THE TAXONOMIC STATUS OF SCILLA BEIRANA SAMP. (HYACINTHACEAE)

ALMEIDA DA SILVA, R.M., F.B. CALDAS & J.A. ROSSELLÓ (1998). The taxonomic status of Scilla beirana Samp. (Hyacinthaceae). Anales Jard. Bot. Madrid 56(2): 253-260. Populations of Scilla beirana Samp. were sampled in NW Portugal and compared with its relatives S. ramburei Boiss, and S. peruviana L. Leaf and scape anatomy, morphology, chromosome number and idiogram were identical in S. beirana and S. ramburei, but differed from S. peruviana. Diagnostic characters previously used to discriminate S. beirana (width of leaves and flower number) showed continuous, but not clinal, variation, and failed to provide a clear-cut basis for identification and no other morphological attributes were found to sepárate the taxa. All available evidence suggests that S. beirana should be put into synonymy with S. ramburei, as was earlier suggested by COUTINHO (1935).


INTRODUCTION
collected in Northwest Portugal (Beira Alta province), in Vila Nova de Paiva (SAMPAIO, One of the least well-known bulbous 1931).This species was first related by the species in Europe is Scilla beirana Samp., an author to 5. peruviana L., from which it was Iberian squill of doubtful affinities.Scilla distinguished by the less vigorous habit, beirana was published on the basis of plants smaller bulb, glabrous leaves and blue anthers.Later SAMPAIO (1936) east doubts about the taxonomic status of S. beirana, treating it as a synonym of S. lusitanica L. and 5. paui Lacaita, and in the same group as S. ramburei Boiss, 5. verna Huds, and S. odorata Link.The view that S. beirana was closely related to 5. ramburei was also shared by COUTINHO (1935) who made S. beirana a synonym of S. ramburei.MCNEILL (1980), in his account of the European squills, retained S. beirana as a distinct species endemic to Portugal and related it to 5. peruviana.This was supported by SPETA (1987) who, when splitting Scilla in several satellite genera, transferred S. beirana under Oncostema Raf.subgenus Oncostema, in which he placed S. peruviana.
Recently, FRANCO & ROCHA AFONSO (1994) included S. beirana at the subspecific rank within S. ramburei.These authors retain the endemic status of 5. beirana, but expanded its range to NW Spain.
Thus the taxonomic status and relationships of S. beirana are by no means clear.In view of this a morphological, anatomical and cytological study was undertaken using S. beirana-like plants from the vicinity of the type locality, which were compared with the related S. ramburei and S. peruviana.

Material
Specimens of 5. beirana, S. peruviana and S. ramburei were studied from the following herbaria: AVE, BC, BCF, COA, COI, ELVE, HVR, ISA, LISU, MA, MAF, PO, SANT, VAB.The type specimen of S. beirana is kept at PO. Original material of 5. peruviana was seen in microfiche (LINN 429.3) whereas no such material or type specimen is available for S. ramburei, at least at the Boissier herbarium at G (BURDET & al., 1982).However, specimens of 5. ramburei revised by Boissier (G) were examined.In addition, living material collected in Portugal and Spain (see Appendix) was cultivated at the Botánica!Garden of Porto University.

