C 1996 The Japan Mendel Society Cytologia 61: 33-39, 1996

Karyotype Evolution of Drymadusini (Decticinae, )

Elzbieta Warchalowska-Sliwa1 and Alexander G. Bugrov2

'Department of Experimental Zoology , Institute of Systematics and Evolution of , Polish Academy of Sciences, Slawkowska 17, 31-016 Krakow, Poland 2Institute of Systematics and Ecology of the Siberian Branch , Russian Academy of Sciences, Frunze 11, 630091 Novosybirsk and Department of Natural Sciences, Novosibirsk State University, 630090 Novosibirsk, Russia

Accepted October 30, 1995

The karyotype variability, especially differences in chromosome numbers and morphology permit the investigation of the manner, rate, and form of karyotype evolution in closely related groups. The chromosome systems of the Decticinae have been described by many authors, e.g., by John and Hewitt (1968), White (1973), Hewitt (1979), Ueshima and Rentz (1979), Camacho et al. (1981a), Warchalowska-Sliwa (1984, 1988), Warchalowska-Sliwa et al. (1992, 1994), Warchalowska-Sliwa and Michailova (1993). However, karyological studies of species of the tribe Drymadusini are so far scare and the chromosome numbers of only three species having been reported (Bugrov 1990). The number of the genera in the Drymadusa group was established by Ramme (1939). This group includes the following genera Ceraecercus Uv., Drymadusa Stein., Afrodrymadusa Rme., Paradrymadusa Herm., Drymadusella Rme., Anadolua Rme., Phytodrymadusa Rme., Scotodrymadusa Rme., Ferganusa Uv., Paratlanticus Rme., Atlanticus Scudd., and Bergiola Stschelk. with question mark. Most of the species belonging to Drymadusini show small restricted ranges. Some of them are endemic in various montain ranges. On the other hand, many species are associated with arid or nemoral areas of the Far East (Sergeev 1993). In this study the karyological analysis of eight species of the Drymadusini group was undertaken. The karyotype analysis includes the number and morphology of chromosomes, C-banding and NOR location patterns.

Materials and methods

Eight species of Drymadusini belonging to the fauna of the maritime region of Russia, Dagestan, Tadzhikistan, Turkmenistan, and North Korea were studied (Table 1). Males were injected with 0.1% colchicine for 1.5-2.0 hr. The testes were fixed in glacial acetic-alcohol (1 : 3), and the fixed materials washed and kept in 70% ethanol or Carnoy solution. Air-dried preparations were made by squashing tissues in 60% acetic acid and then freezing in dry ice. C-bands were obtained by the barium hydroxide denaturation technique (Sumner 1972) with minor original modifications. The silver staining method for NOR regions was performed as previously reported (Warchalowska-Sliwa et al. 1992). Chromosomes were classified according to Levan et al. (1964).

Results

Chromosome number and shape In Table 2 the number and shape of chromosomes in the species analysed are shown. The chromosome numbers in this group range from 2n•‰ =25 to 31. The karyotype of Drymadu- 34 Elibieta Warchalowska-liwa and Alexander G. Bugrov Cytologia 61

Table 1 . List of taxa and collection localities

1

2

3

4

5

6

Figs. 1-6. Karyotypes of: 1. Drymadusella hissarica-2n,Z -=31, NF=31; 2. Atlanticus brun- neri-2n=29, NF=32, the pair M2 with secondary construction; 3. Anadrymadusa robusta-2n= 27, NF=29, with C-bands, small distal C-bands in M2; 4. Bergiola montana-2n=27, NF=30, 5. Tadzhikia pavlovskii-2n = 27, NF =31 with two metacentric pairs (L1, L2); 6. Anatlanticus koreanus-2n = 25, NF = 29 with two metacentric pairs (L1, L2) and secondary constructions in the X chromosome. The X chromosomes are the last elements of the karyotypes. ( X 800). sella hissarica has a basic complement of 2ncll = 31 acrocentrics and NF (Fundamental Number) = 31 (Fig. 1), whereas in Atlanticus brunneri the complement is reduced to 29, NF = 32. Only one pair of autosomes and the X are metacentric (Fig. 2). Four species with 2no' = 27 chromosomes: Anadrymadusa picta, A. robusta, and Paratlanticus ussuriensis possess NF = 29 with one metacentric pair (Fig. 3), while Bergiola monatana possesses NF = 30 with one metacentric pair and metacentric X chromosomes (Fig. 4). The fifth species with the same chromosome number, Tadzhikia pavlovskii (NF = 31), has two metacentric pairs (Fig. 5). The lowest chromosome number in this group was found in Anatlanticus koreanus which possesses 2n♂=25 and NF=29 and is complemented with two metacentric pairs (Fig. 6). Only in 1996 Karyotype Evolution of Drymadusini 35

Fig . 7 . Karyotype evolution scheme in the Drymadusini.

