C 2000 The Japan Mendel Society Cytologia 65:351-358, 2000

C-Banded Karyotypes of Some Podisminae Grasshoppers (, ) from Japan

Alexander G. Bugrov1,*, Elzbieta Warchalowska-Sliwa2, Gen lto3 and Sin-ichi Akimoto3

1 Novosibirsk State University, 630090 Novosibirsk and Institute of Systematics and Ecology of , Siberian Branch of Russian Academy of Sciences, 630091 Novosibirsk, Russia 2 Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, 31-016 Krakow, Poland 3 Hokkaido University, Faculty of Agriculture, Sapporo, 060-8589 Japan

Accepted February 24, 2000

Summary C-banding patterns in Japanese grasshoppers belonging to the subfamily Podisminae:

Podisma sapporensis Shiraki (2n •‰=23, NF=23), Parapodisma subastris Huang, Parapodisma ten-

ryuensis Kobayashi, Parapodisma yamato Tominaga et Storozhenko, Parapodisma mikado (I. Boli-

var), Fruhstorferiola okinawaensis (Shiraki) (2n d=21, NF =21), and Sinopodisma punctata Mist-

shenko (2nd = 21, all chromosomes are two-armed) were studied. Cytogenetic similarities and differ-

ences between particular species are discussed using the C-banding method.

Key words Podisminae grasshoppers, Chromosome, Karyotype, C-banding.

Karyotypes of 8 species of grasshopper belonging to the subfamily Podisminae have been de- scribed from Japan (Inoue 1985). However, the C-banding technique was not commonly used in the cytogenetic studies of Japanese Podisminae. Nevertheless, it has been used extensively with Or- thoptera to discriminate individual chromosomes and their behaviour during meiosis (Gosalvez et al. 1997). The application of this technique has produced new cytogenetic markers for comparative karyology of Podisminae (Westerman and Hewitt 1985, Bugrov et al. 1994, Bugrov 1995, Bugrov and Sergeev 1997). Moreover, the C-banding method gives information about the rates and features of chromosome evolution in groups of closely related species (Cabrero and Camacho 1986). It is important especially for the groups with geographically limited areas of distribution as, for exam- ple, the genus Parapodisma Mistshenko. The aim of the present work was to study unknown karyotypes and C-banding features of some Podisminae species from Japan.

Material and methods

Seven species of Podisminae belonging to the fauna of Japan were studied (Table 1) . Male were injected with 0.1% colchicine for 1.5-2 h. The testes were fixed in glacial acetic alcohol (1 : 3), and kept in 70% ethanol. Air dried preparations were made by squashing the testicles in 45% acetic acid and freezing them in dry ice. They were then stained with C-banding by treating with 0.2 N HCl for 15-20 min, immersed in a saturated solution of Ba(OH)2 at 61•Ž for

3-5 min, rinsed in water, immersed in 2•~ SSC at 61•Ž for 60 min , rinsed, air-dried, and stained with 2% Giemsa. Each portion of the eggs was stored in a separate Petri dish with moist sand and kept in an in- cubator at room temperature. After 15-20 days of incubation, the eggs were placed in a solution of

0.05% colchicine in saline and the tops of the nonmicropylar end were removed . They were then incubated at 30•Ž for 1.5-2 h. The embryos were then dissected out of the eggs into 0.9%

* Corresponding author . 352 A. G. Bugrov et al. Cytologia 65

Table 1. List of taxa and collection localities

sodium citrate solution for 20-30 min at room temperature prior to fixation in glacial acetic alcohol (1 : 3) and air-dried squashed preparations were made. They were stained using the C-method simi- lar to that for testicles, with minor modifications.

