Giemsa C-banded karyotypes in Serapias L. (Orchidaceae)

June 26, 2017 | Author: Domenico Pignone | Category: Plant Biology, Karyotype evolution, Interspecific Variation
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Deskripsi Singkat

Botanical Journal of the Linnean Society (2000), 133: 485–492. With 8 figures doi: 10.1006/bojl.2000.0338, available online at http://www.idealibrary.com on

Giemsa C-banded karyotypes in Serapias L. (Orchidaceae) SAVERIO D’EMERICO1∗, DOMENICO PIGNONE2 AND ANTONIO SCRUGLI3 1

Dipartimento di Biologia e Patologia Vegetale—Sezione di Biologia Vegetale, Universita` di Bari, Via Orabona 4, 70125 Bari, Italy, 2CNR Istituto del Germoplasma, Bari, Italy and 3 Dipartimento di Scienze Botaniche, Universita` di Cagliari, Cagliari, Italy Received October 1999; accepted for publication February 2000

Giemsa C-banding is utilized for the first time to characterize eight taxa of the genus Serapias. Heterochromatin distribution indicated that the Serapias species form a very homogeneous group. All the species possess chromosome pairs with similar heterochromatin patterns. Cbanding showed conspicuous bands located around the centromeres, with some heterochromatic short arms. There was more heterochromatin in S. apulica and S. nurrica than in the other taxa. Extensive centromeric heterochromatin may indicate recent structural rearrangements in the chromosome complement. Taken altogether, karyomorphology indicates a rather recent origin for the genus Serapias, which might also account for the small amount of interspecific variation observed.  2000 The Linnean Society of London

ADDITIONAL KEY WORDS:—heterochromatin distribution – Italy – karyotype evolution – phyletic relationships. CONTENTS

Introduction . . . . . Material and methods . Results . . . . . . Serapias vomeracea subsp. Serapias cordigera . . Serapias apulica . . . Serapias nurrica . . . Serapias parviflora . . Serapias lingua . . . Discussion . . . . . Acknowledgement . . . References . . . . .

. . . . . . . . . vomeracea . . . . . . . . . . . . . . . . . . . . . . . .

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485 486 486 486 487 488 489 489 490 490 491 491

INTRODUCTION

Serapias L. is a genus of Orchidaceae comprising about twenty entities, ten of which are present in Italy (Quentin, 1995). Previous cytological studies have shown ∗ Corresponding author. E-mail: [email protected] 0024–4074/00/080485+08 $35.00/0

485

 2000 The Linnean Society of London

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that in this genus most species possess 2n=2x=36 chromosomes; with only S. lingua having 2n=4x=72 (Heusser, 1938; Scrugli, De Martis & Mulas, 1976; Del Prete, 1977; Mazzola, Crisafi & Romano, 1982; Cauwet-Marc & Balayer, 1986; Bianco et al., 1987). In previous reports (D’Emerico et al., 1990; D’Emerico, Bianco & Medagli, 1992; Bianco et al., 1990, 1992) the karyomorphological characteristics of Serapias vomeracea subsp. vomeracea, S. vomeracea subsp. laxiflora, S.parviflora, S. politisii and S. apulica had been investigated using Feulgen analysis of the chromosome complements. All revealed a complex chromosome morphology and the karyotypes were found to be moderately asymmetrical with a predominance of submetacentric chromosomes. The variation among species of the genus Serapias is rather limited as compared with other Orchidaceae genera, and a recent study based on sequence divergence of the ITS of ribosomal genes showed little differentiation among the species (Pridgeon et al., 1997). In this contribution we report on chromosomal distribution of heterochromatin as revealed by Giemsa C-banding in eight taxa of Serapias, all collected in Italy.

MATERIAL AND METHODS

Preparations of mitotic and meiotic chromosomes were made from immature ovaries. These were pre-treated with 0.3% colchicine at room temperature for 2 h. For Feulgen staining they were fixed for 5 min in 5:1:1:1 (v/v) absolute ethanol, chloroform, glacial acetic acid and formalin (Battaglia, 1957a). Hydrolysis was made at 20°C in 5.5 N HCl for 20 min (Battaglia 1957b). The material was then stained in freshly prepared Feulgen stain. For C-banding, immature ovaries were fixed in ethanol-glacial acetic acid (3:1 v/v) and stored in the deep-freeze for up to several months. Subsequently they were squashed in 45% acetic acid; coverslips were removed by the dry ice method and the preparations air-dried overnight. Slides were then immersed in 0.2 N HCl at 60°C for 3 min, thoroughly rinsed in distilled water and then treated with 4% Ba(OH)2 at 20°C for 4 min. After thorough rinsing they were incubated in 2 × SSC at 60°C for 1 h. The stain used was 3–4% Giemsa (BDH) at pH 7. Chromosome pairs were identified and arranged on the basis of length. The nomenclature used for describing karyotype composition followed Levan, Fredga & Sandberg (1964).