Methods
Anatomy.Fresh leaves and scapes from 5 mature individuáis of each population were collected after anthesis.Epidermal preparations were obtained by peeling off the epidermis of median portions of leaves, clearing in Sodium hypochlorite and staining in Vesuvin.They were then dehydrated through an alcohol series, placed in xylol and mounted in Canada Balsam.Median portions of fresh leaves and scapes were fixed in Formalin-Acetic Acid-Alcohol (FAA) (JOHANSEN, 1940), for 24 h at 0 °C.Fixation was followed by a thorough rinse in running water for 24 h and dehydration in an ethanol series at 0 °C, for 12 h in each step.Material was then transferred to a monomer mixture (GMA), modified from SEMBA (1979), containing 94 % Glycol methacrylate, 3 % 2-Butoxyethanol, 1,5 % Polyethylene Glycol 400, 0,75 % Divinylbenzene, 0,75 % Methyl Methacrylate and 0,72 g of Benzoyl peroxide for each 80 mi of monomer mixture, in two steps of 24 h each, at room temperature.Embedding was carried out in a mixture without PEG, but with 50 pl of 2,2'-azobis [2-methylpropionitrile] for each 3 mi of mixture, polymerizing at room temperature.After trimming, blocks were cut at 1-2 um using glass knives in an adapted rotary microtome (BENNETT & al., 1976;BUTLER, 1979;SEMBA, 1979).GMA sections were stained in two ways: with toluidine blue O in acétate buffer, pH 4.4 (FEDER & O'BRIEN, 1968), and with periodic acid -Schiff's reaction (PAS), using a 30 min aldehyde blockade in 2,4-dinitro-phenylhydrazine in 15 % acetic acid, and omitting the periodic acid oxidation step in controls (O'BRIEN & MCCULLY, 1981), followed by counterstaining with 1 % naphtol blue black val % acetic acid (FISHER, 1968).
Karyology.Mitotic studies were carried out on root tips pre-treated with 0.2 % colchicine for 4 h at room temperature, fixed in ethanol: acetic acid (3:1) for 24 h, hydrolysed in IN HC1 at 60 °C for 5 min, stained with acetic orcein for 24 h and squashed in 45 % acetic acid.Chromosome video image analysis was performed on a Macintosh HCi computer using the public domain NIH Image program.Five individuáis from each population were studied, and at least 10 mitotic plates were used for karyotype and idiogram construction.For centromere position the nomenclature of LEVAN & al. (1964) is followed.In the tables, chromosome parameters are expressed in % of the karyotype (haploid karyotype = 100 %).

Morphology
Plants collected in NW Portugal, including those from the surroundings of the type locality (table 1), closely resemble the type specimen (fig.1A) as well as the original description of S. beirana and were considered to be representative of this taxon.Macroscopic features were compared with 5. ramburei and S. peruviana (table 1).Scilla beirana is very distinct from 5. peruviana in several vegetative and reproductive attributes and no cióse relationship could be firmly established.By contrast, 5. beirana is morphologically very cióse to S. ramburei (fig.IB), and no single feature can differentiate between S. ramburei and 5. beirana.FRANCO & ROCHA AFONSO (1994) separated S. beirana (populations north of Tagus River) from S. ramburei on the basis of its wider leaves (0.3-0.8 and 0.8-1.5 cm, respectively) and more dense inflorescences (8-25 versus 25-45 flowers).We have pursued this avenue to check for such quantitative discontinuities within S. ramburei.Over 150 individuáis of S. ramburei s.l.throughout its range (N África, Spain) showed a continuous variation in both characters, with no discontinuities supporting the recognition of two morphs (fig.2).A clinal (geographic) variation was also not evident from the results when the geographic origin of the samples was plotted on the scatter diagram.The correlation between leaf width and number of flowers is low (r = 0.3505, p s: 0.001), thus supporting the view that most of the variation of these two characters is independent.in anatomical structure with the species belonging to the Scilla verna complex (ALMEIDA DA SILVA, 1997).Some differences exist between S. peruviana and the other two species (fig.3).Trichomes are absent in S. beirana and S. ramburei, bul S. peruviana has long cilia along the leaf margins.The margins show epidemial extensions of 3-5 cells in S. beirana and S. ramburei, and 9-10 cells in S. peruviana.Chlorenchyma is homogeneous and composed of spongy mesophyll in 5. beirana and S. ramburei, while S. peruviana has an heterogeneous mesophyll with a somewhat loosely arranged palisade parenchyma in the subepidermal layers.Large cavities, sometimes surrounded by remnants of parenchyma cells, are found alternating with the vascular bundles in 5. beirana and S. ramburei.Such cavities are not found in S. peruviana.

Anatomy
Scape.With the exception of the wider scapes of S. peruviana, no other internal feature could be used to differentiate the three species.

