Aquatic Botany, 45 (1993) 1–13

Elsevier Science Publishers B.V., Amsterdam

Chromosome numbers and a new model for karyotype evolution in Ruppia L. ( Ruppiaceae) S. Talavera, P. Garcia-Murillo and J. Herrera Departamento de Biologia Vegetal y Ecologia, Universidad de Sevilla, E-41080 Sevilla, Spain

( Accepted 8 October 1992) Go to table of contents

Digitalizado por Biblioteca Botánica Andaluza

ABSTRACT Talavera, S., Garcia-Murillo, P. and Herrera, J., 1993. Chromosome numbers and a new model for karyotype evolution in Ruppia L. (Ruppiaceae). Aquat. Bot., 45: 1-13. Populations of three species in the genus Ruppia that inhabit the western part of the Mediterranean region were studied caryologically. All three species present bimodal karyotypes. Ruppia drepanensis Tineo ex guss. (2n = 20) has a karyotype asymmetry of the 2C type. Ruppia cirrhosa (Petagna) Grande and Ruppia maritima L. (both with 2n =40) present a 2B asymmetry. Observations of meiosis of microspore mother cells revealed that R. drepanensis forms ten bivalents at diakinesis and Metaphase I. The other two species exhibit 20 bivalents. The long arm of subtelocentric, large chromosomes in all three species shows no sign of chiasmata at meiosis.

INTRODUCTION

With only about eight species, the genus Ruppia has a nearly worldwide distribution (Den Hartog, 1981). All of these species are hydrophytes from hypersaline or brackish waters. Studies on species of this genus have dealt with their anatomy (Sauvageau, 1891; Monoyer, 1928; Tomlinson, 1982), morphology (Graves, 1908; Luther, 1947), ecology (e.g. Verhoeven, 1979, 1980; Brock, 1981, 1982; Vollebergh and Congdon, 1986), flower development (Posluszny and Sattler, 1974) and pollination biology (Gamerro, 1968; McConchie, 1982; Cox, 1983, 1988; Haynes, 1988; Les, 1988; Cox and Knox, 1989) . Taxonomic studies have also contributed to the knowledge of this genus (e.g. Mason, 1967; Jacobs and Brock, 1982; Talavera and Garcia-Murillo, 1987). Ruppia seems to be closely related to Potamogeton (including Groenlandia), the main difference being that it bears bisexual flowers with two stamens, while in Potamogeton there are four. Other traits such as stipules fused Correspondence to: S. Talavera, Departamento de Biologia Vegetal y Ecologia, Universidad de Sevilla, E41080, Sevilla, Spain.

© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-3770/93/$06.00

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to the leaf blade, pollination taking place on the surface of the water, and elongated pollen grains, relate Ruppia species to those in the subgenus Coleogeton of Potamogeton. As a result, some authors have included Ruppia in the Potamogetonaceae together with Potamogeton and Groenlandia ( Thorne, 1981; Dahlgren et al., 1985 ), while others refer the genus to a separate family ( Takhtajan, 1980, 1986; Cronquist, 1981, 1988). Information on the cytotaxonomy of the genus has been provided by Reese (1962, 1963), Van Vierssen et al. (1981) and Marchioni-Ortu (1982) for European populations, and by Snoeijs and Van der Ster (1983 ) for Australian plants. Except for Reese's (1962, 1963) observations on the idiogrammatic formula of Ruppia maritima L., there are few data on the karyotypes and behaviour at meiosis of other species. Together with information on the three species studied here, we present a review of the literature on Ruppia. MATERIAL AND METHODS

