C1997The Japan Mendel Society Cytologia 62: 53-60, 1997

Karyotype Variability in the Genus Radacridium (, , Romaleinae )

M. F. Rocha1, M. J. Souza1 and T. Tashiro 2

1 Department of Genetics(CCB) ,2Department of PhysicalEducation(CCS), Federal Universityof Pernambuco, Recife-PE,50732-970, Brazil

Accepted November 28, 1996

The family Romaleidae probably originated in the tropics, although some genera (Brachy- stola, Dracotettix, Litoscirtus, Phrynotettix, , ,Tytthotyle) secondarily in- vaded the Nearctic region (Carbonell 1977). This family comprises more than 80 grasshopper genera distributed in different habitats from the tropical forest to semiarid environments (Kevan 1982). Over the last two decades, several taxonomic and geographic distribution studies have been conducted on various genera of this family, among them Chromacris, Xestotrachelus (Roberts and Carbonell 1982), Agriacris and Staleochlora (Roberts and Carbonell 1992). Radacridium nordestinum is the only species in the genus Radacridium described by Carbonell (1984) as monotypic and endemic in the Brazilian Northeast. A new species denoted R. mariajoseae Carbonell e Mesa, in litteris was collected in the State of Pemambuco, Brazil. R. nordestinum and R. mariajoseae are respectively distributed in the "caatinga" and "agreste" zones of the State, where they cohabit with other romaleids (Brasilacris, Helionotus and Xyleus) that inhibit these regions or nearby regions. The two Rodacridium species have external morphological differences especially of a chromatic nature. The ventral region of the thorax and abdomen and the area of the pinnae are white-yellowish and orange in R. nordestinum and red-orange and ivory-yellowish in R. nariajoseae, respectively. In the phallic complex, the apical valves of the endophallus are narrow and pointy in the former species and wide with a rounded apex in the latter (Carbonell, personal communication) . The use of certain chromosome staining techniques permits the observation of genetic polymorphisms in natural populations, especially in grasshoppers, facilitating karyotype com- parison and the establishment of phylogenetic and evolutionary relations among species. C-banding and silver staining permit the observation of structural and functional aspects of the chromosomes by analysis of the size and location of constitutive heterochromatin blocks, kinetochores, synaptonemal complex, cores, nucleolus and nucleolar organizer regions (King and John 1980, Cabrero and Camacho 1986a, Rufas et al. 1985, Rufas and Gosalvez 1982, Fernandez-Piqueras et al. 1982, Suja et al. 1991). Basic information about the karyotypes of almost 50 romaleid species has been published (Mesa et al. 1982). However, few C-banding studies are available (Vilardi 1986, 1988, Souza and Silva-Filha 1993) and no literature data are available about the use of other techniques for chromosome identification. In the present investigation we studied the karyotypes of the above two Radacridium species using C-banding and silver staining applied to meiotic chromosomes in order to compare some structural features of the chromosomes, numerical and morpholog- ical characteristics, and constitutive heterochromatin patterns, and also to determine the effect of supernumerary chromatin on the frequency and distribution of chiasmata. 54 M . F. Rocha, M. J. Souza and T. Tashiro Cytologia 62

Material and methods

The specimens of R. nordestinum and R. mariajoseae analyzed in the present study were obtained in diurnal collections from natural populations of the zones of the Caatinga (Parna - mirim and Serroldndia) and of the Agreste (Gravata, and Buique ) in the State of Pernambuco , Northeastern Brazil, from 1992 to 1994. A total of 97 individuals were studied in Parnamirim ,

25 in Serrolandia, 89 in Gravatd and 46 in Buique . Cytologic preparations were obtained from the ovarioles of females submitted to treatment with 0.1 % colchicine for 6 hr, and from testes. The gonads were fixed in 3 : 1 ethanol- acetic acid, submitted to the classical squashing technique and stained with 2% lactoacetic orcein .

