera and tric chromosomes (FN=31) (see review P. macedonica, but not in other species of this ge- WARCHA£OWSKA-ŒLIWA 1998). The same is true nus (see WARCHA£OWSKA-ŒLIWA 1998 for re- of the species sampled in this study in the genera view). This character could help the if it Anterastes, Bucephaloptera, Decticus, Eup- is used to define the morphologically difficult P. holidoptera, Parapholidoptera, Platycleis,and aptera group (for characteristics of the song pat- Metrioptera. For these species, karyology gives no tern see HELLER 1988). clues as to their systematic relationships. There For Drymadusa dorsalis limbata a Robertsonian are, however, some species which differ from this fusion and an additional tandem fusion followed basic pattern and which have to be discussed sepa- by an inversion, resulting in 2n%=27 (FN=30) (one rately. Only one of the examined species has a metacentric pair and submeta/subacrocentric X chromosome number higher than the modal chromosome originated by pericentric inversion) number of this subfamily. Psorodonotus illyricus have to be assumed. A very similar karyotype is macedonicus, like its congeneric species P. specu- known from Bergiola montana (WARCHA£OWSKA- laris Fischer de Waldheim, 1839 (WARCHA£OW- ŒLIWA &BUGROV 1996a), another genus of the 168 E. WARCHA£OWSKA-ŒLIWA et al.

Drymadusini. Several other species (from the et al. 1983; CABRERO &CAMACHO 1986a; VISERAS genera , Anatlanticus, Tadzhikia, Ana- et al. 1991). However, C-banding patterns of Euro- drymadusa, Ceraeocercus, Paratlanticus) from pean Tettigoniinae have been described only in a this tribe have karyotypes with a reduced number few genera: Gampsocleis (WARCHA£OWSKA- of chromosomes (2n = 25-29) (WARCHA£OWSKA- ŒLIWA et al. 1992), Montana (WARCHA£OW- -ŒLIWA &BUGROV 1996a). SKA-ŒLIWA et al. 1994), (WARCHA£OW- SKA-ŒLIWA &MARYAÑSKA-NADACHOWSKA 1995), The five species of the genus Pterolepis (for- Anadrymadusa, as well as in some genera of Dry- merly ,HELLER et al. 1998) showed a madusini from Asia (WARCHA£OWSKA-ŒLIWA & karyotype with 2n = 25 (FN=27). In this case, the BUGROV 1996a). In most of these genera, and in first pair L occurs probably as a result of one cen- 1 most described in the present paper, thin paracen- tric fusion between two autosome pairs and two tromeric C-bands were uniformly present in all other pairs as a result of tandem fusions. From autosomes and in the X chromosome. Exception- chromosome data, nothing can be said about the ally, in Parapholidoptera (on three medium-sized phylogenetic relationships of this genus. How- pairs), Gampsocleis (on one small-sized pair) and ever, in no species of the Platycleidini s.str. such the earlier described (WARCHA£OWSKA-ŒLIWA & low chromosome numbers are found, whereas BUGROV 1996a) Tadzhikia (three small pairs), they occur quite frequently in genera character- these C-blocks occupy the region next to the cen- ized morphologically by two spines at the proster- tromere (thick C-blocks). However, in Pterolepis num, e.g. Drymadusini, Pterolepis/Rhacocleis, all chromosomes are characterized by medium- Gampsocleis. sized paracentromeric C-bands, while the metacen- Within the genus Gampsocleis, however, spe- tric X chromosomes in the latter genus have a thick cies with very different karyotypes were found. (double) block