Results

Podisma sapporensis Shiraki. The chromosome complement of P. sapporensis from the popu-

lation under study consists of 2n d= 23. All autosomes are acrocentric and can be divided into 3 size

groups: 2 long (L1, L2), 6 medium (M3—M8), and 3 short (S9—S11). The size of the acrocentric X chromosome is approximately similar to the M4 pair. The paracentromeric C-blocks occur in all chromosomes of the complement, their size varying among autosome pairs. The M4 and M9 pairs of

autosomes were characterised by the presence of distal C-bands. Additionally, the S9 pair of auto- somes has an interstitial C-band between the paracentromeric and telomeric C-blocks (Fig. la). In a few males the X chromosome has in addition to the centromeric C-band a minute interstitial C-band

near the paracentromeric one (Fig. lb). In the prophase of meiosis the long autosomes form 2-4 chiasmata, medium-1-2, and the short autosome only one chiasma. The mean frequency of chias-

mata on a cell is .X=17.64 (-±1.18). Parapodisma subastris Huang. 2n •‰=21. All chromosomes are acrocentric. They may be di-

vided into three size groups; 2 long (L1, L2), 7 medium (M3—M9) and 1 short (S10). The size of the acrocentric X chromosome is approximately similar to the M4 pair. The paracentromeric C-blocks

occur in all the chromosomes of the complement. The chromosomes of the M4 pair have a minute telomeric C-block. The autosomes of the M6 pair have a thick telomeric C-block and interstitial C-

band in heteromorphic state near the telomeric one (Fig. 2). In the prophase of meiosis the long au- tosomes form 2-3 chiasmata, medium-1-2, but the short bivalent only one. The mean frequency of chiasmata on a cell is 12.90 (•}1.00).

Parapodisma yamato Tominaga of Storozhenko. 2n d=21. All chromosomes are acrocentric. They may be divided into three size groups: 2 long (L1, L2), 7 medium (M3—M9), and 1 short (S10).

The size of the acrocentric X chromosome is approximately similar to that of the M4 pair. The para- centrometric C-blocks occur in all the chromosomes of the complement. The chromosomes of the

M5 pair revealed telomeric C-blocks. In the prophase of meiosis the large telomeric C-blocks in the M6 pair of autosomes seem to become double (Fig. 3) similarly to P tenryuensis (see below). In the

prophase of meiosis the long autosomes form 2-3 chiasmata, medium-1-2, and short bivalent only one. The mean frequency of chiasmata on a cell is X=13.06 (•} 1.08). Parapodisma tenryuensis Kobayashi. 2n •‰= 21. In 4 males all chromosomes are acrocentric.

They amy be divided into 3 size groups: 2 long (L1, L2), 7 medium (M3—M9), and 1 short (S10). The size of the acrocentric X chromosome is approximately similar to that of the M4 pair. The paracen-

tromeric C-blocks occur in all the chromosomes of the complement. The L1 pair of chromosomes 2000 C-banded karyotypes of Podisminae grasshoppers 353

Figs. 1-4. I a, b. Podisma sapporensis. Diakinesis. a) M4 with distal C-band (thin arrow) additionally, S9 with interstitial C-band (thick arrow). b) X chromosome with minute intercalar C-band near paracen- tromeric one (thick arrow). 2. Parapodisma subastris. Diakinesis. M4 with minute telomeric C-block (thin arrow), M6 with thick telomeric C-block (thin arrow) and interstitial C-band in heteromorphic state near the telomeric one (thick arrow). 3. Parapodisma yamato. Diakinesis. M5 and M6 with telomeric C- bloks (thin arrows). 4a-d. Parapodisma tentyuenis. a) Diakinesis. L1 with minute telomeric C-block and M6 with thick C-block (arrows). b) Metaphase II. M7 acrocentric with minute interstitial C-band (thick arrow). c) Heteromorphic M7 bivalent (thin arrow). d) Metaphase II with acrocentric (on the right) and metacentric (on the left) M7 bivalent (thin arrows). Bar equals 10 um. 354 A. G. Bugrov et al. Cytologia 65

has a minute telomeric C-block. In the prophase of meiosis the large telomeric C-blocks in the M6 pair of autosomes seem to become double (Fig. 4a). In metaphase II a clearly visible euchromat- ic zone contrasts sharply with the telomeric and near telomeric interstitial C-block in this chro- mosome. The M, pair is acrocentric with a large centromeric C-block and minute interstitial one

(Fig. 4b). In the prophase of meiosis the long au- tosomes form 2-3 chiasmata, medium-1-2, and short bivalent only one. The mean frequency of chiasmata on a cell is x=13.78 (•}1.06).