Root tips and flower buds from plants growing in the western part of the Mediterranean region were used for this study. Buds were fixed for 24 h in Carnoy's solution (100% ethanol/glacial acetic acid 3 : 1), then transferred to 70% ethanol and stored at 4°C. Root tips were pre-treated with 0.002 M 8hydroxyquinoline at 4°C for 3—4 h, then fixed and stored with flower buds until study. Material was stained with hydrochloric acid—alcoholic carmine (Snow, 1963) at 30°C for 4—7 days. Anthers or roots were then placed in a drop of 45% acetic acid on a slide, squashed, and observed. We followed Levan et al. (1965) for the idiogrammatic formula and chromosome nomenclature, and Stebbins (1938, 1971) for karyotype symmetry and chromosome size. Plant vouchers are deposited at the herbaria of the Departamento de Biologia Vegetal y Ecologia, Universidad de Sevilla (SEV and SEVF). RESULTS AND DISCUSSION

Variations in chromosome number As shown in Table 1, western Mediterranean populations of Ruppia are either diploid or tetraploid. Populations of Ruppia drepanensis Tineo ex Guss. are diploid with 2n = 20 (Figs. IC, ID, 2C, 2D) and n =10 (Figs. 3A, 3B, 4A, 4B) . Populations of R. maritima L. var. maritima are tetraploid with 2n = 40 and n =20 (Figs. 1B, 2B ), and the same is true for R. maritima var. brevirostris Ag. and Ruppia cirrhosa (Petagna) Grande (Figs. IA, 2A, 3C—3F, 4C— 4F). Although Murbeck (1902) and Graves (1908) reported 2n= 16 for R. maritima L., all subsequent counts have demonstrated that species in this genus present 2n = 20 or 2n = 40. Values of 2n = 18, 2n = 30, and 2n = 60 have

CHROMOSOME NUMBERS OF RUPPIA

also been reported on occasions (see below). Ruppia polycarpa Mason, Ruppia megacarpa Mason, Ruppia tuberosa J.S.Davis and Tomlinson and R. drepanensis Tineo ex guss. present 2n = 20 (Table 1) . Only a southern Australian population of R. tuberosa was reported by Snoeijs and Van der Ster (1983) to have 2n = 30 (3 x ), and another population in New Zealand studied by Mason (1967) and Carstairs (1982) 2n = 18 and n= 9, respectively. The disagreement between the earlier and these more recent counts might be explained either on the basis of aneuploidy in the studied plants, or because of incorrect counts resulting from the small size of some chromosomes in this genus since small chromosomes may often remain undetected if they are under larger ones (Reese, 1962 ). Some populations of R. maritima L. are diploid (2n = 20) and others are tetraploids (2n = 40) . All studies on R. cirrhosa (Petagna) Grande (Table 1) have reported n =20 and 2n = 40, so it seems to be mainly a tetraploid. Only Reese (1962) has reported hexaploid populations of this taxon from northern Germany. According to the literature and to our own data, the basic chromosome number in the genus Ruppia is x= 10. Among the species for which data are available, four are diploid, one is a tetraploid, and one ( R. maritima) presents both diploid and tetraploid populations. Diploid populations occur in cool regions of northern Europe and Canada, while tetraploids inhabit warm or temperate areas from the Mediterranean region and Japan. Hexaploids (of R. cirrhosa from Germany with 2n = 60; Reese (1962)) and triploids (of R. tuberosa from southern Australia with 2n = 30; Snoeijs and Van der Ster (1983)) are very rare. Size and morphology of chromosomes, and karyotype asymmetry Reese (1962) established that R. maritima has a pair of chromosomes much larger (about 4 ,um long) than the remaining pairs (ranging from 0.5 to 2.5 ,u m) . He also indicated that large chromosomes had the centromere near the distal end, and a satellite on the longest arm. Among the small pairs, five had the centromere at a submedian position, and the remaining four were so small that they were termed `punktformig' (i.e. dot-like). Furthermore, Reese (1962) reported that tetraploids and hexaploids had two and three pairs of relatively large chromosomes, respectively. These observations have been verified by Gamerro (1968) and Van Vierssen et al. (1981) . The same pattern has also been observed in R. megacarpa and R. tuberosa by Snoeijs and Van der Ster (1983 ). The karyotype of R. drepanensis (Figs. 1C–1E ) shows a pair of subtelocentric chromosomes (the ratio of the long arm to the short one is 3.3) with a secondary constriction at the long arm (Fig. 1 D ). According to Stebbins (1938 ), this pair should be considered medium-to-large (4.5–5 ,um) . All the remaining pairs are small, their sizes ranging from 1.2 ,um to 2 ,u m. In two

