Ustulata (Orthoptera: Tettigoniidae) Analysed by C and G Banding
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Heredity 56 (1986) 161—165 The Genetical Society of Great Britain Received 21 June 1985 H ete roch romati n variants in Baetica ustulata (Orthoptera: Tettigoniidae) analysed by C and G banding J. Navas-Castillo, Departamento de Genética, Facultad de Ciencias, J. Cabrero and Universidad de Granada, 18071 Granada, Spain J. P. M. Camacho Extensive variation in heterochromatin exists in natural populations of Baetica ustulata, as deduced following the application of C and 6 banding techniques. These techniques enable us to describe nine C-heterochromatin variants for the M chromosomes (M3, M4 and M5) and to distinguish between the response of several classes of heterochromatin to G-banding. Different populations of this species are very different for the C-heterochromatin variants, which is due to their geographical isolation, the low mobility of these insects and the extensive intrapopulational variation which, as we propose, may be increased by meiotic recombination. Three classes of response to C banding show the different C-heterochromatic regions analysed. Firstly, a contrary response is shown by the nucleolar associated heterochromatin interstitially located in the long arm of L2 chromosome so that it C-bands darkly but G-bands lightly. Secondly, a parallel response to both techniques is demonstrated by centromeric heterochromatin. Thirdly, the large heterochromatic blocks in the M chromosomes show dark C-banding but "grey" G-banding, being those in M3 and M4 surrounded by small dark G-bands. INTRODUCTION of Warramaba Virgo(Webb et a!., 1978) and Melanoplussanguinipes(Zhan et a!., 1984). IntraspecificC-heterochromatin variation has Baetica ustulata isthe only species of this been observed in many species of plants and monospecific genus, and is endemic in the Iberian animals. One of the possible significations of the Peninsula, living only in Sierra Nevada (Granada) polymorphisms for C-heterochromatin content at altitudes ranging from 2500 m to 3450 m rests on the influence that C-positive blocks have (Pascual, 1978). This species has been studied on both chiasma formation in the nearby regions cytologically by Fernández-Piqueras et a!. (1984) to their location in the chromosome and mean cell in populations from the North side of Sierra chiasma frequency (Hewitt, 1979; John, 1983; Nevada, and they found an extensive polymorph- Navas Castillo et a!., 1985). However, the charac- ism for C-heterochromatin. In this paper we terisation of the different C-band variants is a analyse one population sample from the South side necessary previous condition before generalising of Sierra Nevada using C and G banding tech- the results obtained from particular cases. In this niques. sense, the heterochromatin of the standard comple- ment seems to be heterogeneous (see Rocchi, 1982), and the supernumerary heterochromatin MATERIALSAND METHODS may show different responses to the same C- banding technique (Camacho et aL, 1984). In order Elevenmales and seven females were caught in to characterise the nature of heterochromatin Alto del Chorrillo (2700 m altitude), which is situ- variants it is advisable to use several different tech- ated at the South side of Sierra Nevada (Granada, niques which may discriminate between them. Spain). Testes were fixed in ethanol-acetic acid Although C-banding is easily obtained in orthop- (3: 1). Females were injected with 005 per cent teran chromosomes, G-banding has only been well colchicine in insect saline for 6 hours prior to defined for the B chromosomes of Chortoicetes fixation of the ovarioles. C-banding was conducted terminfera (Webb, 1976; Webb and Nehaus, 1979) as described by Camacho et aL (1984) and G- and, with less definition, in standard chromosomes banding with trypsin followed by a brief treatment 162 J. NAVAS-CASTILLO, J. CABRERO AND J. P. M. CAMACHO with 2 x SSC at 60°C. This G-banding technique Some are similar and others are different from was recently been put forward for mammal those observed by Fernández-Piqueras et a!. (bc chromosomes by our colleagues M. Burgos, R. cit.,) in North side populations. The C-hetero- Jiménez and Dr R. DIaz de la Guardia (submitted chromatin variants for the M autosomes found in for publication). our population are shown in fig. 3. They were two for the M3, three for the M4 and four for the M5. The most frequent type of M3 (A-variant) carries RESULTS two C-blocks, one subterminal and the other distal. This latter block is slightly larger than the subter- Thechromosome complement of the ephippigerid minal one. The other M3 variant (B) carries only Baetica ustulata consists of 2n =24+XO/XX the subterminal C-band. chromosomes(fig. 1) and