One male demonstrated chromosome het- eromorphism in the M7 pair. At prophase I the M7 bivalent consists of one acrocentric homo- logue with the large centromeric C-block and a minute interstitial one. The other homologue in this bivalent is of the same size, metacentric with very small centromeric and telomeric C-blocks in one of the arms. This heteromorphic bivalent forms only one terminal or subterminal chiasma

(Fig. 4c). At first anaphase the M7 bivalent un- dergoes divisions as in other autosomes. At sec- ond metaphase the cells with the acrocentric M7 and cells with the metacentric M7 were clearly seen (Fig. 4d). Parapodisma mikado (I. Bolivar). 2n d = 21.

The karyological analysis was performed on neuroblast mitosis of embryos. The chromosome complement 2n =21, with all acrocentrics, possi- bly shows the male karyotype (2n d=21, Fig.

5a). They may be divided into 3 size groups: 2 long (L1, L2), 7 medium (M3—M9), and 1 short

(S10). The size of the acrocentric X chromosome is approximately similar to the M5 pair. The paracentromeric C-blocks occur in all the chro- mosomes of the complement. The M5 chromo- Figs. 5-6. 5a-c. Parapodisma mikado. a) C-banded karyotype, b) mitotic metaphase. M5 pair with large inter- some pair has a small paracentromeric C-block, stitial C-bands near paracentromeric and telomeric regions a large interstitial one near the telomeric region (thick arrow). c) Thin C-bands between paracentromeric and a telomeric C-block (Fig. 5a, b). In addition, and thick interstitial C-blocks (thick arrows). 6. Fruhstorfe- there are a few thin C-bands between paracen- riola okinawensis. Mitotic metaphase, M4 with telomeric block (thin arrows). Bar equals 10 ƒÊm. tromeric and thick interstitial C-blocks, which are more prominent in early metaphase (Fig. 5c). Fruhstorferiola okinawaensis (Shiraki). 2nd=21. The karyological analysis was performed on neuroblast mitosis of embryos. The chromosome complement consisting of 2n =21, with all acro- centrics, possibly shows the male karyotype (2n 6=21, Fig. 6). They may be divided into three size groups: 2 long (L1, L2), 7 medium (M3—M9),and 1 short (S10).The size of the acrocentric X chro- mosome is approximately similar to that of the M3 pair. The paracentromeric C-blocks occur in all the chromosomes of the complement. Chromosomes of the M4 pair revealed a telomeric block (Fig. 2000 C-banded karyotypes of Podisminae grasshoppers 355

Fig. 7. Sinopodisma punctata. a) C-banded karyotype, b) mitotic metaphase. c) Mitotic metaphase is heterozygous in the presence and absent second arms in S10 (thin arrows). Bar equals 10 ƒÊm.

6).

Sinopodisma punctata Mistshenko. 2n 6=21. The karyological analysis was performed on neuroblast mitosis of embryos. The chromosome complement shows 2n = 22 in which all chromo- somes were two-armed. With two X chromosomes, the karyotype is of the female (2n •Š=22, Fig.

7a, b). This implies 2n =21 in the males. They may be divided into three size groups: 1 long (L1), 7 medium (M2—M8), and 2 short (S9, S10). The size of the acrocentric X chromosome is approximately

similar to the L1 pair (Fig. 7a). The L1, M4, M8 autosome pairs have very short second arms and may be considered as subacrocentric chromosomes. The remaining autosomes are submetacentric

and metacentric (Fig. 7a, b). One embryo is heterozygous in the presence of the second arm in the Slo autosome (Fig. 7c). The paracentromeric C-blocks occur in all the chromosomes of the comple-

ment. The second arms (short arms) of L1, M2, M3, M5, M6, and M7 chromosome pairs are he- terochromatic with quantity of C-heterochromatin, which varies among the chromosomes (Fig. 7a).

