CARYOLOGIA Vol. 57, no. 1: 25-29, 2004

Karyotype and C-banding patterns of the katydid elongata (L.) (, , ) from Amami Is. (Japan) and Borneo (Malaysia)

A.G. BUGROV1, E. WARCHALOWSKA-SLIWA2, *, ITO G.3, A. TCHERNYKH4 and MARYATI M.5 1 Novosibirsk State University, 630090 Novosibirsk, Russia and Institute for 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, Slawkowska 17, Poland. 3 Systematic Entomology, Department of Ecology and Systematics, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan. 4 Ju¯ras Ve¯jsˇ, Riga, 1045, Latvia. 5 Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Locked bag 2073, 88999 Kota Kinabalu, Sabah, Malaysia.

Abstract - Two types of the karyomorphs in the katydid (L.) from Borneo (Malaysia) and Amami Is. (Japan) were found. The chromosomal comple- ment of M. elongata from Amami Is. and one karyomorph from Borneo (Kota Kinabalu – song 1) consists of 28 autosomes plus the X in the male (2n=29). In two long pairs, pair L1 is metacentric, whereas pair L2 is subacrocentric. Other autosomes are meta or submetacentric. The size and shape of the X chromosome is similar to the L1 pair. “Kota Kinabalu - song-2” karyomorph has the same chromosome num- ber as “KK song-1” and “Amami”. However, in ”KK song-2” the autosomes belong- ing to L2 pair are metacentric. The size of these chromosomes is approximately sim- ilar to the L1 pair and the X chromosome. The discrete chromosome differentiation in M. elongata suggest that this species consists of several biological species. In M. elongata from Borneo, the assumption of a belonging of two groups of the kary- omorphs to different species proves to be true also in differences in crepitating. Key words: karyotypes, C-banding, evolution, Orthoptera.

INTRODUCTION ine the exact taxonomic position of different populations of this species because several Mecopoda elongata (L.) is one of the more types of song are observed in among them. The widely distributed katydids. The area of this present authors used cytogenetic analysis of species includes the greater part of the Orien- three song populations of M. elongata from tal region and Northern part of the Aus- Japan and Malaysia for the first step in revising tralasian one. At present two subspecies are this species. recognized in the species as a consequence that There are very few cytogenetic data con- many species, subspecies, and variations have cerning the Mecopoda. At first HAREYAMA been synonymized as M. elongata (OTTE 1997). (1932) reported a diploid number of 27 in the These invalid taxa different only slightly in male of M. elongata nipponensis from Japan. morphology. However, it is necessary to exam- Later, ASANA et al. (1938) described a kary- otype of the M. elongata from Northern India with 2n = 27 consisting of one pair submeta- * Corresponding author: e-mail: [email protected]. krakow.pl centric, one pair subacrocentric, 11 pairs of 26 BUGROV, WARCHALOWSKA-SLIWA, ITO, TCHERNYKH and MARYATI

Fig. 1a-d. – Mecopoda elongata of individual from Amami Is.: (a) spermatogonial metaphase; (b) male karyotype – in L2 C-bands heteromorphic in size; (c) metaphase I; (d) spermatogonial metaphase – in L2 C-bands homomorphic on size. acrocentric chromosomes, and the metacentric elongata from the same site, in the vicinities of X-chromosome. Relatively recently ASWATHA- Mysore city, which differ from the karyotype of NARAYANA and ASHWATH (1994) described the Mecopoda sp. in chromosome number and karyotypes of two species belonging to the morphology of the second autosome pair. Un- genus Mecopoda and reported the C-banding fortunately, they did not mention any morpho- pattern of one of those species named as logical or crepitating differences between M. Mecopoda sp. They confirmed the possesing of elongata and Mecopoda sp. a 27 chromosome form in Mecopoda on the ma- The level of diversity of chromosome com- terial from South West India. However, they plements may suggest that karyotypic diversifi- described the 29 chromosome karyotype in M. cation is one of the features of the speciation in KARYOTYPE AND C-BANDING IN MECOPODA ELONGATA 27