RESULTS

The provenance and chromosome numbers of each studied taxon are reported in Table 1. Serapias vomeracea (N.L. Burm.) Briq. subsp. vomeracea The observed chromosome number 2n=2x=36 in all the investigated populations agrees with previous reports (Heusser, 1938; Del Prete, 1977; Mazzola et al., 1982;

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T 1. Provenance and chromosome numbers of the species studied Taxon

Provenance

Chromosome number 2n

Serapias vomeracea (N.L. Burm.) Briq. subsp. vomeracea

: Potenza, Matera. : Gargano Peninsula (Foggia), Cassano Murge (Bari), Martina Franca (Taranto)

36

S. vomeracea (N.L. Burm.) Briq. subsp. laxiflora (Soo`) Go¨lz & Reinh.

: Gargano Peninsula (Foggia)

36

S. cordigera L.

: Potenza, Matera. : Gargano Peninsula (Foggia), Cassano Murge (Bari), Martina Franca (Taranto) : Muravera

36

S. parviflora Parl.

: Potenza, Matera. : Gargano Peninsula (Foggia), Cassano Murge (Bari), Martina Franca (Taranto) : Portoscuso, Castiadas

36

S. politisii Renz

: Lecce

36

S. apulica (Baumann & Ku¨nkele) P. Delforge

: Torre Canne (Brindisi), Mattinata (Foggia)

36

S. nurrica Corrias

: Cortoghiana

36

S. lingua L.

: Potenza, Matera : Gargano Peninsula (Foggia), Cassano Murge (Bari), Martina Franca (Taranto) : Portoscuso, Muravera

72

Bianco et al., 1987). This species possesses a moderately asymmetrical karyotype, consisting of mainly submetacentric chromosomes (D’Emerico et al., 1992). Giemsa C-banding analysis showed very conspicuous bands located at centromeric positions on many chromosomes, together with euchromatic telomeric regions. Only pair 12 has a completely heterochromatic short arm. Pairs 15 to 18 show small amounts of centromeric heterochromatin (Figs 1, 8A). The karyomorphology and banding pattern in S. vomeracea (N.L. Burm.) Briq. subsp. laxiflora (Soo`) Go¨lz & Reinhard were identical to those of S. vomeracea subsp. vomeracea.

Serapias cordigera L. In this species somatic chromosome counts showed 2n=36 chromosomes in agreement with previous reports (Scrugli et al., 1976; Mazzola et al., 1982; CauwetMarc & Balayer, 1986). The karyotype morphology, consisting of 16(18)m+18(16)sm+2st chromosomes, is reported here for the first time. The banding pattern of S. cordigera appears to be similar to that of S. vomeracea in having large centromeric bands on many chromosomes. Pairs 10 and 12 showed large centromeric bands and heterochromatic short arms. Pairs 13, 14, 16, and 18 showed small centromeric bands (Figs 2, 8B).

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Figures 1–4. C-banded metaphases. Fig. 1. Serapias vomeracea. Fig. 2. S. cordigera. Fig. 3. S. parviflora. Fig. 4. S. apulica. Scale bar: 5 m.

Serapias apulica (Baumann & Ku¨nkele) P. Delforge Somatic cells showed 2n=36 chromosomes as reported by D’Emerico et al., (1990). With respect to the previously-described taxa, this species showed a more asymmetrical karyotype, comprising mainly submetacentric and subtelocentric chromosomes (D’Emerico et al., 1990). S. apulica showed stronger heterochromatin bands than in the other taxa. Pairs 13, 14 and 18 displayed large centromeric bands and heterochromatic short arms (Figs 4, 8C).

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Figures 5–7. Fig. 5. Serapias nurrica, somatic C-banded metaphase. Fig. 6. S. lingua: somatic C-banded metaphase. Fig. 7. S. lingua: Feulgen stained metaphase I in EMC showing 36 bivalents. Scale bar: 5 m.