Part of the male preparations were treated by the technique of Sumner (1972) for C-banding . The slides were exposed to an acid solution (0.2 N HC1) followed by basic solutions (Ba(OH)2 ) and saline (2 X SSC), the last two at a temperature of 60•Ž. For silver staining (Rufas et al . 1987), the preparations were pretreated with 2 X SSC and then stained with a silver nitrate solution (1 g/ 1 ml), with the pH 3.0 adjusted with formic acid , and incubated at 70-80 •Ž . Photomicrographs were obtained with a Zeiss III microscope using Agfa Copex Pan Film .

Photographic copies were made using Agfa 3 Brovira paper .

Results

R. nordestinum (Carbonell 1984) and R. mariajoseae Carbonell e Mesa, in litteris are described here chromosomally for the first time. Both had 2n = 24 , XX karyotypes for females and 2n = 23, XO karyotypes for males. All autosomes were acrocentric and were grouped into three large pairs (L1-L3), six medium pairs (M4-M9) and two small pairs (S1o-Si 1) (Fig. 1) .

The X chromosome is large in both species and is approximately equivalent to pair 3. In both species the M9 pair is heteropycnotic positive during prophase I and was considered to be the megameric chromosome, serving as a marker within the karyotype of this genus .

Fig . 1. Spermatogonial metaphase of Radacridium nordestinum (2n=23, XO ) . 1997 Karyotype Variability in the Genus Radacridium 55

The constitutive heterochromatin of R. mariajoseae, visualized by C-banding, is located in only a few chromosomes in the complement. Small constitutive heterochromatin blocks are found in the pericentromeric region of pair L1 and in the proximal region of pair L2. In turn, pairs Sioand S11presented minute pericentromeric bands of difficult visualization. Chromosome X showed three constitutive heterochromatin blocks, a pericentromeric one and a proximal one of medium size, and a third small distal block (Fig. 2A). The megameric bivalent M9 presented a more complex C-banding pattern, with proximal and distal bands and a narrower interstitial band. This pattern corresponds to what is observed in terms of the positive heteropycnosis of prophase I during meiosis. All chromosomes in the complement of R. nordestinum showed small pericentromeric blocks of constitutive heterochromatin (Fig. 2B). Interstitial heterochromatin blocks were identified in at least two bivalents (probably M5 and L2). These blocks are visualized in the earlier phases of condensation during meiosis. The C-banding pattern of the megameric chromosome M9 most of the times was ill defined due to the high extent of heteropycnosis of

A B

C D

Fig . 2. Representatives C-banded chromosomes of male (A) R. mariajoseae (B-D) R. nordest- inum. Note the bivalents presented supernumerary heterochromatic segments (bivalents 3, 4 and 7) and the megameric bivalent 9. 56 M. F. Rocha, M. J. Souza and T. Tashiro Cytologia 62 this chromosome however, it was possible to observe pericentromeric heterochromatin blocks in this chromosome. This heterochromatin distribution was considered to be the normal pattern for R. nordestinum. On the other hand, some individuals from different R. nordestinum populations presented supernumerary heterochromatin segments (SS). Of the sample of 12 male specimens from the Serrolandia population, 3 were heterozygous for an interstitial SS in the M4 bivalent. Of the 75 male specimens from Parnamirim, 12 were heterozygous for heterochromatin SS in the following bivalents: L3; M4 and M7 (one specimen) and M4 ( 11 specimens) (Fig. 2B-D ). Analysis of the mean chiasma frequency and distribution was carried out on six of the 12

Table 1 . Comparison of chiasma distribution between the standard karyotype and the karyotype with a supernumerary segment in chromosome M4 in R. nordestinum from the Parnamirim population

Fig . 3. Representatives Ag-stained pachytene cells (A) R. nordestinum; (B) R. mariajoseae. Note selected pachytene bivalents showing nucleoli attached to specific positions: (ab) R. nordestinum; (c-d) R. mariajoseae. 1997 Karyotype Variability in the Genus Radacridium 57

A B

Fig. 4. Ag-stained bivalents of male R. nordestinum (A) 'core' in metaphase I; (B) kinetochore in metaphase II. Arrow indicate the X chromosome.