in this region. Similarly to the above Several species (see WARCHA£OWSKA-ŒLIWA mentioned species, most genera and species of 1998 for review) show chromosomes which do Phaneropterinae (Poecilimon, Isophya, Metaplastes, not differ in number nor in shape from the typical Polysarcus, and Andreiniimon) possess thick para- ones of the subfamily (2n =31, FN=31). On the centromeric C-bands on one or on almost all chro- other hand, two species, G. glabra and G. abbrevi- mosomes (WARCHA£OWSKA-ŒLIWA &BUGROV ata, have only 23 chromosomes with FN=26. 1998; WARCHA£OWSKA-ŒLIWA &HELLER 1998; Since these two species are the most western rep- WARCHA£OWSKA-ŒLIWA et al. 2000). resentatives of the genus, a common origin of this The C-banding patterns and distribution of inter- highly derived karyotype can be assumed and the stitial and telomeric C-bands in autosomes and the reduction in chromosome number happened cer- X chromosomes are usually found to vary among tainly independently from that in other genera genera and between species of one genus. For in- mentioned above. stance, qualitative and quantitative variation of C- An interesting feature of the Pterolepis ferdi- bands was observed in Metrioptera and Eup- nandi karyotype is the sporadic occurrence of holidoptera (present paper), as well as in Montana B chromosomes. According to many authors (e.g. (WARCHA£OWSKA-ŒLIWA et al. 1994). Only Pholido- BATTAGLIA 1964; JOHN &LEVIS 1968; WHITE ptera frivaldsky and P. griseoaptera as well as 1973; HEWITT 1979; CAMACHO et al. 1981) the Pterolepis ferdinandi and P. germanica, possess existence of B chromosomes, previously noted in the same C-banding patterns similar to Anadry- few species of tettigoniids, indicates that these madusa picta and A. robusta (WARCHA£OWSKA- elements are rare in this group and often unstable ŒLIWA &BUGROV 1996a). (WARCHA£OWSKA-ŒLIWA et al. 1992). Intraspecific polymorphism mainly connected In tettigoniids (similar to other Orthoptera) the with different numbers of additional heterochro- C-banding technique is used, among others, in matin blocks (indicated in Table 1 as *) was de- comparative studies of populations, species and scribed in a few species as in earlier studies of genera of the subfamilies Bradyporinae (NAVAS representatives of the genus Gampsocleis (WARCHA- CASTILLO et al. 1986; WARCHA£OWSKA-ŒLIWA £OWSKA-ŒLIWA et al. 1992). &BUGROV 1996b, 1998) and Phaneropterinae The silver Ag-staining of the NORs is one of (WARCHA£OWSKA-ŒLIWA &HELLER 1998; WAR- methods used for demonstrating the position of the CHA£OWSKA-ŒLIWA &MARYAÑSKA-NADACHOW- gene complex at 18S and 28S ribosomal DNA in SKA 1992; WARCHA£OWSKA-ŒLIWA et al. 1992, the chromosome set (GOODPASTURE &BLOOM 1995, 1996, 2000). Differences between species 1975). In mammals and plants NOR studies are belonging to the same genus and between differ- provided on mitotic divisions, however, in Orthop- ent genera are the result of the heterochromatin tera these regions may be stained only in meiotic differentation as is reflected by the existence of cells. The location of NORs in six genera of tettigo- intraspecific variation for many species (SANTOS niids is presented in Table 1. The distribution of Cytogenetic of European Tettigoniinae 169