Serapias nurrica Corrias The number 2n=36 confirms a previous finding (Scrugli, 1982) for the species. The banding pattern in S. nurrica appears to be similar to that of S. apulica in heterochromatin distribution (Fig. 5). Serapias parviflora Parl. Specimens from southern Italy and Sardinia showed 2n=36, in accordance with Del Prete (1977), Scrugli (1978) and Mazzola et al. (1981). Among the species considered here, S. parviflora shows the least asymmetrical karyotype (Bianco et al., 1990). C-banding analysis revealed that this is the species of this complex having

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Figure 8. Haploid idiograms showing heterochromatin distribution. a, Serapias vomeracea. b, S. cordigera. c, S. apulica. d, S. parviflora.

the smallest amount of heterochromatin, with centromeric bands present on fewer chromosomes than in the other taxa. Pairs 6, 7, 10, 12 and 16 showed large centromeric bands and heterochromatic short arms. Pairs 11, 15, 17 and 18 showed thin centromeric bands (Figs 3, 8D). S. politisii Renz, recently reported from Apulia (Bianco et al., 1992), shows a banding pattern identical to that of S. parviflora.

Serapias lingua L. In this species somatic metaphases showed 2n=4x=72 chromosomes, as already reported (Scrugli, 1978; Mazzola et al., 1982; Cauwet-Marc & Balayer, 1986). Meiotic studies revealed 36 bivalents at metaphase I in EMCs (Fig. 7). The banding pattern of S. lingua appears to be similar to the other taxa in having chromosomes with large blocks of centromeric heterochromatin (Fig. 6).

DISCUSSION

The genus Serapias encompasses a group of taxa of difficult taxonomy due to their highly complex morphology and similarity. Some features of the karyotypes have been described in previous reports (D’Emerico et al., 1990, 1992; Bianco et al., 1992) and the general overall resemblance is in line with the small morphological variation. In this paper, the study of the chromosomal distribution of Giemsa C-bands in the different taxa of this genus has revealed some interesting aspects of their phyletic relationships. C-banding showed conspicuous bands in numerous chromosome pairs, located at centromeric positions, with some pairs also characterized by C-bands at the telomeric position on the short arms or heterochromatic short arms. The heterochromatin occupied the entire extent of the chromosomes, with euchromatin being limited to the extremities. This heterochromatin distribution has been observed

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only in a few other cases, e.g. Cicer, Melipona (Galasso et al., 1996; Rocha & Pompolo, 1998). The investigated taxa were characterized by different amounts of constitutive heterochromatin. In spite of the large number and particular position of C-bands, it is still difficult to recognize homologies between chromosome pairs of different taxa. Nevertheless, the analysis of C-band distribution, along with variation of morphological characters, suggests that the taxa of Serapias form a rather homogeneous group. In some cases the apparent karyomorphological homology reaches a high degree, as in the case of S. vomeracea subsp. vomeracea and S. vomeracea subsp. laxiflora. In consideration of this high level of karyomorphological and heterochromatin resemblance, it is conceivable that these two taxa are better considered as different morphotypes of the same species. Similar conclusions, based on the remarkable resemblance of their C-banded karyotypes, may be drawn for S. parviflora and S. politisii. It is interesting to note that S. apulica displayed a more asymmetrical karyotype and a higher amount of heterochromatin bands than the other taxa. In this connection, S. nurrica shows a close similarity to S. apulica in heterochromatin distribution. Karyological analysis in the tetraploid S. lingua failed to identify all chromosomes precisely, due to the complexity of its karyotype. Nevertheless, based on heterochromatin amount and distribution, it was possible to show that the complement of this species shows some chromosome pairs with smaller amounts of heterochromatin. This feature is a characteristic of the chromosomes of S. parviflora, which indeed resemble many of those of S. lingua. It is therefore possible to suggest that S. parviflora has played a role in the evolution of S. lingua. Whether this latter species is to be considered an autotetraploid or not is still an open question. Bernard & Miklos (1979) suggested that an increase in centromeric heterochromatin is produced as a consequence of structural chromosome rearrangements. On these grounds, the presence of large centromeric heterochromatin blocks in Serapias might indicate recent structural rearrangements in the chromosomal complement, as also suggested by the asymmetry of the karyotype as a whole (Stebbins, 1971). Taken altogether, these indications might indicate a rather recent origin for Serapias, and the reduced time scale might account for the small interspecific variation of nuclear and morphological characters that are observed in the genus.

