The chromosome banding of some Italian Amaryllidaceae

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1 CARYOLOGIA Vol. 52, n. 1-2: 87-92, 1999 The chromosome banding of some Italian Amaryllidaceae G.F. D'AMATO * and G. BIANCHI Dipartimento di Biologia Vegetale, Universita "La Sapienza", P.le Aldo Moro, Roma, Italia Abstract Cytological investigations of some representative species of Amaryllidaceae belonging to the Italian flora were carried out using banding techniques and silver nitrate staining for nucleoli and NORs. The occurrence of diverse quality and amount of heterochroma-tin was observed in metaphase chromosomes but in general the patterns of C-bands are very simple. Mean karyotype length and Simmetry index for each investigated species are also reported. Key words: Amaryllidaceae, cytogenetics, C-bands, heterochromatin, NORs. INTRODUCTION Amaryllidaceae is one of the largest and most important families of Monocots, mainly of warmtemperate and subtropical regions. The species of the Italian flora are grouped in five genera (PIGNATTI 1982). Among them, Leucojum and Narcissus comprise most species. Pancratium and Sternbergia comprise two species each, while the genus Galanthus is represented only by the snowdrop G, nivalis. Most of these plants are distributed throughout the Mediterranean region to the Balkans and East Europe often displaying overlapping areas. Current information concerning chromosome banding in this family has shown no significant heterochromatin content or very low C-band numbers on metaphase chromosomes (VosA and MARCHI 1979; KURITA 1986; SINHA and ROY 1986; D'AMATO and DE DOMINICIS 1996; YUZBASIOGLU et al. 1996). A more substantial polymorphic pattern, consisting of interstitial C-bands, was found by SVESHNIKOVA (1991) in analysing 30 populations of Galanthus species from Russia and Bulgaria. To date, however, information is still scanty. In the present investigation we examined the banding patterns and heterochromatin composition of species of Galanthus, Leucojum and Sternbergia by means of Giemsa, fluorochrome banding techniques and silver nitrate staining of NORs and nucleoli. MATERIAL AND METHODS The plants used in this study were collected from natural populations in localities near Rome or in the botanical garden of the University "La Sapienza" (Rome). The list of species, the number of specimens and their provenance are reported in the Table 1. Chromosome preparations were obtained from root tips pretreated with colchicine (0.5%, 4-5 h) at room temperature and then fixed in 3/1 alcohol-acetic solution. Maceration of root tips before squashing was performed in a 50% acetic acid solution at room temperature for mins. C-banding and Quinacrine staining followed the technique of VOSA and MARCHI (1972). DAPI and chromomicine A 3 (CMA 3 ) were used according to the method described by SCHWEIZER (1980). Staining of nucleoli and NORs was made according to MEHERA et al. (1984). For each of the examined species, five Feul-gen stained metaphases were photographed, magnified at 2000x and measured to draw the ideograms. The nomenclature of LEVAN et al. (1964) was used. * Corresponding author: fax ; damatoe@axrma.uniromal.it