NORs often characterizes species within one ge- BLACKMAN R. L. 1980. Chromosome numbers in the Aphidi- nus, but sometimes shows variation even between dae and their taxonomical significance. Systematics Ento- mology 5: 7-25. specimens of the same species (WARCHA£OWSKA- BUGROV A. G. 1990. Karyotypes of some rare katydids (Or- ŒLIWA &MARYAÑSKA-NA- DACHOWSKA 1995). thoptera, Tettigoniidae) from Siberia. (In: Redkije Gelminty In some Tettigoniinae the NOR occurs in the large Kleshchi i Nasekomyje. Nauka, Novosibirsk): 54-60. (In bivalents as in the genus Tettigonia andinrepre- Russian). CABRERO J., CAMACHO J. P. M. 1986a. Cytogenetics studies sentatives of Drymadusini (WARCHA£OWSKA- in gomphocerine grasshoppers. I. Comparative analysis of ŒLIWA &MARYAÑSKA-NADACHOWSKA 1995; chromosome C-banding pattern. Heredity 56: 365-372. WARCHA£OWSKA-ŒLIWA &BUGROV 1996a, re- CABRERO J., CAMACHO J. P. M. 1986b. Cytogenetic studies spectively), or in the middle-sized bivalents, e.g. in in gomphocerine grasshoppers. II. Chromosomal location of active nucleolar organizing region. Canadian Journal of Ge- Decticus, Eupholidoptera, Metrioptera (the present netics and Cytology 28: 540-544. paper) as well as in Gampsocleis and Montana (the CAMACHO J. P. M., OROZCO J. C., PASCUAL F. 1981. Chro- present paper and WARCHA£OWSKA-ŒLIWA et al. mosomal rearrangements and karyotypic evolution in Dec- 1992, 1994). In species from the genera Eup- ticinae (Orthoptera: Tettigonioidea). Cytologia 46: 209-215. FONTANA F., RUBINI M. 1990. Chromosomal evolution in holidoptera, Pholidoptera (only with 2n=31), or Cervidae. BioSystems 24: 157-174. Gampsocleis (the present paper and WARCHA£OW- GOODPASTURE C., BLOOM S. E. 1975. Visualization of nu- SKA-ŒLIWA et al. 1992), the NORs are located in cleolar organizer regions in mammalian chromosomes using most cases probably on the same bivalent (at least silver staining. Chromosoma 53: 37-50 according to size). HELLER K.-G. 1988. Bioakustik der europäischen Laub- heuschrecken (Ökologie in Forschung und Anwendung (1). The heterochromatin associated with NORs Verlag Josef Margraf, Weikersheim. 358 pp. sometimes shows a positive C-banding response. HELLER K.-G., KORSUNOVSKAYA O., RAGGE D. R., VEDENINA V., WILLEMSE F., ZHANTIEV R. D., FRANTSEVICH L. 1988. In ten species described in this paper there are Check-list of European Orthoptera. Articulata 7, Beiheft: NORs which correspond to a C-band in the equiva- 1-61. lent position (Table 1). On the other hand, NORs in HEWITT G. M. 1979. Grasshoppers and crickets. Cy- Gampsocleis abbreviata do not show correspon- togenetics, 3. Insecta I. Orthoptera. Borntraeger, Berlin, Stuttgart. dence with C-bands; the nature of heterochromatin JOHN B., HEWITT G. M. 1968. Patterns and pathways of chro- associated with these NORs requires further inves- mosome evolution the Orthoptera. Chromosoma 25: 40-47. tigation using other banding techniques (CMA3 JOHN B., LEVIS K. R. 1968. The chromosome complement. and FISH, e.g. SOUZA et al. 2003). The present Protoplasmatologia. Vol. 6. Springer, Vienna, New York. study demonstrates a high degree of conservatism PETITPIERRE E. 1997. The value of cytogenetics for taxonomy of the NOR location pattern, agreeing with obser- and evolution of Leaf Beetles (Coleoptera, Chrysomelidae). Miscellania Zoologica 20: 9-18. vations on the genera