ACKNOWLEDGEMENT

This research was supported by a grant from the Ministry of the University and Research and Technological Science (M.U.R.S.T. 60%).

REFERENCES

Battaglia E. 1957a. A new ‘‘5 minutes-fixation using cold hydrolysis’’. Caryologia 9: 368–370. Battaglia E. 1957b. A simplified Feulgen method using cold hydrolysis. Caryologia 9: 372–373. Bernard J, Miklos GLG. 1979. Functional aspects of satellite DNA and heterochromatin. International Review of Cytology 58: 1–14.

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Bianco P, D’Emerico S, Medagli P, Ruggiero L. 1990. Orchiserapias nelsoniana Bianco, D’Emerico, Medagli et Ruggiero, hybr. nat. nov. della Puglia. Webbia 44: 315–322. Bianco P, D’Emerico S, Medagli P, Ruggiero L, Liverani P. 1992. Serapias politisii Renz (Orchidaceae), nuova per la Flora Italiana. Webbia 46: 219–223. Bianco P, Medagli P, D’Emerico S, Ruggiero L. 1987. Numeri cromosomici per la flora italiana. Informatore Botanico Italiano 19: 322–332. Cauwet-Marc AM, Balayer M. 1986. ‘‘Les orchide´es du bassin me´diterrane´en’’. Contribution a` l’e´tude caryologique des espe`ces des pyre´ne´es-orientales (France) et contre´es limitrophes. II. Tribu des Ophrydeae Lindl. Bulletin de la Socie´te´ Botanique de France, Lettres Botaniques 133: 265–277. D’Emerico S, Bianco P, Medagli P. 1992. Karyological studies on Orchidaceae. Tribe Ophrydeae, subtribe Serapiadinae. Caryologia 45: 301–311. D’Emerico S, Bianco P, Medagli P, Ruggiero L. 1990. Karyological studies of some taxa of the genera Himantoglossum, Orchis, Serapias and Spiranthes (Orchidaceae) from Apulia (Italy). Caryologia 43: 267–276. Del Prete C. 1977. Numeri cromosomici per la flora italiana: 358-365. Informatore Botanico Italiano 9: 135–140. Galasso I, Frediani M, Maggiani M, Cremonini R, Pignone D. 1996. Chromatin characterization by banding techniques, in situ hybridization, and nuclear DNA content in Cicer L. (Leguminosae). Genome 39: 258–265. Heusser C. 1938. Chromosomenverhaltnisse bei schweizerischen basitonen Orchideen. Berichte der Schweizerischen Botanischen Gesellschaft 48: 562–599. Levan A, Fredga K, Sandberg AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52: 201–220. Mazzola P, Crisafi F, Romano S. 1981. Numeri cromosomici per la flora italiana. Informatore Botanico Italiano 13: 182–188. Mazzola P, Miceli G, Not R. 1982. Numeri cromosomici per la flora italiana. Informatore Botanico Italiano 14: 275–279. Pridgeon AM, Bateman RM, Cox AV, Hapeman JR, Chase MW. 1997. Phylogenetics of subtribe Orchidinae (Orchidoideae, Orchidaceae) based on nuclear ITS sequences. 1. Intergeneric relationships and polyphyly of Orchis sensu lato. Lindleyana 12: 89–109. Quentin P. 1995. Synopsis des Orchide´es Europe´ennes. Cahiers de la S.F.O., no. 2. Paris. Rocha MP, Pompolo SD. 1998. Karyotypes and heterochromatin variation (C-bands) in Melipona species (Hymenoptera, Apidae, Meliponinae). Genetics and Molecular Biology 21: 41–45. Scrugli A. 1978. Numeri cromosomici per la flora italiana. Informatore Botanico Italiano 10: 414–421. Scrugli A. 1982. Numeri cromosomici per la flora italiana. Informatore Botanico Italiano 14: 229–233. Scrugli A, De Martis B, Mulas B. 1976. Numeri cromosomici per la flora italiana. Informatore Botanico Italiano 8: 82–91. Stebbins GL. 1971. Chromosomal evolution in higher plants. London: Arnold.