2 88 D'AMATO and BIANCHI Galanthus RESULTS Investigation of 25 individuals of G. nivalis showed 2n=24 chromosomes. The set consisted of two long metacentric pairs, seven sm-st medium size chromosome pairs and three short median pairs; one of the st pairs showed sizable satellites. Mean basic karyotype length was 110,5m + 3,7 and Symmetry index Syi=50,24. In some plants we found 2n=24+lB chromosomes. The occurrence of accessory chromosomes is very common in Galanthus. Normally 4-8% or more individuals displayed accessory chromosomes and in some populations up to 40%; in particular it appairs that unfavourable environmental conditions increase their presence in the population (SVESHNIKOVA 1991). Giemsa C-banding has shown a variable individual pattern of interstitial bands on both arms of both the long metacentric pairs of chromosomes and on the long arm of some medium sized subtelocentric chromosomes. The satellited pair showed constantly a dark band on the long arm and, with one exception, on the whole short arm and satellite (Fig. 1 A). The haploid set of an individual is represented in Fig. 3 A. In this representation for each chromosome pair, the most banded homologous was selected to represent the haploid ideogram. The position of all banded sites was calculated by measuring their distance from the centromere and was termed with alphabetic letters. All the variants of the banded sites found in the population are represented in Fig. 3 B. The banded sites of metacentric chromosomes, A, D and B, C, respectively, are at the same distance from the centromere. In our specimens, site F has no corresponding site on the other arm. The banded sites of subtelocentric chromosomes, E, are at 2/3 of the long arm. The maximum C- band number was generally found on the second pair of the long chromosomes. Comparing banding patterns of the Italian populations analysed in the present paper with those found in Russian and Bulgarian populations by SVESHNIKOVA (1991), no remarkable difference can be pointed out. Interstitial bands were only moderately DAPF or Q + -. and CMA 3 DAPI-chromomi-cine A 3 double staining gave the same results. The nucleolus organizer regions were slightly enhanced or almost indifferent to CMA 3 and negative to DAPI fluorescence (Fig. 2 A,B). The moderate chromomicine A 3 fluorescence in correspondence of NORs seems to be a characteristic of the individual plant, at least in our populations. The centromeric region appeared slightly bright when Quinacrine was used (Fig. 2 A). The maximum number of nucleoli was two, thus equal to the number of satellited chromosomes. The data are summarized in Table 2. Leucojum The chromosome number in L. aestivum was 2n=22. The mean basic karyotype length and Symmetry index were respectively 89,65 µm ±2,9 and 32,47. The karyotype comprises one long pair of metacentric (m) and 10 medium sized pairs of st chromosomes; two st pairs were satellited. After Giemsa banding, interstitial heterochromatic segments were found on the short arms of the first pair of metacentric chromosomes. The NOR of satellited pairs was also banded (Fig. IB). Fluorochrome bands consisted only of the CMA + 3 DAPI - fluorescence of the NOR heterochromatin, while the interstitial C- band of the metacentric pair was not evident (Fig. 2 C, D). Two large and two small nucleoli were found in telophase nuclei stained with AgNO 3. Sternbergia Examination of six bulbs of S. colchiciflora showed 2n=20 chromosomes. Of these, 3 pairs were long metacentric and 7 intermediate sized subtelocentric. Mean basic karyotype length was 82,38 µm +3,07; Symmetry index was 38,58. The position of satellites was hardly evident in

3 CHROMOSOME BANDING IN AMARYLLIDACEAE 89 Fig.l. A, above: the C-banded chromosomes of a metaphase of G. nivalis; bottom: silver stained telophase nuclei. B, above: C- banded chromosome set in L. aestivum; bottom: silver stained nucleoli. C-E: S. colchiciflora: (C), Giemsa banded metaphase; (D), telophase nucleus; (E), NORs stained by silver nitrate. Arrows point to NOR.

4 90 D'AMATO and BIANCHI Fig. 2. A-B: G. nivalis. (A), Quinacrine fluorescence; (B), CMA 3 sat chromosome (arrow). OD: L. aestivum metaphase; (C), DAPI; (D), CMA 3 fluorescence. E-F: S. colchtaflora metaphase; (E), DAPI; (F), CMA, stained.

5 CHROMOSOME BANDING IN AMARYLLIDACEAE 91 Fig. 3. A-B: Giemsa banded karyograms. (A), G. nivalis, maximum banded haploid set (see text); (B), schematic representation of all C-banded chromosome types observed. C-D: C-banded karyograms. (C), L. aestivum. (D), S. colchiciflora. Feulgen preparations. The Giemsa banding showed, as in Leucojum, only one intercalary band on the long arm of a sub-metacentric pair. In addition small C-bands were detected in two pairs of st chromosomes which should correspond to the NORs. These bands were often visible as thin spots, or were totally absent (Fig. 1C). After fluorochrome staining, no interstitial bands resulted evident. There were CMA 3 + DAPI - bands which occurred in correspondence of the NORs. However as the heterochromatin in these regions was in small amounts, fluorescence resulted poorly expressed as well (Fig. 2E,F). To better define position and number of NORs, colchicine treated chromosomes were stained by silver nitrate. With this technique four dark NORs were detected in metaphase plate (Fig. IE). Silver stained nucleolar organizer regions correspond, as it is known (HUBBELL 1985), to the active ribosomal RNA transcription regions.