Montana and Gampsocleis REDI C. A., GARAGNA S., ZACHARIAS H., ZUCCOTTI M., (WARCHA£OWSKA-ŒLIWA et al. 1992, 1994). CAPANNA E. 2001. The other chromatin. Chromosoma 110: Thus, NORs are useful chromosome markers for 136-147. interspecific comparison in the Tettigoniinae, like RENTZ D.C. F. COLLESS D. H. 1990. A classification of the shield-backed katydids (Tettigoniinae) of the world. (In: The in the acridid subfamilies Oedipodinae (VISERAS et Tettigoniidae: biology, systematics and evolution. Bailey al. 1991) and Gomphocerinae (CAMACHO & W. J. & Rentz D. C. F. eds. Bathurst, Crawford House Press): CABRERO 1986a,b). 352–377. RUMPLER Y. 2000. What cytogenetic studies may tell us about In summary, the present results show some simi- species diversity and speciation of lemurs. Int. J. Primatol- larities and differences between karyotypes, C- ogy 21: 865-881. banding patterns, and NOR patterns within Euro- SANTOS J. L., ARANA P., GIRALDEZ R. 1983. Chromosome pean species of Tettiginiinae. It is worth stressing C-banding patterns in Spanish Acrididae. Genetica 61: 65-74. SHAPOSHNIKOV G., KUZNETSOVA V., STEKOLSHCHIKOV A. that modification of morphology of chromosomes 1988. (In: Aphids Natural and Managed Ekosystems. Nieto caused a decrease in chromosome number in the Nafria J. M., Dixon A. F. G. eds): 481-487. genus Pterolepis and in some species of Dry- SERGEEV M. G., BUGROV A. G. 1988. Katydids of subfamily madusa, Gampsocleis and Pholidoptera. The fur- Deracanthinae (Orthoptera, Bradyporidae) from Siberia. (In: Taksonomiya Zhivotnych Sybiri, Russian Acad. Sci., ther employment of molecular techniques will Novosibirsk): 46-53. (In Russian). probably provide additional information on the SOUTHERN D. I. 1967. Pseudo-multiple formation as a conse- evolution of the genome in e.g. Pterolepis, Gamp- quence of prolonged non-homologous chromosome asso- ciation in Metrioptera brachyptera. Chromosoma 21: socleis, Pholidoptera, and Drymadusini. 272-284. SMITH S. A. 1990. Cytosystematic evidence agsinst mono- phyly of Peromysces boylii species group (Rodentia: Cricet- ida). J. Mammal. 71: 645-667. References SOUZA M. J., O. HAVER P. R., MELO N. F. 2003. Karyotype, C- and fluorescence banding patterns, NOR location and AREFJEV V. A. 1989. Karyotypes of three acridid species and FISH in the grasshopper Xestotrschelus robustus (Romalei- one grasshopper species from the south European part of the dae). Caryologia 56: 261-267. USSR. Citologia 31: 582-591. (In Russian). STOROZHENKO S. Yu. 2004. Long-horned orthopterans (Or- BATTAGLIA E. 1964. Cytogenetics of B-chromosomes. Cary- thoptera: ) of the Asiatic part of Russia. Vladivistok: ologia 17: 245-299. Dalnauka. 280 pp. 170 E. WARCHA£OWSKA-ŒLIWA et al.

SUMNER S. G. 1972. A simple technique for demonstrating WARCHA£OWSKA-ŒLIWA E., BUGROV A. G., MARYAÑ- centromere heterochromatin. Exp. Cell Res. 75: 304-306. SKA-NADACHOWSKA A. 1996. Karyotypes and C-banding patterns of some species of Phaneropterinae (Orthoptera, UESHIMA N. 1986. Chromosome systems of some Japanese Tettigoniidae (Orthoptera). J. Matsusaka Univ. 4: 13-20. Tettigonioidea). Folia biol. (Kraków) 44: 5-10. WARCHA£OWSKA-ŒLIWA E., HELLER K.