Deskripsi

Botanical Journal of the Linnean Society (2000), 133: 485–492. With 8 figures doi: 10.1006/bojl.2000.0338, available online at http://www.idealibrary.com on

Giemsa C-banded karyotypes in Serapias L. (Orchidaceae) SAVERIO D’EMERICO1∗, DOMENICO PIGNONE2 AND ANTONIO SCRUGLI3 1

Dipartimento di Biologia e Patologia Vegetale—Sezione di Biologia Vegetale, Universita` di Bari, Via Orabona 4, 70125 Bari, Italy, 2CNR Istituto del Germoplasma, Bari, Italy and 3 Dipartimento di Scienze Botaniche, Universita` di Cagliari, Cagliari, Italy Received October 1999; accepted for publication February 2000

Giemsa C-banding is utilized for the first time to characterize eight taxa of the genus Serapias. Heterochromatin distribution indicated that the Serapias species form a very homogeneous group. All the species possess chromosome pairs with similar heterochromatin patterns. Cbanding showed conspicuous bands located around the centromeres, with some heterochromatic short arms. There was more heterochromatin in S. apulica and S. nurrica than in the other taxa. Extensive centromeric heterochromatin may indicate recent structural rearrangements in the chromosome complement. Taken altogether, karyomorphology indicates a rather recent origin for the genus Serapias, which might also account for the small amount of interspecific variation observed.  2000 The Linnean Society of London

ADDITIONAL KEY WORDS:—heterochromatin distribution – Italy – karyotype evolution – phyletic relationships. CONTENTS

Introduction . . . . . Material and methods . Results . . . . . . Serapias vomeracea subsp. Serapias cordigera . . Serapias apulica . . . Serapias nurrica . . . Serapias parviflora . . Serapias lingua . . . Discussion . . . . . Acknowledgement . . . References . . . . .

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485 486 486 486 487 488 489 489 490 490 491 491

INTRODUCTION

Serapias L. is a genus of Orchidaceae comprising about twenty entities, ten of which are present in Italy (Quentin, 1995). Previous cytological studies have shown ∗ Corresponding author. E-mail: [email protected] 0024–4074/00/080485+08 $35.00/0

485

 2000 The Linnean Society of London

486

S. D’EMERICO ET AL.

that in this genus most species possess 2n=2x=36 chromosomes; with only S. lingua having 2n=4x=72 (Heusser, 1938; Scrugli, De Martis & Mulas, 1976; Del Prete, 1977; Mazzola, Crisafi & Romano, 1982; Cauwet-Marc & Balayer, 1986; Bianco et al., 1987). In previous reports (D’Emerico et al., 1990; D’Emerico, Bianco & Medagli, 1992; Bianco et al., 1990, 1992) the karyomorphological characteristics of Serapias vomeracea subsp. vomeracea, S. vomeracea subsp. laxiflora, S.parviflora, S. politisii and S. apulica had been investigated using Feulgen analysis of the chromosome complements. All revealed a complex chromosome morphology and the karyotypes were found to be moderately asymmetrical with a predominance of submetacentric chromosomes. The variation among species of the genus Serapias is rather limited as compared with other Orchidaceae genera, and a recent study based on sequence divergence of the ITS of ribosomal genes showed little differentiation among the species (Pridgeon et al., 1997). In this contribution we report on chromosomal distribution of heterochromatin as revealed by Giemsa C-banding in eight taxa of Serapias, all collected in Italy.

MATERIAL AND METHODS

Preparations of mitotic and meiotic chromosomes were made from immature ovaries. These were pre-treated with 0.3% colchicine at room temperature for 2 h. For Feulgen staining they were fixed for 5 min in 5:1:1:1 (v/v) absolute ethanol, chloroform, glacial acetic acid and formalin (Battaglia, 1957a). Hydrolysis was made at 20°C in 5.5 N HCl for 20 min (Battaglia 1957b). The material was then stained in freshly prepared Feulgen stain. For C-banding, immature ovaries were fixed in ethanol-glacial acetic acid (3:1 v/v) and stored in the deep-freeze for up to several months. Subsequently they were squashed in 45% acetic acid; coverslips were removed by the dry ice method and the preparations air-dried overnight. Slides were then immersed in 0.2 N HCl at 60°C for 3 min, thoroughly rinsed in distilled water and then treated with 4% Ba(OH)2 at 20°C for 4 min. After thorough rinsing they were incubated in 2 × SSC at 60°C for 1 h. The stain used was 3–4% Giemsa (BDH) at pH 7. Chromosome pairs were identified and arranged on the basis of length. The nomenclature used for describing karyotype composition followed Levan, Fredga & Sandberg (1964).