Fig. 2a-c. – Mecopoda elongata of individual from Borneo: (a) male karyotype from individual of “KK song-2”, pair L2 is meta- centric ; (b) metaphase I and (c) metaphase II with two metacentric bivalents L1 and L2 (arrows). this species. This suggestion was the reason for excised, incubated in hypotonic solution (0.9% sodi- studying the karyotypes of M. elongata from um citrate), fixed in ethanol:glacial acetic acid (3:1), new localities. In the present paper two kary- and kept in 70% ethanol. Air dried preparations were omorphs of M. elongata from Japan and made by squashing the testicles in 45% acetic acid and Malaysia are reported. freezing them in dry ice. They were then stained with C-banding by treatment with 0.2 N HCL for 15 - 20 min, immersed in a saturated solution of Ba(OH)2 at 61°C for 3-5 min, rinsed in water, immersed in 2xSSC MATERIALS AND METHODS at 61°C for 60 min, rinsed, air-dried, and stained with 2% Giemsa. In early September 2000, 10 male nymphs and 2 adult males were collected in Amami Is., Ryukyu Arch- ipelago, Japan (“Amami”). In late November 2001, 8 RESULTS adult males were collected in Kota Kinabalu, Sabah, Malaysia on a song at night. In Kota Kinabalu two dis- The chromosomal complement of “Amami” tinct types of song were observed (“KK song-1” and consists of 28 autosomes plus the X in the male “KK song-2”). “Song-1” is a continuous monotonous (2n=29). Autosomes can be divided into three crepitating. “Song-2” consists of separate portions of size groups: 2 large (L1 and L2), 12 medium, and the crepitating. Both songs differed from the song of small (M – S ) (Fig. 1). In two long pairs, pair “Amami”. But the continuous crepitating of “Amami” 3 14 that follows after several short crepitating is almost the L1 is metacentric, whereas pair L2 is subacro- same as “KK song-1”. The males were placed in two centric. Other autosomes are meta, submeta- or separate plastic boxes according to their songs. subacrocentric, gradually decreasing in size. The males and male nymphs were injected with The size of submetacentric X chromosome is 0.1% colchicine for 1.5 - 2.0 hours. The testes were approximately similar to the L1 pair (Fig. 1a,b). 28 BUGROV, WARCHALOWSKA-SLIWA, ITO, TCHERNYKH and MARYATI