-G. 1998. C- VISERAS E., CABRERO J., TALAVERA M., CAMACHO J. P. M. banding patterns of some species of Phaneropterinae (Or- 1991. C-banding and NOR location variations in oedipodi- thoptera, Tettigoniidae) of Europe. Folia biol. (Kraków) 46: nae grasshoppers. Cytobios 68: 165-177. 177-181. VOLLETH M., HELLER K.-G. 1994. Phylogenetic relation- ships of vespertilionid genera (Mammalia: Chiroptera) as re- WARCHA£OWSKA-ŒLIWA E., HELLER K.-G., MARYAÑ- SKA-NADACHOWSKA A., LEHMANN A. 2000. Chromosome vealed by karyological analysis. Z. zool. Evolut.-forsh. 32: evolution in the genus Poecilimon (Orthoptera, Tettigonioi- 11-34. dea, Phaneropteridae). Folia biol. (Kraków) 48: 127-136. WARCHA£OWSKA-ŒLIWA E. 1984. Karyological studies on polish Orthoptera species of the Tettigonioidea superfamily. WARCHA£OWSKA-ŒLIWA E., MARYAÑSKA-NADACHOW- SKA A. 1992. Karyotypes, C-bands, NORs location in sper- II. Karyotypes of families Tettigoniidea and Decticidae. Fo- matogenesis of Isophya brevipennis Brunner (Orthoptera: lia biol. (Kraków)32: 311-325. Phaneropteridae). Caryologia 45: 83-89. WARCHA£OWSKA-ŒLIWA E. 1988. Karyotype of Pholidop- tera aptera karnyi Ebner. from Bulgaria and its comparison WARCHA£OWSKA-ŒLIWA E., MARYAÑSKA-NADACHOW- SKA A. 1995. Cytogenetic studies of the genus Tettigonia with that of Pholidoptera aptera aptera (Fabr.) from Poland (Orthoptera, Tettigonioidea, Tettigoniinae). II. Heteromor- (Orthoptera, Decticidae). Caryologia 41: 161-168. phism of C-bands. Folia biol. (Kraków) 43: 99-106. WARCHA£OWSKA-ŒLIWA E. 1998. Karyotype characteristics of katydid orthopterans (Ensifera, Tettigoniidae) and re- WARCHA£OWSKA-ŒLIWA E., MICHAILOVA P. 1993. Cyto- marks on their evolution at different taxonomic levels. Folia logical study of Pholidoptera frivaldsky (Herm.) (Dectici- biol. (Kraków) 46: 143-176. dae, Orthoptera). Cytobios 74: 155-162. WARCHA£OWSKA-ŒLIWA E. MICHAILOVA P., PESHEV G. WARCHA£OWSKA-ŒLIWA E., BUGROV A. G. 1996a. Karyo- 1987. Karyological studies of Metrioptera arnoldi Rme type evolution of Drymadusini (Decticinae, Orthoptera). (Decticidae) and Ancistrura nigrovittata (Br.) (Phaneropte- Cytologia 61: 33-39. ridae: Tettigonioidea: Orthoptera). Caryologia 40: 369-380. WARCHA£OWSKA-ŒLIWA E., BUGROV A. G. 1996b. Karyo- types and C-banding patterns of Bradyporinae (Orthoptera, WARCHA£OWSKA-ŒLIWA E., MARYAÑSKA-NADACHOW- SKA A., BUGROV A. G. 1992. Karyotypes, C- heterochroma- Tettigoniidae) Folia biol. (Kraków) 44: 95-98. tin, and NOR in three species of the genus Gampsocleis Fieb. WARCHA£OWSKA-ŒLIWA E., BUGROV A. G. 1998. Karyo- (Orthoptera: Tettigonioidea: Decticinae). Folia biol. (Kraków) types and C-banding patterns of some Phaneropterinae katy- 40: 119-127. dids (Orthoptera, Tettigonioidea) with special attention to a post-reductional division of the neo-X and the neo-Y sex WARCHA£OWSKA-ŒLIWA E., BUGROV A. G., MARYAÑ- SKA-NADACHOWSKA A. 1994. Karyotypes, C- banding pat- chromosomes in Isophya hemiptera. Folia biol. (Kraków) tern, and NORs of the genus Montana Zeuner 1941 (Orthop- 46: 47-54. tera, Tettigonioidea, Decticinae). Folia biol. (Kraków) 42: WARCHA£OWSKA-ŒLIWA E., BUGROV A. G., MARYAÑ- 89-94. SKA-NADACHOWSKA A. 1995. Karyotypes of three species of the genera Poecilimon Fisch. and Isophya Br.-W. (Or- WHITE M. J. D. 1973. Animal Cytology and Evolution, 3rd ed. thoptera, Tettigonioidea, Phaneropterinae) from the North Cambridge Univ. Press, London. Caucasus. Caryologia 48: 27-34. Cytogenetic of European Tettigoniinae 171

