Botanical Journal of the Linnean Society, 2008, 158, 762–774. With 7 figures

A new hexaploid species of Centaurea section Acrolophus (Asteraceae) from Evvia Island, Greece P. TRIGAS1,2*, TH. CONSTANTINIDIS2 and T. TOULOUMENIDOU3 1

National Agricultural Research Foundation (N.AG.RE.F.), Forest Research Institute, Terma Alkmanos str., Ilisia, GR – 115 28 Athens, Greece 2 Laboratory of Systematic Botany, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, GR – 118 55 Athens, Greece 3 Laboratory of Plant Breeding and Biometry, Department of Crop Science, Agricultural University of Athens, Iera Odos 75, GR – 118 55 Athens, Greece Received 31 May 2006; accepted for publication 21 April 2008

Centaurea carystea Trigas & Constantin., a new yellow-flowered species of Centaurea from Mt. Ochi on Evvia Island (Greece), is described and illustrated. It is a member of the polymorphic C. section Acrolophus and allied to taxa of the C. attica aggregate, C. pelia and C. mantoudii. The new species appears to be a local and threatened endemic, with the total number of individuals known being less than 500. A karyological examination revealed that it is hexaploid, with 2n = 6x = 54, an unusual number in Centaurea, which may indicate a hybrid origin. To further clarify the taxonomic position of C. carystea, we used random amplification of polymorphic DNA markers of 25 plants belonging to nine species, subspecies and varieties morphologically related to the new species, together with two reference taxa. Clustering using the unweighted pair group method with arithmetic mean indicated a discrete position for C. carystea, close to but distinct from the yellow-flowered C. mantoudii. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 762–774.

ADDITIONAL KEYWORDS: chromosome number – compositae – conservation – endemic species – Flora Hellenica – morphology – RAPD – taxonomy.

INTRODUCTION The polymorphic genus Centaurea L. comprises about 200–700 species depending on the classification adopted (Dittrich, 1977; Bremer, 1994; Wagenitz & Hellwig, 1996; Greuter, 2003; Hellwig, 2004). The distribution of the genus is mainly Eurasian with a centre confined to the eastern Mediterranean– Turanian region (Wagenitz, 1955). In Greece, it comprises c. 140 taxa (species and subspecies of the genus Centaurea s.l.), and is one of the largest genera in the Greek flora. Although particular taxa of the genus are

*Corresponding author. E-mail: [email protected], [email protected]

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morphologically distinct, species delimitation may nevertheless be difficult in several sections represented in Greece, with intermediate populations and a pronounced variation existing in some species. Centaurea section Acrolophus (Cass.) DC. comprises about 115 species worldwide (Ochsmann, 2000). It is a taxonomically difficult group because some species hybridize easily, followed in certain cases by introgression (Georgiadis, 1981; Ochsmann, 2000). The section is represented by c. 30 species in Greece, to which several infraspecific taxa should be added (Georgiadis, 1980). A considerable number (c. 19 species or 63%) are endemic to the country and often geographically restricted to a small area, such as a single mountain or island. In 2003, the first author observed and collected specimens of a Centaurea species belonging to section

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NEW HEXAPLOID SPECIES OF CENTAUREA FROM GREECE Acrolophus, close to the summit area of Mt. Ochi in southern Evvia Island. The plants were in flower and the pale-yellow colour of the corolla indicated an interesting discovery. The species was linked to C. mantoudii T.Georgiadis, another yellow-flowered member of section Acrolophus endemic to Evvia and to the unrelated shrubby and thorny C. spinosa L. These were the only yellow members of section Acrolophus known from Evvia at that time. The area on Mt. Ochi was revisited the following summer and a few herbarium specimens were collected. It was particularly difficult to find intact plants in flower because of the goats that had grazed the habitat of the species. After examination, it was realized that the plants on Mt. Ochi constituted a new, undescribed member of section Acrolophus. This species is described and illustrated here. It is the fourth in a series of species described from southern Evvia over the last decade (Brullo, Pavone & Salmeri, 1997; Trigas & Tzanoudakis, 2000; Trigas & Iatrou, 2003).

MATERIAL AND METHODS Dry specimens of the new species were compared with reference material kept in the herbaria ACA, ATH and UPA. The herbarium acronyms follow Holmgren, Holmgren & Barnet (1990). Two living plants were

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cultivated for two years in Athens, in order to check the morphological stability and to study the chromosome number and morphology. For karyological investigations, fresh root tips were collected and pretreated with an aqueous solution of 8-hydroxyquinoline (0.3 g L-1) for 3.5 h at room temperature. The roots were then hydrolysed in 1 M HCl at 60 °C, stained in Fuelgen’s reagent for 3 h and squashed over a glass microscope slide in a drop of 45% acetic acid solution. Metaphase plates of good quality were recorded and photographed. Total genomic DNA of 27 plants belonging to 11 Centaurea taxa (species, subspecies or varieties, Table 1) was extracted from young leaves preserved in silica gel for a few days. A DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) was used, according to the manufacturer’s protocol. Thirty different 10-mer primers were tested, 13 of which were subsequently selected for further analysis (Table 2) based on the number of reproducible polymorphic bands. PCR was performed in a total volume of 25 mL containing 50 ng of genomic DNA, 0.625 mM primer, 150 mM of each deoxynucleoside triphosphate (dNTP) and 1 U Taq polymerase DNA (Promega) in the incubation buffer provided by the manufacturer. The amplification reactions were carried out in an MJ-Research PTC-200 Peltier Thermal Cycler with an initial denaturation

Table 1. Alphabetical list of the Centaurea taxa used in the molecular analysis with provenance and chromosome number