Table 2. General karyotypical features and C-heterochromatin location in the species of Drymadusini analysed (m-metacentric, a-acrocentric, * variation of C-heterochromatin)

Drymadusella hissarica and Tadzhikia pavlovskii are the X chromosome the largest element, in the remaining species analysed the X chromosome is the second element in size (Figs. 1-6). At mitotic metaphase with Giemsa staining in the X chromosome of Anatlanticus koreanus and the M2 autosome of Atlanticus brunneri secondary constructions were observed (Figs. 2, 6). The scheme of karyotype evolution in Drymadusini is proposed in Fig. 7.

Chromosome C-banding patterns Table 2 shows the C-banding patterns of 7 species analysed, and Figs. 1, 3, 5, 8 and 9 give 36 Elibieta Warchalowska-Sliwa and Alexander G. Bugrov Cytologia 61

8 9

10 11 12 Figs. 8-12. 8, Mitotic metaphase of Tadzhikia pavlovskii with C-bands; thick C-bands in the four small-sized bivalent (arrows). 9, Metaphase I of Anatlanticus koreanus with C-bands, the heteromorphic small bivalent (arrow). 10, Diplotene/diakinesis of Atlanticus brunneri with NOR (arrow). 11, Diplotene of Paratlanticus ussuriensis preserves NORs of the L2 and S bivalents (arrows). 12, Diakinesis of Tadzhikia pavlovskii preserves NORs of the L2 or L3 and S bivalents (arrows). ( X 1,000). some examples of the results. C-bands may show three different locations: paracentromeric, interstitial and distal. Most of the species analysed have paracentromeric C-bands in the vicinity of the centromeric regions. However, in Tadzhikia pavlovskii in two metacentric autosomes (L1, L3) C-bands are not visible (Fig. 5). In most cases, the paracentromeric C-bands are restricted to the centromeric region (thin C-bands), e.g. in the whole autosomes complement of Drymadusella hissarica (Fig. 1), Atlanticus brunneri, Paratlanticus ussuriensis, and Anatlanticus koreanus. In other cases, they occupy the region next to the centromere (thick C-bands) as in the X chromosome of Drymadusella hissarica, Anadrymadusa picta, A. robusta (Figs. 1, 3) and in the four small-sized pairs of Tadzhikia pavlovskii (Fig. 8), whereas the remaining pairs possess thin C-bands. Four of the seven species shown in Table 2 have interstitial C-bands on one or more chromosomes. Interstitial C-bands are located near the paracentromeric region (the X in Anatlaticus koreanus) or the interstitial region (pair M3 in Anadrymadusa picta and A. robusta Fig. 3, pairs M4-M6 in Drymadusella hissarica, and M2 in Atlaticus brunneri). When distal C-bands are present they are located in chromosomes of different size, and are thin in size (Table 2, Figs. 1, 3). It is also worth mentioning that sometimes in one small pair of Anatlanticus koreanus there occurs a variation between the distal C-bands of two homolo- gous chromosomes (Fig. 9). One small pair of Tadzhikia pavlovskii is heteromorphic, but it is very difficult to decide whether this is connected with the occurrence of a supernumerary segment or submetacentric chromosome. 1996 Karyotype Evolution of Drymadusini 37

NOR location The chromosome NOR location is known only in five of the eight species analysed in this work. For Drymadusella hissarica, Anatlanticus koreanus and Bergiola monatna the NOR analysis was not undertaken owing to the absence of diplotene-diakinesis in the studied cells. Three species, namely Atlanticus brunneri, Anadrymadusa picta, and A. robusta, showed a single active NOR located on the M2 bivalent in an interstitial position (Fig. 10). Paratlanticus ussuriensis and Tadzhikia pavlivskii showed two active NORs. However, only one of them showed primary NOR (this activity in the majority of cells of each individual) located on M2 in Paratlanticus ussuriensis and on L2 or L3 in Tadzhikia pavlovskii. The secondary NORs in these species are detected in one of the small bivalents (Figs. 11, 12).