Appendix:

Species in alphabetical order: Anterastes serbicus Brunner Mountain, Tatrzañska Kotlina, near Lendak, (49°14’N, v. Wattenwyl, 1882, CH6403-5, males, CH6407, female, 20°2’E), 20 vii 2001, coll. E. Warcha³owska-Œliwa, L. Œliwa; GREECE: Kavalla, Mt. Pangaion (40°53’N, 24°3’E), 1700 Metrioptera () roeselii ambitiosa Uvarov, 1923, m, 21 vii 2004, coll. K.-G. & M. Heller; Bucephaloptera CH6344, male, GREECE: Ioannina, lake near Metsovon bucephala (Brunner v. Wattenwyl, 1882), CH6073, male, (39°49’N, 21°8’E), 27 vii 2004, coll. K.-G. & M. Heller; TURKEY: Mersin, Anamourion (near Anamur) (36°2’N, CH6345, female, GREECE: Thesprotia, lake of Kefalovriso, 32°47’E), 3 m, 9 vii 2002, coll. K.-G. Heller; Decticus verru- western shore (39°29’N, 20°27’E), 28 vii 2004, coll. K.-G. civorus (Linnaeus, 1758), 2 males, POLAND: Pieniny Na- Heller; Metrioptera (Roeseliana) roeselii roeselii (Hagen- tional Park, Palenica, (49°25’N, 20°30’E), 13 vii 1996, coll. bach, 1822), 6 males, POLAND: Ojców National Park, E. Warcha³owska-Œliwa, L. Œliwa; Drymadusa dorsalis lim- S¹spowska valley, (50°15’N, 19°5’E), 10 vii 1997, coll. bata Brunner v. Wattenwyl, 1882, CH6080, male, TURKEY: E. Warcha³owska-Œliwa, A. Maryañska-Nadachowska; Met- Antalya, above Gazipasa (near Ilýca) (36°24’N, 32°23’E), rioptera (Vichetia) oblongicollis (Brunner v. Wattenwyl, 800 m, 10 vii 2002, coll. K.-G. Heller; Eupholidoptera ana- 1882), CH6343, male, GREECE: Drama, 3 km south- south- tolica (Ramme, 1930), CH5380, CH5715, males, TURKEY: west of Perithori (41°18’N, 23°46’E), 24 vii 2004, coll. K.-G. Antalya, Termessos (36°58’N, 30°30’E), 13 vii 2002, coll. & M. Heller; CH6396, male, GREECE: Ioannina, lake near K.-G. .Heller; Eupho- lidoptera annulipes (Brunner v. Wat- Metsovon (39°49’N, 21°8’E), 27 vii 2004, coll. K.-G. & M. tenwyl, 1882), CH5387, male, TURKEY: Mersin, below Heller; Parapholidoptera signata (Brunner v. Wattenwyl, Güzeloluk (22 km to Erdemli) (36°44’N, 34°9’E), 1000 m, 1861), CH5394, male, TURKEY: Mersin, below Güzeloluk 6 vii 2002, coll. K.-G. Heller; CH5720, male, TURKEY: (22 km to Erdemli) (36°44’N, 34°9’E), 1000 m, 6 vii 2002, Mersin, below Güzeloluk, near Arslanlý (36°41’N, 34°11’E), coll. K.-G. Heller; Pholidoptera aptera aptera (Fabricius, 500 m, 6 vii 2002, coll. K.-G. Heller; Eupholidoptera chabri- 1793), 4 males, POLAND, Pieniny Mountains, polana Walu- eri garganica (La Greca, 1959), CH6337-8, males, siówka, alt. 724 m (49°25’N, 20°28’E), 17 viii 1998 coll. GREECE: Kerkyra, near Agios Spiridon (in the north of E. Warcha³owska-Œliwa, L. Œliwa; Pholidoptera frivaldskyi Kerkyra) (39°48’N, 19°50’E), 5 m, 15 vii 2004, coll. K.-G. (Herman, 1871), CH6341, male, GREECE: Drama, ca. 5 km Heller, M. Heller; CH3200, male, GREECE: Ioannina, Do- north of Elatia (41°30’N, 24°18’E), 23 vii 2004, coll. K.-G. & dona (39°33’N, 20°45’E), 9 vi 1998, coll. Heller & Volleth; M. Heller; Pholidoptera griseoaptera (De Geer, 1773), 6 Eupholidoptera epirotica (Ramme, 1927), CH6141-6, males, POLAND, Ojców National Park, S¹spowska valley, males, CH6255, female, GREECE: Aitolia-Akarnania (Cen- (50°15’N, 19°50’E), 1-30 vii 1999, coll. E. War- tral Greece), Mt. Akarnania above Thirion (38°48’N, cha³owska-Œliwa, A. Maryañska-Nadachowska; Pholidop- 20°58’E), 1400 m, 3 viii 2003, coll. K.