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TABLE 1

n

20 10

20

40

20 40 20 40 40

40 40 40 20 20

20 40 20 20 40 40

2n

Spain, Cadiz

N. Germany S. France, Camargue Italy, Sardinia Italy, Sardinia France, Corse

Canada Japan N. Germany The Netherlands S. France Spain, Cadiz (SEVF) Spain, Almeria (SEV114537) Spain, Sevilla (SEV113519) Spain, Sevilla (SEV113512) Spain, Guadalajara (MA346158) Spain Italy, Sardinia

Locality

Van Vierssen et al. (1981)

Reese (1961, 1962, 1963) Van Vierssen et al. (1981) Marchioni-Ortu (1982 ) Marchioni-Ortu (1982) This study

Taylor and Mulligan (1968) Harada (1956) Reese (1961, 1962, 1963) Van Vierssen et al. (1981) Van Vierssen et al. (1981) This study This study This study This study Cirujano (S. Cirujano, unpublished data) Aedo and Fernandez Casado (1988 ) Marchioni-Ortu (1982)

Reference

Chromosome numbers in species of the genus Ruppia Taxon Ruppia maritima var. maritima

var. brevirostris

var. longipes Hagstrom

Ruppia cirrhosa

var. occidentalis

Ruppia drepanensis (sub R. cirrhosa) (sub R. cirrhosa) (sub R. cirrhosa)

Ruppia polycarpa

Ruppia tuberosa

Ruppia megacarpa

20

10 10

9

20 20, 30

20 18, 20

20

20 20 20 20 20

40 39

40

New Zealand S. Australia S. Australia S. Australia

S. Australia S. Australia

New Zealand S. Australia S. Australia

Spain Spain Spain Italy, Sardinia Spain, Cadiz (SEV113597) Spain, Cadiz (SEV113595) Spain, Huelva (SEV113585) Spain, Malaga (SEVF)

Canada Argentine, Patagonia

N. Germany N. Germany S. France, Camargue Spain, Cadiz (SEV 113555) Spain, Huelva (SEV 113552) Morocco, Tanger (SEV128702) Italy, Sardinia

Mason (1967) Brock (1982) Carstairs (1982 ) Snoeijs and Van der Ster (1983)

Brock (1982) Snoeijs and Van der Ster (1983 )

Mason (1967) Brock (1982) Carstairs (1982 )

Cirujano (1982) Castroviejo (1983) Cirujano (1986) Marchioni-Ortu (1982) This study This study This study This study

Love and Love (1981) Gamerro (1968)

Reese (1961, 1962, 1963) Reese (1962) Van Vierssen et al. (1981) This study This study This study Marchioni-Ortu (1982)

40 60 40 40 40

20 20 20 20

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Fig. 1. Metaphase configurations in root apices of Ruppia species. ( A) R. cirrhosa (Spain, Huelva, Odiel River, SEV 113552; 217=40). (B) R. maritima var. maritima (Spain, Sevilla, Las Cabezas, SEV113512; 2n=40). (C—E) R. drepanensis: ( C) Spain, Huelva, Donana, SEV113585; 2n = 20; ( D) Spain, Cadiz, Chiclana, SEV 113 5 97; 2n= 20; (E) caryogram from C. Scale bars, 2.5 pm. The secondary construction of a subtelocentric chromosome is marked with an arrow in (D).