RESULTS

The provenance and chromosome numbers of each studied taxon are reported in Table 1. Serapias vomeracea (N.L. Burm.) Briq. subsp. vomeracea The observed chromosome number 2n=2x=36 in all the investigated populations agrees with previous reports (Heusser, 1938; Del Prete, 1977; Mazzola et al., 1982;

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T 1. Provenance and chromosome numbers of the species studied Taxon

Provenance

Chromosome number 2n

Serapias vomeracea (N.L. Burm.) Briq. subsp. vomeracea

: Potenza, Matera. : Gargano Peninsula (Foggia), Cassano Murge (Bari), Martina Franca (Taranto)

36

S. vomeracea (N.L. Burm.) Briq. subsp. laxiflora (Soo`) Go¨lz & Reinh.

: Gargano Peninsula (Foggia)

36

S. cordigera L.

: Potenza, Matera. : Gargano Peninsula (Foggia), Cassano Murge (Bari), Martina Franca (Taranto) : Muravera

36

S. parviflora Parl.

: Potenza, Matera. : Gargano Peninsula (Foggia), Cassano Murge (Bari), Martina Franca (Taranto) : Portoscuso, Castiadas

36

S. politisii Renz

: Lecce

36

S. apulica (Baumann & Ku¨nkele) P. Delforge

: Torre Canne (Brindisi), Mattinata (Foggia)

36

S. nurrica Corrias

: Cortoghiana

36

S. lingua L.

: Potenza, Matera : Gargano Peninsula (Foggia), Cassano Murge (Bari), Martina Franca (Taranto) : Portoscuso, Muravera

72

Bianco et al., 1987). This species possesses a moderately asymmetrical karyotype, consisting of mainly submetacentric chromosomes (D’Emerico et al., 1992). Giemsa C-banding analysis showed very conspicuous bands located at centromeric positions on many chromosomes, together with euchromatic telomeric regions. Only pair 12 has a completely heterochromatic short arm. Pairs 15 to 18 show small amounts of centromeric heterochromatin (Figs 1, 8A). The karyomorphology and banding pattern in S. vomeracea (N.L. Burm.) Briq. subsp. laxiflora (Soo`) Go¨lz & Reinhard were identical to those of S. vomeracea subsp. vomeracea.

Serapias cordigera L. In this species somatic chromosome counts showed 2n=36 chromosomes in agreement with previous reports (Scrugli et al., 1976; Mazzola et al., 1982; CauwetMarc & Balayer, 1986). The karyotype morphology, consisting of 16(18)m+18(16)sm+2st chromosomes, is reported here for the first time. The banding pattern of S. cordigera appears to be similar to that of S. vomeracea in having large centromeric bands on many chromosomes. Pairs 10 and 12 showed large centromeric bands and heterochromatic short arms. Pairs 13, 14, 16, and 18 showed small centromeric bands (Figs 2, 8B).

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Figures 1–4. C-banded metaphases. Fig. 1. Serapias vomeracea. Fig. 2. S. cordigera. Fig. 3. S. parviflora. Fig. 4. S. apulica. Scale bar: 5 m.

Serapias apulica (Baumann & Ku¨nkele) P. Delforge Somatic cells showed 2n=36 chromosomes as reported by D’Emerico et al., (1990). With respect to the previously-described taxa, this species showed a more asymmetrical karyotype, comprising mainly submetacentric and subtelocentric chromosomes (D’Emerico et al., 1990). S. apulica showed stronger heterochromatin bands than in the other taxa. Pairs 13, 14 and 18 displayed large centromeric bands and heterochromatic short arms (Figs 4, 8C).

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Figures 5–7. Fig. 5. Serapias nurrica, somatic C-banded metaphase. Fig. 6. S. lingua: somatic C-banded metaphase. Fig. 7. S. lingua: Feulgen stained metaphase I in EMC showing 36 bivalents. Scale bar: 5 m.