-G. Heller, M. Heller, tera macedonica Ramme, 1928 (or a related species), M. Volleth; Eupholidoptera sp., CH6151-2, males, CH6154-5, CH6342, CH6399, males, GREECE: Drama, near Elatia females, GREECE: Kikladhes: Andros, north of Vourkoti (41°30’N, 24°18’E), 23 vii 2004, coll. K.-G. & M. Heller; (37°52’N, 24°52’E), 30-31 vii 2003, coll. K.-G. Heller, M. Platycleis (Parnassiana) tenuis Heller et Willemse, 1989, Volleth; Eupholidoptera icariensis Willemse, 1980, CH3204, CH6275, male, GREECE: Arta, Mt. Tsoumerka above Vour- male, GREECE: Samos, Ikaria (37°36’N, 26°9’E), 23 v 1998, gareli, Grat (39°24’N, 21°9’E), 1850 m, 6-7 viii 2003, coll. coll. Heller & Volleth; Eupholidoptera karabagi Salman, K.-G. Heller, M. Heller, M. Volleth; Psorodonotus fieberi 1983, CH5381-2, CH5722, males, TURKEY: Antalya, Ter- macedonicus Ramme, 1931, CH6349-52, males, GREECE: messos (36°58’N, 30°30’E), 13 vii 2002, coll. K.-G. Heller; Ioannina, lake near Metsovon (39°49’N, 21°8’E), 27 vii 2004, Eupholidoptera megastyla (Ramme, 1939), CH6148-50, coll. K.-G. & M. Heller; Pterolepis edentata (Willemse, CH6297, males, GREECE: Arta, Mt. Tsoumerka above 1982), CH6251, male, CH6174, male, CH6260, male, Theodoriana (39°25’N, 21°10’E), 5-6 viii 2003, coll. K.-G. GREECE: Aitolia-Akarnania (Central Greece), Mt. Akar- Heller, M. Heller, M. Volleth; Eupholidoptera mersinensis nania above Voustrio (38°48’N, 20°59’E), 1100 m, 3-4 viii Salman, 1983, CH5391, male, TURKEY: Mersin, Fýndýkpýnarý 2003, coll. K.-G. Heller, M. Heller, M. Volleth; Pterolepis (36°50’N, 34°20’E), 4 vii 2002, coll. K.-G. Heller; CH5390, ferdinandi (L.Willemse & Tilmans 1986), CH6173, male, male, TURKEY: Mersin, below Güzeloluk (near Köserlý) GREECE: Aitolia-Akarnania (Central Greece), near Plakoti (36°45’N, 34°7’E), 1400 m, 6 vii 2002, coll. K.-G. Heller; (39°0’N, 21°9’E), 4 viii 2003, coll. K.-G. Heller, M. Heller, Eupholidoptera prasina (Brunner v. Wattenwyl, 1882), M. Volleth; Pterolepis germanica (Herrich-Schaffer, 1840), CH5383-5, CH5388, CH5729, males, TURKEY: Mersin, CH6172, male, GREECE: Arta, Mt. Gavrogo (39°13’N, Kasyayla (above Anamur) (36°15’N, 32°54’E), 9 vii 2002, 21°15’E), 1600-1800 m, 4-5 viii 2003, coll. K.-G. Heller, coll. K.-G. Heller; Eupholidoptera tauricola (Ramme, 1930), M. Heller, M. Volleth; CH6268, male, GREECE: Arta, CH5386, CH5734, males, TURKEY: Mersin, below Güzelo- Mt. Tsoumerka above Vourgareli, southern slope (39°23’N, luk (22 km to Erdemli) (36°44’N, 34°9’E), 1000 m, 6 vii 2002, 21°9’E), 1500 m, 6 viii 2003, coll. K.-G. Heller, M. Heller, coll. K.-G. Heller; Gampsocleis abbreviata Herman, 1874, M. Volleth; Pterolepis insularis (Ramme, 1928), CH6169-70, CH4784-6, males, GREECE: Fokis, 4 km north east of CH6234, males, GREECE: Kikladhes: Andros, above Apikia Gravia (38°40’N, 22°24’E), 21 vi 1997, coll. K.-G. Heller; (37°52’N, 24°55’E), 30 vii 2003, coll. K.-G. Heller, M. Heller, (Philippi, 1830), 2 males, POLAND: M. Volleth; Pterolepis n. sp., CH6175-6, males, CH6178, fe- Wieprzecka Mountain, (23°10’N, 50°35’E), 30-31 vii 1998, male (paratypes), GREECE: Arta, Mt. Gavrogo (39°13’N, coll. E. Warcha³owska-Œliwa, M. Jeleñ; Metrioptera bra- 21°15’E), 1600-1800 m, 4-5 viii 2003, coll. K.-G. Heller, chyptera (Linnaeus, 1761), 5 males, SLOVAKIA: Tatra M. Heller, M. Volleth.