CHROMOSOME NUMBERS OF RUPPIA

7

Fig. 2. Drawings of metaphase configurations in root apices of Ruppia species appearing in Fig. 1.

pairs the centromere is at the middle point, while in the remaining it is at the middle region (Fig. 1E). All these small chromosomes are clearly metacentric. Thus, the idiogrammatic formula would be: 1

STsat,

2M, 7m

Since the ratio of the largest to the smallest chromosome pair is 4.3, and given that the proportion of acrocentric chromosomes is 20%, it can be estab -

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S. TALAVERA ET AL.

Fig. 3. Meiosis in pollen mother-cells of Ruppia species. (A) and (B) R. drepanensis (Spain, Huelva, Donana, SEV 113 5 8 5; n=10) at Metaphase I. (C–F) R. cirrhosa ( Morocco, Tanger, SEV128702; n=20); (C) diakinesis; (D) and (E) Metaphase I; (F) anaphase to telophase. Scale bars, 2.5 µm. Arrows mark either subtelocentric chromosomes ((A) and (C)) or lagging bivalents (F).

CHROMOSOME NUMBERS OF RUPPIA

Fig. 4. Drawings of meiotic configurations of Ruppia species appearing in Fig. 3.

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lished according to Stebbins (1971) that the karyotype asymmetry in R. drepanensis is of the 2C type. In the present study, populations of R. cirrhosa and R. maritima were tetraploid with two pairs of large chromosomes evident at somatic metaphase (Figs. IA, 1B, 2A, 2B). The ratio of the largest to the smallest chromosome is near 4 in both taxa. On the other hand, the large chromosomes are not identical, since one pair is metacentric and the other is submetacentric (the ratios of the long arm to the short one are respectively, 1.5 and 2) . This holds for both R. cirrhosa and R. maritima, so that the karyotypic asymmetry is of the 2B type. According to Stebbins (1971) bimodal karyotypes result either from pericentric inversions or from translocations of chromosome segments that accumulate into the largest pairs. We suggest that translocations are responsible for the bimodal karyotype exhibited by Ruppia, which is supported by the fact that the long arm of the subtelocentric chromosomes is satellised. However, and given that the small chromosomes are metacentric, it is unlikely that these pairs have contributed to the increase in size of the subtelocentric pair and, consequently, to karyotype asymmetry. In stead, our hypothesis is that the subtelocentric pair includes residual material that was once present in other, now missing chromosomes. The transfer of such material would have left minute chromosomes which were subsequently lost, a suggestion implying that the extant basic chromosome number in Ruppia is lower than it was originally. Bimodal karyotypes occur in the genus Muscari (Liliaceae) (Ruiz-Rejon et al., 1986 ), the Dracaenaceae–Noliniaceae group of the Asparagales (Stebbins, 1971), as well as in some genera in the Alismatales and Hydrocharitales (Sharma and Chatterjee, 1967). Recently, Kuo et al. (1990) have demonstrated that Posidonia species have 2n= 20, with a bimodal karyotype with five pairs of large and five pairs of small chromosomes. The main difference with Ruppia is that in Posidonia the large chromosomes are not acrocentric, its karyotype being thus relatively more symmetric. Chromosome pairing at meiosis The study of meiosis in anthers of R. drepanensis revealed the existence of ten bivalents at Metaphase I (Figs. 3A, 3B, 4A, 4B). This is in accordance with observations made by Reese (1962) on diploid populations of R. mariti ma from northern Germany. Furthermore, we have observed in R. drepanensis that chromosomes from the largest pair are linked,near the centromeric region, while their long arms remain free of chiasmata (Figs. 3A, 4A). Regarding the smaller chromosomes, only three link by their tips (thus giving a typical ring-like appearance) and the remainder seem to link only by the tip of one of their arms.