R. nordestinum specimens from Parnamirim that were heterozygous for the SS of the M4 pair, and the data were compared to those obtained for a standard sample (basic karyotype) of the same population. A mean number of 20 diplotene chromosomes per individual from the two samples were analyzed. A comparison of the mean chiasma frequency per cell between samples did not reveal a significant difference (T = 0.1901, P > 0.05). When the two samples were compared in terms of chiasma distribution, the sample with SS presented a significant reduction in the frequency of proximal chiasmata in the group of medium bivalents, an increase in the frequency of interstitial chiasmata in all three groups, and a decrease in distal chiasmata in the group, of small chromosomes (Table 1). Silver nitrate staining permitted the differentiation of nucleoli, which stained black or dark brown, in contrast to the chromatin of the chromosomes which stained yellow. Only one nucleolar organizer pair was observed in each species. In R. nordestinum the NOR was located in the interstitial area of chromosome L2 (Fig. 3A), and in R. mariajoseae in the area proximal to chromosome X (Fig. 3B). No secondary NORs were observed. Other differentially staining silver nitrate-labelled structures were adjunct centrioles (ACs), the cores and the kinetochores. The kinetochores presented a rounded shape during pachytene. Starting from diakinesis, the homologous kinetochores were individualized and at metaphase II they became flattened (Fig. 4A, B). In some metaphases and in anaphases I it was possible to observe the association of kinetochores with the cores. In general, it was possible to clearly visualize only one core per bivalent however, these cores are not continuous and are interrupted by the chiasmata that occur between the homologues. It is quite clear that in the univalent X each chromatid has its own core (Fig. 4A, arrow).

Discussion The genus Radcridium (Carbonell 1984) is endemic in Northeastern Brazil and is strongly adapted to the adverse conditions of aridity prevailing in this Region. The two species of this genus present great similarity in outer morphology, a fact that makes their distinction difficult. 58 M. F. Rocha, M. J. Souza and T. Tashiro Cytologia 62

Both present strong color mimetism with the environment. The color pattern varies from rust to marble in its different tonalities. Although they are morphologically close and have similar diploid numbers and chromo- some shapes, the two Radacridium species have different C-banding patterns. This divergence indicates that heterochromatin rearrangements, losses and acquisition may be involved in the karyotypic evolution of the genus Radacridium. Pericentromeric constitutive heterochromatin occurs in all chromosomes of R. nordestinum and interstitial blocks occur in at least two chromosome pairs. Furthermore, there is a large number of extra segments involving several chromosomes in the complement. R. mariajoseae, in turn, has C bands only in chromosome X and in five autosome pairs, and these bands are predominantly located in the pericentromeric region. In R. nordestinum there is wide polymorphic variability for supernumerary heterochroma- tin segments. If we consider that chiasmata are not produced in the heterochromatin regions (Fox et al. 1973), it may be assumed that the presence of segments in the homo- or heterozygous form affects the pattern of chiasma distribution, at least in the bivalent involved. This effect has been observed in several species of grasshoppers (Santos and Giraldez 1982, Camacho et al. 1984, Souza and Silva-Filha 1993). On the other hand, it has been observed that heterochromatin segments usually involve the dislocation of chiasmata from their proximity. This effect is independent of the location and nature of the segment (Camacho et al. 1984, Navas-Castilho et al. 1987). In R. nordestinum, the interstitial heterochromatin segment of the M4 pair causes a redistribution of chiasmata in this bivalent but does not alter the mean frequency of chiasmata per cell. Depending on their chromosomal location, the chiasmata may have different genetic and functional meaning. Considering that proximal or distal chiasmata only involve the recombi - nation of a few genes and basically serve to guarantee correct chromosome segregation, the interstitial chiasmata present a greater potential for recombination (Zarchi et al. 1972). This suggests that the interstitial heterochromatin block of chromosome M4 of R. nordestinum may be of high adaptive value for this species since it changes drastically the frequency of interstitial chiasmata. Because of its karyotypic variability and chiasma redistribution, R. nordestinum seems to be a species with high genomic plasticity, greatly differing from R. mariajoseae. It is possible that the redistribution of chiasmata produced by SS has important consequences for the formation and conservation of coadapted gene complexes. Camacho et al. (1989) suggest that the gene combinations present in the proximity of SS may remain intact due to to the absence of recombination. These assumptions are based on the need for primary genetic variability in a population (Camacho et al. 1989). In the family Romaleidae, like in Acrididae, there is a predominance of species with uniform karyotypes of the type having 2n = 23 chromsomes in males. This apparent uniformity contrasts with the high variability detected in the C-banding and NOR patterns observed in acridids (King and John 1980, Santos et al. 1983, Rufas et al. 1985). On the other hand, although few C-banding studies are currently available within Romaleidae (Vilardi 1986, 1988, Souza and Silva-Filha 1993) and NOR data are scarce, a significant variability in NORs and in C-banding patterns has been detected in Xyleus angulatus, Brasilacris gigas and Chromacris speciosa (Souza and Kido 1995) and in the Radacridium species analyzed in the present study. The two Radacridium species, in addition to differing in C-banding pattern, also differ in NOR position (autosomal in R. nordestinum and allosomal in R. mariajoseae). The chromo- some position and distribution of the NORs have been analyzed in many grasshopper species. Rufas et al. (1985) proposed that chromosome X and the megameric chromosome represent the ancestral NOR location in the family Acrididae. Even though the X corresponds to a 1997 Karyotype Variability in the Genus Radacridium 59