Second arms in M4, M8 are euchromatic. Second arms of the S9 chromosome pair have a large para- centromeric C-block and a thin telomeric one (Fig. 7a). Telomeric C-blocks in the long arms of

chromosomes were found in the L1 autosome pair only (Fig. 7a-c).

Discussion

The analysis of chromosome complements of some Japanese grasshoppers belonging to the

subfamily Podisminae supported the view that there are 2 karyotypically different groups in this subfamily, namely a 23-chromosome group and a 21-chromosome one (White 1973, Fontana and

Vickery 1976, Hewitt 1979, Inoue 1985, Bugrov et al. 1994). 356 A. G. Bugrov et al. Cytologia 65

Podisma sapporensis has the 23-chromosome karyotype (all chromosomes are acrocentric) as in other species of the genus Podisma: P.pedestris (L.) (the vast majority of Palearctic populations; John and Hewitt 1972, Westerman and Hewitt 1985), P aberrans Ikonn. (Bugrov et al. 1994), P ty- atiensis Bugrov et Sergeev (Bugrov and Sergeev 1997), P teberdina Rme. (unpublished data), and in the species of the genera Primnoa, Melanoplus, and Eirenephilus (Inoue 1985, Bugrov et al. 1994). On the other hand, the males of P sapporrensis from Sapporo differs from the Kunashiri population of this species, which consist of 2n d=22 (20AA+neo-X+neo-Y), as the result of a rec- iprocal translocation of the X chromosome and an autosome (Bugrov 1995). The Sakhalin popula- tion of P sapporensis is more similar to the population from Sapporo but differs from it in the mor- phology of the X chromosome, which is subacrocentric in Sakhalin (Krylion population) (Bugrov 1995). Differences among the karyomorphs of Sakhalin, Kunashiri, and Hokkaido of P sapporensis could be good material for an experimental verification of the chromosomal speciation model and for the determination of the taxonomic status of island populations of P sapporensis. C-banding in P sapporensis from Sapporo is similar to that of P sapporensis from Sakhalin and the Kunashiri Islands but differs from the latter population in the presence of intercalar and telomeric C-bands in small (S9) autosome pairs. Other species of Podisma: P pedestris, P aberrans and P tyatiensis in which C-banding was examined, have the obligate telomeric block in the X chro- mosome (Westerman and Hewitt 1985, Bugrov et al. 1994, Bugrov and Sergeev 1977). Besides, P pedestris and P aberrans have the megameric M7 pair, which is variable in size and has the inter- calar and distal heterochromatin (Westerman and Hewitt 1985, Bugrov et al., 1994). The megamer- ic M7 pair was not found in a chromosome set of P sapporensis from Hokkaido, Sakhalin and Ku- nashiri Islands. Four species of the genus Parapodisma: P subastris, P tenryuensis, P yamato and P mikado analysed in this work, like the reported chromosomes of P.mikado (Bolivar), P subaptera (Hebard), P.fauriei (Bolivar), P setouchiensis Inoue, and P niihamensis Inoue (Inoue 1985) have the chromo- some number 2n 6=21. This is the same as that in species of the genus Miramella, Zubovskya, Anapodisma and Boonacris (Powers 1942, Helwig 1958, Fontana and Vickery 1976, Bugrov et al. 1994). The evolution of the 21-chromosome karyotype (2n (3=21, NF =21) of some Podisminae grasshoppers from the basic karyotype of Acrididae with the 23-chromosome one (2n d=23, NF= 23) is a traditional problem in Orthoptera cytogenetics. Various interpretations have been proposed to solve this problem (Powers 1942, Helwig 1958, White 1973, Fontana and Vickery 1976, Hewitt 1979, Bugrov et al. 1994). Using the C-banding technique, Bugrov et al. (1994) found the presence of the interstitial C-block in a medium sized autosome of some 21-chromosome species, not typical of the 23-chromosome ones. The observed interstitial blocks in one of medium