CHROMOSOME NUMBERS OF RUPPIA

11

The studied population of R. cirrhosa from Morocco showed marked meiotic abnormalities. Twenty bivalents could be seen at diakinesis (Figs. 3C, 4C ), although the smallest ones were often associated with another bivalent. The long arms of the two largest chromosome pairs show no sign of chiasmata (Figs. 3C, 4C ), which is similar to what happens in R. drepanensis. Abnormalities are even clearer at Metaphase I and Anaphase I, during which synapsis and lagging bivalents are common (Figs. 3D-3F, 4D-4F). Harada (1956) also observed 20 bivalents at meiosis in R. maritima, and Reese (1962) reports the same for R. cirrhosa. The existence of 20 bivalents at diakinesis supports the notion of an allopolyploid origin for tetraploid races of R. maritima and R. cirrhosa. The observation that, in Ruppia, long arms in large chromosomes do not recombine during meiosis suggests that these regions of the genome may be of some adaptive value (Stebbins, 1971) . ACKNOWLEDGEMENT

The authors are most grateful to P.E. Gibbs for critically reading the manuscript.

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Snoeijs, P.J.M. and van der Ster, H.E., 1983. Some notes on the cytotaxonomy of two Ruppia species in South Australia. Aquat. Bot., 16: 297-302. Snow, R., 1963. Alcoholic hydrochloric acid-carmine as a stain for chromosomes in squash preparations. Stain Technol., 38: 9-13. Stebbins, G.L., 1938. Cytological characteristics associated with the different growth habits in the dicotyledons. Am. J. Bot., 25: 189-198. Stebbins, G.L., 1971. Chromosomal Evolution in Higher Plants. Edward Arnold, London, 216 PPTakhtajan, A.L., 1980. Outline of the Classification of Flowering Plants (Magnoliophyta). Bot. Rev., 46: 225-359. Takhtajan, A.L., 1986. Floristic Regions of the World. University of California Press, Berkeley, CA, 356 pp. Talavera, S. and Garcia-Murillo, P., 1987. Ruppia L. In: B. Valdes, S. Talavera and E.F. Galiano (Editors ), Flora Vascular de Andalucia Occidental. 3. Ketres, Barcelona, pp. 194-195. Taylor, R.L. and Mulligan, G.A., 1968. Flora of the Queen Charlotte Islands. II. Cytological aspects of the vascular plants. Queen's Printer, Ottawa, 148 pp. Thorne, R.F., 1981. A summary statement. In: D.A. Young and D.S. Seiger (Editors), Phytochemistry and Angiosperm Phylogeny. Praeger, New York, pp. 233-295. Tomlinson, P.B., 1982. Potamogetonaceae. In: C.R. Metcalfe (Editor), Anatomy of the Monocotyledons. VII. Helobiae (Alismatidae). Clarendon Press, Oxford, pp. 270-335. Van Vierssen, W., van Wijk, R.J. and van der Zee, J.R., 1981. Some additional notes on the cytotaxonomy of Ruppia taxa in western Europe. Aquat. Bot., 11: 297-301. Verhoeven, J.T.A., 1979. The ecology of Ruppia dominated communities in Western Europe. I. Distribution of Ruppia representatives in relation to their autecology. Aquat. Bot., 6: 197267. Verhoeven, J.T.A., 1980. The ecology of Ruppia dominated communities in Western Europe. II. Synecological classification. Structure and dynamics of the macroflora and macrofauna communities. Aquat. Bot., 8: 1-85. Vollebergh, P.J. and Congdon, R.A., 1986. Germination and growth of Ruppia polycarpa and Lepilaena cylindrocarpa in ephemeral saltmarsh pools, Westernport Bay, Victoria. Aquat. Bot., 26: 165-196.

Introduction Material and methods Results and discussion Variations in chromosome numbers Size and morphology of chromosomes, and karyotype asymmetry Chromosome pairing at meiosis Acknowledgments References Table 1: Chromosome numbers Figure 1: Metaphase configurations Figure 2: Drawing metaphase configurations Figure 3: Meiosis in pollen Figure 4: Drawing meiotic configurations