Discussion

Most of the Old World Decticinae have been recorded as having 2n c71= 31 (NF = 31) (John and Hewitt 1968, White 1973, Hewitt 1979, Warchaiowska-Sliwa et al. 1992, 1994). However, the New World decticine genera, with one or more pairs of metacentrics, diverge from this standard karyotype, thus the chromosome numbers of these species range from 2n = 22 to 31 in the male (Ueshima and Rentz 1979). The results obtained in the cytological analysis of the eight species of Drymadusini described in this paper, indicate the presence of a more intensive karyotype evolution than that in other genera of the Old World Decticinae. The basic karyotype 2n = 31 is present in Drymadusella hissarica, which coincides with the earlier described species Uvarovina daurica and Eulithoxenus mongolicus (Bugrov 1990). On the other hand, the seven species possess karyotypes different from the basic one. This variation is mainly caused by chromosomal rearrangement such as centric fusions and pericentric inversions. In species with other karyotypes, may be distinguished two groups: 1. when NF is similar to the basic number (NF = 31), and 2. when NF is different from it, and some explanation of this change should be sought. A hypothesis of the karyotype evolution of Drymadusini is shown in Fig. 7. Atlanticus brunneri and Tadzhikia pavlovskii belong to the first group, while Atlanticus brunneri shows 2n = 29, with one pair of the metacentric autosomes caused by centric fusion of two pairs of acrocentrics. In such a way, the autosomes are reduced in Metrioptera saussureana (John and Hewitt 1968), aptera (Warchatowska-liwa 1988), Montana deghestanica (Warchalowska-liwa et al. 1994), and some of the North American Decticinae (Ueshima and Rentz 1979). Besides, Atlanticus brunneri also has the metacentric X chromosome, this change perhaps being due to pericentromeric inversion. On the other hand, the chromosome number of Tadzhikia pavloskii has been reduced to 2n = 27 (NF = 31), thus two pairs of autosomes are metacentric. However, centric fusion has taken place between four pairs of medium sized acrocentrics, while the first pair of the complement remains acrocentric. As a rule centric fusion involves large acrocentrics (e.g. Pholidoptera aptera Warcha/owska-Sliwa 1984). Four species in the second group Anadrymadusa picta, A. robusta, Paratlanticus ussuriensis (this paper), and Ceraeocercus fuscipennis (Bugrov 1990) show 2n = 27 (NF = 29), with one metacentric pair. Bergiola montana possesses the same number of chromosomes (NF = 30) as other studied species. It is suggested that on the basis of karyological data the genus Bergiola indubitably belongs to Drymadusini though Ramme (1939) included this species with a question mark. Of the remaining species of this group Anatlanticus koreanus has 2n= 25 (NF = 29) with two metacentrics, while Atlanticus pachymerus reported by White (1941) and A. testaceus (Ueshima and Rentz 1979) have 2n = 25 (NF = 30) with two metacentric autosomes and a metacentric X chromosome. Therefore, a pair of small acrocentrics in all the species of 38 Elibieta Warchalowska-Sliwa and Alexander G. Bugrov Cytologia 61 this group was lost during chromosome evolution, and may have been incorporated in other chromosomes. A similar interpretation was proposed by Ueshima and Rentz (1979) for some North American Decticinae. Within Decticinae of the Old World Anatlanticus koreanus with 2n = 25 is the most advanced in structural evolution of the karyotype. The present authors' cytological investigations permit a discussion to be undertaken on the present taxonomic status of the Drymadusini. Ramme (1939) established the species compo- sition of the Drymadusa group, Storozhenko (1986) reduced the species number of the group only to forms which possess a prosternum without two spines. The karyotypes analysis of nine genera of the Drymadusa group (sensu Ramme) showed that the genera Uvarovina, Eulithoxe- nus are karyologically more closely related with Platycleini. Perhaps the Drymadusini is a separate group within Decticinae. Unfortunalely, the authors have had no opportunity to study the chromosome numbers of other representatives of the Drymadusini genera. Therefore, in order to clarify the taxonomic situation further karyotype as well as taxonomic studies are desirable. In Decticinae, C-bands have been described in the genera Gampsocleis (Warchalowska- Shwa et al. 1992) and Montana (Warchalowska-Sliwa et al. 1994) so far. In these genera the paracentromeric C-bands were uniformly present in all the autosomes and the X chromosome. However, these C-bands were not observed in Tadzhikia pavlovskii. Similarly, Camacho et al. (1981b) noted the absence of C-bands in three pairs of autosomes and the X in Eumigus monticulus. C-banding distribution of distal and interstitial position are found to vary among species belonging to different genera. Only Anadrymadusa picta and A. robusta possessed the same C-banding patterns. Moreover, these two species are characterized by the largest (thickest) paracentromeric C-bands in comparison with others. Differences in C-banding patterns between genera of the same subfamily have been attributed to the transformation of euchromatin to heterochromatin or vice versa, or to duplication or deletion (King and John 1980, Camacho et al. 1985). Changes in the distribution and the amount of heterochromatin may have played a role in speciation (White 1973). Active NORs (primary) during meiotic prophase in five species of Drymadusini studied in the present work, were located on the same bivalent. This demonstrates, similarly to the genus Montana and Gampsocleis (Warchatowska-Sliwa et al. 1992, 1994), a high degee of conserva- tism of the NOR location in the four genera of Drymadusini.

Summary

Karyotypes (chromosome number and shape) of eight species of the Drymadusini group were studied. The chromosome numbers in this group range from 2n,Z = 25 to 31. Cytological analysis of these species indicate the presence of a more intensive karyotype evolution than in other genera of the Old World Decticinae. Additionally, C-band distribution and chromosomal location of the NORs were studied. Paracentromeric, interstitial, and distal C-banding in this species was observed. Three of the five analysed species showed a single active NOR located on the M2 bivalent, whereas two active NORs (primary and secondary) were observed in the two remaining species.

References

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