autosome pairs probably suggest that this C-band corresponds to the place of tandem translocation of small auto- somes. C-banding patterns of P subastris, P tenryuensis, P yamato and P mikado confirm the pres- ence of an intercalar C-block in a middle size (M5) chromosome in 21-chromosome species of the subfamily Podisminae. As a whole the size and localization of the C-bands in examined Parapodis- ma species are generally similar to one another and differ from other 21-chromosome species be- longing to the genera Zubovskya, Anapodisma and Miramella in the presence of the large telomeric block in the M5 autosome pair. Variations in constitutive heterochromatin even among closely relat- ed species (Santos et al. 1983, Cabrero and Camacho 1986, Gosalvez et al. 1997) and populations (John 1981) suggest a slow rate of cytogenetic differentiation between species in the genus Para- podisma. A change in the position of the centromere of M8 in one male of P tenryuensis may hap- pen in 2 ways: 1) pericentric inversion and 2) functional transposition. Pericentric inversions lead to a change in the position of the centromeres in the originally acrocentric resulting in the second chromosomal arms in karyotypes of many species of Acrididae (White 1973, Hewitt 1979). The heteromorphism in M7 in one male P tenryuensis is more similar to functional transposition of the centromere. Undoubtedly, more detailed investigations are needed to demonstrate whether the pop- 2000 C-banded karyotypes of Podisminae grasshoppers 357 ulation is polymorphic for the centromere shift. Karyotype and C-banding patterns of Fruhstorferiola okinawaensis are similar to Parapodisma species but differ from them in the absence of interstitial and telomeric C-blocks in M5. It is diffi- cult to conduct comparative karyological analysis for Fruhstorferiola okinawaensis because other species of the genus Fruhstorferiola have not been karyotypically studied so far. Sinopodisma punctata have a unique karyotype among the above species observed because of two-armed chromosomes. Helwig (1958) described the karyotype of a similar type for Indopodis- ma. On this basis White (1973) suggested that multiple pericentric inversion occurred in the phy- logeny of the Podisminae. White's hypothesis may be correct for those chromosomes of S. punctata which have the euchromatic second arms (M4, M8) However, by using the C-banding technique to define heterochromatic sites it was revealed that most second arms in S. punctata are heterochro- matic. They may be interpreted as supernumerary segments in a homozygous or in a heterozygous state. In grasshoppers supernumerary heterochromatic segments are probably even more common than supernumerary chromosomes (John 1983). Polymorphism for the presence or absence of the second arm in the S10of S. punctata is a phenomenon similar to the polymorphism in supernumer- ary segments in some grasshoppers (Nur 1961, John 1973, Shaw 1971, Kayano 1973). This corrob- orates the possibility that the second heterochromatic arms in S. punctata arise owing to amplifica- tion of the particular near-centromeric chromosome segments. Apparently, the approaches for solv- ing the problem of the origin the heterochromatic second arms in S. punctata chromosomes may be successful through the study of polymorphic populations in Sinopodisma.

Acknowledgements

The authors thank to Mr. N. Murayama (Okinawa Pref.) for his kind help in collecting the indi- viduals of Sinopodisma punctata and Fruhstorferiola okinawaensis. The study was partly supported by a grant of the Russian Federal Programme "The Integration" and Polish grant KBN No. 6 PO4C 03516. We are very grateful to the anonymous reviewer provided constructive suggestion and criti- cism of the manuscript.