NOR-bearing chromosome in several species, most of the times the NOR is also associated with one or more autosomes (Rufas et al. 1985, Cabrero and Camacho 1986b). R. mariajoseae, therefore, represents one of the few species having only the X as a nucleolar organizer. Viseras and Camacho (1984) suggested that this association may be important for NOR activity. In Radacridium, as in some other grasshopper species, the NOR location apparently coincides with constitutive heterochromatin blocks. The observation of cores and kinetochores in Radacridium by the same technique as reported for Acrididae (Rufas et al. 1987) suggests a greater structural chromosomal similarity between these two families. More primitive families among grasshoppers, such as Proscopiidae, did not yield clear results with this technique (Moura 1995). As a whole, chromosome analysis revealed that, despite the apparently superficial mor - phoanatomic differentiation of the two Radacridium species, they present a considerable evolutionary divergence at the cytogenetic level, supporting their identifies at the species level.

Summary The genus Radacridium (Romaleidae) is endemic in the Northeast region of Brazil and is chromosomally described here using specimens collected in the Agreste (R. mariajoseae) and in the Caatinga (R. nordestinum), State of Pernambuco. Both species presented 2n = 23, XO in males and acrocentric chromosomes. The two species can be distinguished on the basis of C -banding patterns and location of the nucleolar organizer regions (NORs). R. mariajoseae showed C -bands only in the pericentromeric regions of autosomes L1, M9, S10 and S11and in the proximal regions of L2. Chromsome X presented proximal and distal pericentromeric bands. R. nordestinum, in turn, has pericentromeric bands in all chromosomes and interstitial bands in pairs L2 and M5. The bivalent M9 corresponds to the megameric chromosome for the two species. Furthermore, R. nordestinum presented four chromosome pairs (L3, M4, M6, M7) bearing supernumerary heterochromatin segments that were absent in R. mariajoseae. In R. nordestinum a comparative analysis of standard karyotypes and karyotypes with an interstitial supernumerary segment in pair 4 revealed chiasma redistribution in all chromosome groups, with no change in mean chiasma frequency per cell. The nucleolar organizer chromosome was the X in R. mariajoseae and the L2 pair in R. nordestinum. Cores and kinetochores stained identically in the two species. Taken together, the chromosome data show that these species are cytogenetically well distinct .

Acknowledgments The authors are grateful to Prof. Carlos S. Carbonell for the taxonomic determinations and to Prof. Marcelo Guerra for valuable suggestions and comments about the manusciript. This research was supported by Fundacao de Amparo a Ciencia e Tecnologia do Ertado de Pernambuco (FACEPE) and by Conselho Nacional de Desenvolvimento Cientifico e Tecnolo - gico ( CNPq ) .

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