The paracentromeric C-blocks occur in all ciation is connected with the stasipatric model chromosomes of the complement, their size of speciation (WHITE 1968, 1974). One of the varying among autosomes. The first pair of the important features of the stasipatric model is autosomes has a thick centromeric C-block that certain chromosomal changes play a key with an interstitial one near centromeric posi- role in the initial stages of speciation (WHITE tion, and telomeric C-blocks in both arms 1968). The conception of chromosomal speci- which vary in size. The second pair has a thick ation stimulated a development of the evolution paracentromeric C-band, heterochromatic cytogenetic investigation in different groups of short arms, and telomeric C-band in the long animals. As to Orthopteran , more later arm (Fig. 1a-d). Sometimes both C-blocks were researches showed that karyotipic differences heteromorphic in size (Fig. 1a,b). The auto- are only a weakly selected marker for more fun- somes belonging to the M3 and S4 pairs have damental changes in genome (SHAW and paracentromeric C-bands and telomeric ones WILKINSON 1980; BARTON and HEWITT 1981). in long and short arms. Other autosomes are Nevertheless, the discussions about the role of characterised by the presence of paracen- chromosome changes in evolution have contin- tromeric C-bands and telomeric ones in some ued to the present (KING 1993). In spite of the of them (Fig. 1b). Short arms in five small pairs, discussion concerning the concept of chromo- (S5, S6, S7, S8, S9), are heterochromatic. In oth- some speciation in Orthoptera there are very er small pairs both the arms are euchromatic. few models connected with this problem. Prob- The X chromosome has a thick paracen- ably, the Mecopoda elongata (L.) will become tromeric C-block; sometimes it is clearly seen, one more model in this respect. such as a double, interstitial one near the telom- In the area of distribution in South and eric end in the shorter arm, and interstitial and South East Asia M. elongata presented some telomeric ones in the longer arm (Fig. 1a-d). chromosomal races. One karyomorph from The chromosome complement of “KK South India (ASWATHANARAYANA and ASH- song-1” has a karyotype very similar to “Ama- WATH 1994), Amami Is., Japan (“Amami”, pre- mi” as on morphology of the chromosomes, sent paper), and Borneo (“KK song-1”, present and on localities of the C-banding regions. Un- paper) have common chromosome number like those from the “Amami” population, the (2n=29). “KK song-1” and “Amami” differ telomeric C-blocks in the X chromosome of from the Indian 29 chromosome M. elongata in “KK song-1” population show a sporadically the presence of a short second arm in L2 auto- variability in size . some. At the same time, “KK song-1” and “KK song-2” has the same chromosome “Amami” have identical karyotypes. C-band- number as “KK song-1” and “Amami”. How- ing patterns in chromosomes of Japanese and ever, in ”KK song-2” the autosomes belonging Bornean 29 chromosomal races also are very to L2 pair are metacentric (Fig. 2a-c). The size similar. Another Mecopoda species from India of these chromosomes is approximately similar named as Mecopoda sp. has a different type of to the L1 pair and the X chromosome. The re- chromosome complement consisting of 26 au- sults showed some differences in C-banding tosomes plus the X in a male (ASWATHA- patterns in the two song types. The second NARAYANA and ASHWATH 1994). The first pair metacentric pair, such as L1, is characterised by of autosomes is submetacentric with a minor thick-double paracentromeric C-blocks. Addi- centromeric C-block, minor telomeric C-block tionally, in third medium size pair (M3) of au- in the long arm, and a thick telomeric one in tosomes the euchromatic region in short arm the short arm. The X-chromosome resembles and telomeric C-blocks in long arm were ab- the first chromosome pair except for the pres- sent (Fig. 2a). ence of the interstitial band near the cen- tromere and the absence of the small telomer- ic C-band in the long arm. The second pair of DISCUSSION the autosome is subacrocentric with polymor- phic in the size of centromeric C-block. There There is great interest among evolutionary are two types of the small chromosome pairs in biologists in the role of chromosome changes in Mecopoda sp. Six pairs of them are meta- and speciation. The discussion on chromosome spe- submetacentric, while other pairs are acrocen- KARYOTYPE AND C-BANDING IN MECOPODA ELONGATA 29 tric. In common, C-banding patterns of this ASWATHANARAYANA N.V., ASHWATH S.K., 1994 – karyomorph are similar to those of M. elongata Karyotypes of two Indian Mecopodinae (Or- from Amami and from Borneo. thoptera – Tettigoniidae). Cytologia, 59: 285-287. “KK song-2” has a more peculiar structure BARTON N.H., HEWITT G.M., 1981 – The genetic of karyotype in the presence of a large second basis of hybrid inviability in the grasshopper metacentric pair between the researched pop- Podisma pedestris. Heredity, 47: 367-383. ulations of M. elongata. The karyotype of such HAREYAMA S., 1932 – On the chromosomes of some insects belonging to Locustidae. Zool. Mag. morphology was not previously described in M. (Japan), 44: 83-84. elongata. KING M., 1993 – Species Evolution: The Role of The high level of chromosome differentia- Chromosome Change. Cambridge University tion among populations in M. elongata argue Press, Cambridge. the suggestion that this species consists of sev- MRONGOVIOUS M.J., 1979 – Cytogenetics of the hy- eral biological species, since karyotypic diver- brids of three members of the grasshopper genus sification often leads to reproductive isolation Vandiemenella (Orthoptera: Eumastacidae: Mora- owing to hybrid incompatibility (WHITE et al. binae). Chromosoma, 71: 81-107. 1967; MRONGOVIUS 1979; BARTON and HEWITT OTTE D., 1997 – Tettigonioidea. Orthoptera Species 1981). As to M. elongata from Borneo, the as- file, n. 7. 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