the 12 autosomal pairs The three types of M4 chromosomes carry two may be divided into three size groups: Two long C-blocks, one of which is subterminal the other (L1 and L2), three medium (M3 —M5)and seven distal. The A-variant possesses two C-blocks of short pairs (S6—S12). Both pairs of L autosomes similar size separated by an euchromatic short are metacentric while the remainder, including the region, the B-variant is similar but with larger X, are telocentric. The size of the X chromosome C-blocks, and the C-variant has a small subter- is intermediate between that of the L and M auto- minal C-block plus a larger distal one. somes. The C-banding pattern consists of small The M5 is the most variable chromosome in paracentromeric C-bands in all chromosomes, and our population caused by the presence of supernu- there is an interstitial C-band in the long arm of merary chromosome segments. We found four L2 chrosome which coincides with the only active different types: One lacking any distal C-hetero- NOR in this species (fig. 2). In addition there are chromatin (A), one carrying a small distal C-band prominent C-blocks located interstitially, subter- (B), the third carrying a large distal C-block (C), minally and distally in the M chromosomes (fig. 1). and the fourth (D) carrying a distal C-block double Fernández-Piqueras eta!. (1984) have reported the size of that found in C. extensive variation for the C-blocks in the M The G-banding technique gives a characteristic chromosomes. In our popftlation sample, collected G-banding pattern in the L and M autosomes (fig. from the South side of Sierra Nevada, there is also 4). The two L1 chromosomes show a similar pattern variation for the C-blocks in these chromosomes. of dark bands and light interbands which coincide L2 X 9 fr Is.. M3 S6 S7 *1 • 0 'I'•1 *4 L2 44' $9 5io 5ii 12 :8 - 2 Figures land 2 C-banded karyotype of a M4—A/M4— B heterozygous male (1), and silver stained pachytene cell showing the location (arrowhead) of the nucleolus organiser region (NOR) in Baeticaustulata. NU:nucleolus. HETEROCHROMATIN VARIANTS IN BAETICA USTULATA 163 mentis less condensed a series of small bands and interbands are apparent (see fig. 7). In an individual carrying two different M4 chromosome variants (A and C) we observed incurved pairing configuration in pachytene cells (fig. 10), which indicates that homologous pairing is specific for the dark G-bands, but inspecific for the light interbands. a Meiotic segregation of the distal C-block in M5 0 heteromorphic bivalents is always equational due nSA to the formation of one chiasma in the region between the C-blocks and the centromere (figs. 11 and 12). In the M3 bivalents we have occasionally observed chiasma formation in the euchromatic region between the C-blocks (see fig. 6), which a might be a source of new chromosome variants a when they occur in heteromorphic M3 (and per- (n haps M4) bivalents. r.c DISCUSSION The present report analyses a population sample from the South side of Sierra Nevada, while Fernández-Piqueraset al. (1984) studied individuals from the North side of Sierra Nevada. Figure 3C-heterochromatin variants for M3, M4 and M5 Both localities are sited very far and completely chromosomes. isolated one another, given the low mobility of these apterous insects. In the North side popula- in their position and sequence. The same is tions, Fernandez-Piqueras et a!. (1984) found one apparent in the L2 chromosomes but, here, one of type of M3 and four types of M4 and M5 chromo- the light interbands in the long arm coincides in somes on the basis of C-banding pattern. In the position with the dark C-band associated to the South side population, we have found two types NOR (see also fig. 5). The C-heterochromatin of M3, three types of M4 and four types of M5 blocks in the M chromosomes show differential chromosomes, some of which are similar but others response to G-banding, so that some C-regions are different to those in the North side populations: G-band darkly and others only lightly (figs. 6-10). The two types of M3 in the South side are different The M3 shows in the region of the C-heterochro- to the only type in the North side (compare our matic blocks, a G-banding pattern consisting of fig. 3 with fig. la,b in Fernández-Piqueras et al. two unequal "grey" interbands surrounded by bc cit.,). The M4 type A in South side population three small dark bands, one being proximal, one and that type A in North side populations seems sited between the two interbands (which presum- to be the same but, however, the two other M4 ably coincides with the euchromatic region variants in the former are different to the three between both C-blocks) and one distally located other ones in the latter. Two out of the four M5 (figs. 6 and 7).