6 92 D'AMATO and BIANCHI DISCUSSION It is generally assumed that Galanthus and Leucojum represent two strictly related genera sharing many morpho-ecological traits. Their karyotypes may have risen by a common ancestor with a basic chromosome number x=7 originated, according to STERN (1956), in central Europe or more probably in the Mediterranean region, after hybridization processes, polyploidy and karyotype repatterning (SVESHNIKOVA 1975). The two taxa have evolved in almost the same area and while Galanthus maintained the same basic number x=12 (see FEDOROV 1969), Leucojum produced different disploid numbers (BARROS NEVES 1939). Given the observed differences in amount and staining properties of the heterochromatin, C-bands cannot represent at this moment a chromosome marker documenting any possible common origin. However a more extended analysis on other species of Leucojum is needed to support this possibility. While in some cases heterochromatin amount and distribution were considered as a valid element to compare karyotypes of related genera (D'AMATO et al. 1975), in most cases banding characteristics are of little utility in analysing taxonomic ranks above and often also below generic level. In Narcissus we found CMA 3 + interstitial bands in some species, and C-bands indifferent to fluorochromes in other species, irrespectively of their systematic position within the Genus (our unpublished data). On the other hand, quantitative and qualitative variation of heterochromatin seems to occur in a relatively short time during chromosome evolution and there are examples of changes in the properties of heterochromatin connected to Robertsonian mutations and new bands may also originate as saltatory DNA replication (see SCHWEIZER and LOIDL 1987). In Sternbergia, however, where the simple pattern found in S. colchiciflora is consistent with the patterns of S. lutea and S. sicula, reported by YUZBASIOGLU et al. (1997) heterochromatin represents a conserved nucleotypic character. REFERENCES BARROS NEVES J., Contribution a I'etude caryologique du genre Leucojum L. Bol. Soc. Brot. Ser. 2, 13: D'AMATO G.F., CAPINERI R., MARCHI P., Analisi della fluorescenza da quinacrina nel complemento cromosomico di Tragopogon porrifolius L., T. eriospermus Ten., T. crocifolius L. e Geropogon glaber L. (Compositae). Ann. Bot. (Roma), 34: D'AMATO G.F., DE DOMINICIS R.I., Heterochromatin pattern, nucleolar organizer regions and rdna in Pancratium illyricum and P. maritimum (Amaryllidaceae). Cytobios, 85: FEDOROV A. A., Chromosome numbers of flowering plants. Leningrad. HUBBELL H.R., Silver staining as an indicator of active ribosomalgenes. Stain Techn., 60: KURITA S., Variation and evolution in the karyotype of Lycoris, Amaryllidaceae. I. General karyomorphological characteristics of the genus. Cytologia, 51: LEV AN A., FREDGA K., SANDBERG A. A., Nomenclature for centromenc position on chromosomes. Hereditas, 52: PIGNATTI S., Flora d'ltalia. Edagricole, Bologna. SCHWEIZER D., Reverse fluorescent chromosome banding with chromomycin anddapl. Chromosoma (Berl.), 58: SCHWEIZER D. and LOIDL J., A model for heterochromatin dispersion and the evolution of C-band patterns. Chromosomes Today, 9: SINHA U.K. and ROY R.P., Q-banding in three species ofcrinum (Amaryllidaceae). Caryologia, 39: STERN F.C., Snowdrop and Snow/lakes. A study of the genera Galanthus and Leucojum. London Royal Hort. Soc. SVESHNIKOVA L.I., On the origin of karyotype of the genus Galanthus L. Bot. Zhurn., 60: , The chromosome polymorphism in natural populations of Galanthus nivalis (Amaryllidaceae). Bot. Zhurn., 76: VOSA C.G. and MARCHI P., On the Quinacrine fluorescence and Giemsa staining patterns of the chromosomes of Vitiafaba. Giorn. Bot. Ital., 106: , Chromosome analysis of Haemanthus and Scadoxus (Amaryllidaceae). Pi. Syst. Evol., 135: YUZBASIOGLU D., UNAL F., DUMAN H., Giemsa C- banding analysis of Sternbergia lutea (L.) Ker-Gawl. ex Sprengl and S. sicula Tineo ex Guss. from Turkey. Cytologia, 62: 1-6. Received 28 June 1999; accepted 18 August 1999