References

Bugrov, A. G. 1995. Interpopulation sex-chromosome polymorphism in the grasshopper Podisma sapporensis Shit from Sakhalin and Kurile Islands. Folia Biol. (Krakow) 43: 51-53. ―and Sergeev, M. G. 1997. A new grasshopper species of the genus Podisma Bertold (Orthoptera, Acrididae) from the Southern Kuril Island and its karyotypic features. Acta Zool. Cracov. 40: 47-52. ― , Warchalowska-Sliwa, E. and Maryanska-Nadachowska, A. 1994. Karyotype evolution and chromosome C-banding pat- terns in some Podismini grasshoppers (Orthoptera, Acrididae). Caryologia 40: 183-191. Cabrero, J. and Camacho, J. P. M. 1986. Cytogenetic studies in gomphocerine grasshoppers. I. Comparative analysis of chromosome C-banding pattern. Heredity 56: 365-372. Fontana, P. G. and Vickery, V. R. 1976. Cytotaxonomic studies on the genus Boonacris. I. The "eastern" taxa and a compari- son with the related genera Dendrotettix and Appalachia (Orthoptera: Catantopidae: Podismini). Can. J. Genet. Cytol. 18: 625-652. Gosalvez, J., Mason, P. L. and Lopez-Fernandez, C. 1997. Differentiation of Individuals, Populations and Species of Or- thoptera: the Past, Present and Future of Chromosome Markers. In: Gangwere, S. K. (ed.). The Bionomics of Grasshoppers, Katydids and their Kin. M.C. Muralirangan and Muralirangan. Cambridge University Press. pp. 355-383. Helwig, E. R. 1958. Cytology and taxonomy. Bios (Madison, N.J) 24: 59-72. Hewitt, G. M. 1979. Grasshoppers and Crickets. In: Borntraeger, G. (ed.). Cytogenetics Vol. 3: Insecta I. Or- thoptera. Berlin, Stuttgart. Inoue, M. 1985. A taxonomic revision of Japanese Acridoidea (Orthoptera) with special reference to their karyomorpholo- gy. Trans. Shikoku Ent. Soc. 17: 103-183. John, B. 1973. The cytogenetic systems of grasshopers and locusts. II. The origin and evolution of supernumerary segments. 358 A. G. Bugrov et al. Cytologia 65

Chromosoma 44: 123-146. •\ 1981. Heterochromatin Variation in Natural Population. In: Bennet, M. D., Bobrow, M. and Hewitt, G. M. (eds.). Chro-

mosomes Today 7. George Allen and Uniwin, London. pp. 128-137.

1983. The Role of Chromosome Change in the Evolution of Orthopteroid•\ Insects. In: Sharma, A . K. and Sharma, A.

(eds.). Chromosomes in Evolution of Eukaryotic Groups. 1: 1-110.

and Hewitt, G. M. 1972. Interpopulation sex chromosome polymorphism•\ in the grasshopper Podisma pedestris . I. Fun- damental facts. Chromosoma 37: 291-308. Kayano, H. 1973. Polymorphism for a supernumerary segments in natural populations of Mecostethus magister (Or-

thoptera). Japan. J. Genetics 48: 151-153.

Nur, U. 1961. Meiotic behavior of an unequal bivalent in the grasshopper Calliptamus palaestinensis Bdhr . Chromosoma 12: 272-279.

Powers, P. B. A. 1942. Metrical studies on spermatogonial chromosomes of Acrididae. J . Morphol. 71: 523-676. Santos, J. L., Arana, P. and Giraldez, R. 1983. Chromosome C-banding patterns in Spanish Acridoidea. Genetica 61:

65-74. Shaw, D. D. 1971. The supernumerary segment system of Stethophyma. II. Heterochromatin polymorphism and chiasma

variation. Chromosoma 34: 19-39. Westerman, M. and Hewitt, G. M. 1985. Chromosome banding in Podisma pedestris. Heredity 55: 157-161.

White, M. J. D. 1973. Animal Cytology and Evolution, 3 ed. Cambridge University Press.