Karyology
Chromosome numbers and the statistics of the haploid complement of all taxa are depicted in table 2 and figure 4. Populations of S. peruviana sampled from the western Iberian península have 2« = 16, with three metacentric (no.1-3) and five telocentric (no.4-8) pairs.Secondary constrictions were difficult to observe in every cell and therefore were not considered.The somatic chromosome number was 2n = 20 for S. beirana and S. ramburei, with very similar karyotypes comprising two metacentric (no.1-2), three submetacentrics (no.3-5), four subtelocentrics (no.6-9) and one telocentric pair (no.10).This striking similarity is further supported by the presence of satellites located on the short arm of a subtelocentric chromosome pair.

DISCUSSION
The Scilla verna complex is a group of western mediterranean taxa (S. verna, S. ramburei, S. beirana, S. odorata, S. merinoi and S. paui) of controversial taxonomic relationships at the generic and specific level.Overall morphology is very similar and without clear conspicuous gaps between all taxa, matering the delimitation of species borderlines difficult without a previous knowledge of the geographic origin of the plants.Individuáis from near the type locality of S. beirana were indistinguishable from those from other Iberian populations of S. ramburei.Vigour and associated morphological characters (length and width of leaves, length of scape, number of flowers) are highly variable in this complex, and the type (S.beirana) and Boissier's revised specimens (S. ramburei) of these taxa are at  the opposite ends of a continuous variation.In addition, no geographic or ecological pattern has been observed.However, the taller and more vigorous individuáis usually come from rainy or wet áreas.Scilla ramburei grows in a high diversity of habitats, from flxed coastal dunes under a Pinus pinea L. canopy to mountain grasslands, from sea level to 1600 m.In the absence of common garden experiments or molecular data we cannot determine the nature of morphological variability, but it is possible that part of it is due to phenotypic plasticity.A cióse inspection of the S. beirana type has not revealed any feature, other than its size, which could link this taxon to S. peruviana.Apparently, SAMPAIO (1931) described S. beirana from plants cultivated at the Botanical Garden of Porto.Therefore, some characters, including the habit, might be expected to be modified thus mimicking the 5. peruviana habit.The type specimen, which has two scapes, was prepared at the onset of flowering so the shape of its inflorescence (subcorymbose) is wider than usual, a diagnostic feature of S. peruviana.This may explain why such a relationship has been suggested (MCNEILL, 1980;SPETA, 1987).From our results, 5. ramburei s.l. is fairly homogenous at the anatomical and chromosomal level throughout its range (north African populations were not available for this study).Anatomical data, although of restricted taxonomic valué within the S. verna complex (ALMEIDA DA SILVA, 1997), support the identity of S. ramburei-S.beirana and also point to their distinctiveness from S. peruviana, which differs from the S. verna complex by its hairy leaves, heterogeneous mesophyll and absence of cavities in the chlorenchyma.
Chromosome counts and karyotypes of S. peruviana were in accordance with previous records from Spain and Portugal (BATTAGLIA, 1949a(BATTAGLIA, , 1949b;;BARROS NEVES, 1973;CARMONA & ai, 1984;PASTOR, 1985;LUQUE & al., 1988).Chromosome numbers of S. beirana and S. ramburei confirmed earlier reports (FERNANDES & al, 1948;GIMÉNEZ MARTÍN, 1959;BARROS NEVES, 1973;PASTOR, 1985;SPETA, 1987).PARKER (1981) attributed the same chromosome number (2n = 20) to S. beirana based on the record of S. ramburei by BARROS NEVES (1973) from Serra da Lapa, which is very cióse to the type locality of S. beirana.No karyotypic variation was detected when the idiograms of both taxa were compared.In conclusión, morphological, anatomical and karyological evidence show that the distinction of S. beirana and S. ramburei, even at the infraspecific level (FRANCO & ROCHA AFONSO, 1994), has no sound basis.Not a single character or combination of consistent characters can be used to differentiate between them throughout their range.From a taxonomic point of view, S. beirana is a mere synonym of the older ñame S. ramburei.

Fig. 2 .
Fig. 2.-Scatter plot showing individuáis of Scilla ramburei s.l. in relation to flower number and width of leaves (mm).• populations situated to the North of river Tagus; O populations situated to the South of river Tagus.