Serapias nurrica Corrias The number 2n=36 confirms a previous finding (Scrugli, 1982) for the species. The banding pattern in S. nurrica appears to be similar to that of S. apulica in heterochromatin distribution (Fig. 5). Serapias parviflora Parl. Specimens from southern Italy and Sardinia showed 2n=36, in accordance with Del Prete (1977), Scrugli (1978) and Mazzola et al. (1981). Among the species considered here, S. parviflora shows the least asymmetrical karyotype (Bianco et al., 1990). C-banding analysis revealed that this is the species of this complex having

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Figure 8. Haploid idiograms showing heterochromatin distribution. a, Serapias vomeracea. b, S. cordigera. c, S. apulica. d, S. parviflora.

the smallest amount of heterochromatin, with centromeric bands present on fewer chromosomes than in the other taxa. Pairs 6, 7, 10, 12 and 16 showed large centromeric bands and heterochromatic short arms. Pairs 11, 15, 17 and 18 showed thin centromeric bands (Figs 3, 8D). S. politisii Renz, recently reported from Apulia (Bianco et al., 1992), shows a banding pattern identical to that of S. parviflora.

Serapias lingua L. In this species somatic metaphases showed 2n=4x=72 chromosomes, as already reported (Scrugli, 1978; Mazzola et al., 1982; Cauwet-Marc & Balayer, 1986). Meiotic studies revealed 36 bivalents at metaphase I in EMCs (Fig. 7). The banding pattern of S. lingua appears to be similar to the other taxa in having chromosomes with large blocks of centromeric heterochromatin (Fig. 6).

DISCUSSION

The genus Serapias encompasses a group of taxa of difficult taxonomy due to their highly complex morphology and similarity. Some features of the karyotypes have been described in previous reports (D’Emerico et al., 1990, 1992; Bianco et al., 1992) and the general overall resemblance is in line with the small morphological variation. In this paper, the study of the chromosomal distribution of Giemsa C-bands in the different taxa of this genus has revealed some interesting aspects of their phyletic relationships. C-banding showed conspicuous bands in numerous chromosome pairs, located at centromeric positions, with some pairs also characterized by C-bands at the telomeric position on the short arms or heterochromatic short arms. The heterochromatin occupied the entire extent of the chromosomes, with euchromatin being limited to the extremities. This heterochromatin distribution has been observed

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only in a few other cases, e.g. Cicer, Melipona (Galasso et al., 1996; Rocha & Pompolo, 1998). The investigated taxa were characterized by different amounts of constitutive heterochromatin. In spite of the large number and particular position of C-bands, it is still difficult to recognize homologies between chromosome pairs of different taxa. Nevertheless, the analysis of C-band distribution, along with variation of morphological characters, suggests that the taxa of Serapias form a rather homogeneous group. In some cases the apparent karyomorphological homology reaches a high degree, as in the case of S. vomeracea subsp. vomeracea and S. vomeracea subsp. laxiflora. In consideration of this high level of karyomorphological and heterochromatin resemblance, it is conceivable that these two taxa are better considered as different morphotypes of the same species. Similar conclusions, based on the remarkable resemblance of their C-banded karyotypes, may be drawn for S. parviflora and S. politisii. It is interesting to note that S. apulica displayed a more asymmetrical karyotype and a higher amount of heterochromatin bands than the other taxa. In this connection, S. nurrica shows a close similarity to S. apulica in heterochromatin distribution. Karyological analysis in the tetraploid S. lingua failed to identify all chromosomes precisely, due to the complexity of its karyotype. Nevertheless, based on heterochromatin amount and distribution, it was possible to show that the complement of this species shows some chromosome pairs with smaller amounts of heterochromatin. This feature is a characteristic of the chromosomes of S. parviflora, which indeed resemble many of those of S. lingua. It is therefore possible to suggest that S. parviflora has played a role in the evolution of S. lingua. Whether this latter species is to be considered an autotetraploid or not is still an open question. Bernard & Miklos (1979) suggested that an increase in centromeric heterochromatin is produced as a consequence of structural chromosome rearrangements. On these grounds, the presence of large centromeric heterochromatin blocks in Serapias might indicate recent structural rearrangements in the chromosomal complement, as also suggested by the asymmetry of the karyotype as a whole (Stebbins, 1971). Taken altogether, these indications might indicate a rather recent origin for Serapias, and the reduced time scale might account for the small interspecific variation of nuclear and morphological characters that are observed in the genus.

ACKNOWLEDGEMENT

This research was supported by a grant from the Ministry of the University and Research and Technological Science (M.U.R.S.T. 60%).

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