Taxon

Provenance

C. attica Nyman subsp. attica

Attiki, Mt. Imittos, close to Ioannis Kareas monastery Attiki, Mt. Gerania, west parts of Korifi summit Attiki, Mt. Pateras, east parts of Agios Ilias summit Attiki, Mt. Pendeli, c. 3 km north-east of Pendeli settlement Evvia, Mt. Ochi, west of Profitis Ilias summit Attiki, c. 1 km north-west of Agios Konstantinos (Kamariza) village Evvia, c. 1–2 km east of Mantoudi village Attiki, north-west of Megara, close to Agios Ierotheos monastery Viotia, north of Aliki settlement Viotia, Mt. Parnassos, north of Arachova village Viotia, Mt. Kitheronas, north of the mountain shelter Attiki, Mt. Pateras, upper parts of Mikri Kolosoura summit

C. attica subsp. megarensis (Halácsy & Hayek) Dostál C. attica subsp. pateraea (Halácsy) T.Georgiadis C. attica subsp. pentelica (Hausskn.) Dostál C. carystea Trigas & Constantin. C. laureotica Heldr. ex Halácsy C. mantoudii T.Georgiadis C. pelia DC. var. gerania T.Georgiadis C. pelia var. pelia C. pichleri Boiss. [Cyanus pichleri (Boiss.) Holub] C. raphanina Sm. subsp. mixta (DC.) Runemark C. subsericans Halácsy

Number of plants used in the analysis

Chromosome number (2n)

3

18, 36

3

36

2

18

3

36

1

54

3

36

3

36

2

18

3 1

18 ?

1

20

3

36

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Table 2. DNA sequences of the 13 primers used in the rapid amplified polymorphic DNA (RAPD) analysis

Primer

Sequences (5′ → 3′)

Number of polymorphic bands

OPF-6 OPF-7 OPG-2 OPG-5 OPG-6 OPH-13 OPH-16 OPH-17 RAPD-3 RAPD-13 RAPD-14 RAPD-16 RAPD-18

GGGAATTCGG CCGATATCCC GGCACTGAGG CTGAGACGGA GTGCCTAACC TGAGTCCGCA CAAGGTGGGT CAGTGGGGAG AGAACCGAGG GACGCGAACC ACGCTGCGAC GACACAGCCC GAAACGGGTG

6 13 12 13 17 8 15 14 11 17 17 16 14

step at 94 °C for 2 min, followed by 35 cycles of 1 min at 94 °C, 1 min at 37 °C and 1 min at 72 °C, and a final extension step of 10 min at 72 °C. The PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. The reproducibility of the banding patterns was checked by repeating the reactions twice and by running samples lacking any plant DNA. The reproducible polymorphic amplified fragments were scored as present (1) or absent (0) for each marker, and were assembled in a data matrix table. Genetic similarities between taxa were calculated using the Jaccard and Dice similarity coefficients with the SIMQUAL program. Cluster analyses were performed using the unweighted pair group method with arithmetic mean (UPGMA) and neighbor joining (NJ) method. The correlation between the similarity and cophenetic matrices for each similarity coefficient and clustering method was computed.

RESULTS CENTAUREA CARYSTEA P.TRIGAS & TH.CONSTANTINIDIS SP. NOV. (FIGS 1, 2) Type: GREECE: Nomos Evvias, Eparchia Karistias, Evvia Island, Mt. Ochi. Western parts of Profitis Ilias summit, north of the mountain shelter. Stony plain with rocks in open, steppe-like vegetation, 38°03′N, 24°28′E, c. 1200 m a.s.l., 26.vi.2004, leg. P. Trigas 3117 (holotype: ACA; isotype: UPA).

Additional specimens seen: Trigas 3104 (UPA), collected at the same locality on 5.vii.2003, and cultivated material.

Diagnosis: Planta perennis, caules decumbentes (4–)8–24 cm longi. Folia omnia tomentosa, inferiora petiolata, bipinnatisecta, 1.0–6.5 ¥ 0.5–25 cm longa, lobulis oblanceolatis vel obovatis; folia caulina apicem versus deminuta, superiora 0.6–1.2 ¥ 0.2–0.4 cm, integra vel pinnatifida, segmentis utrinque 1–2. Involucrum angusteovatum vel semiglobosum 1.0– 1.2 ¥ 0.5–1.0 cm. Phylla lanuginosa vel glabra, 3-nervia, media 6.0–9.5 ¥ 5.0–7.5 mm; appendices phyllorum triangulares vel rhombiformes, decurruntes, 2.8–3.2 mm longae, 5.0–7.5 mm latae, pectinato-ciliatae, ciliis ad 3 mm longis, in spinam terminalem 0.3–1.2 mm longam excurrens. Flores flavi, glabri, marginales steriles. Achenia sparsim hirsuta vel glabra, c. 3 mm longa. Pappus ad 1.5 mm longus. A C. mantoudii T.Georgiadis caulibus procumbentibus, capitulis 1–4 non 5–30, indumento foliorum lanato-tomentoso, acheniis et pappo minoribus specifice differt. A C. attica Nyman s.l. capitulis paucioribus, appendicibus phyllorum obscurioribus, mucrone plerumque minore, flosculis luteis specifice differt. Description: Perennial herb with rosettes and a woody rootstock. ROSETTES one to three per individual, usually giving rise to one decumbent, tomentose flowering stem, (4–)8–24 cm long, emerging from the centre of a rosette and bearing one to four capitula. BASAL LEAVES sparsely tomentose when young, rather densely whitish lanate-tomentose when mature, narrowly oblong to elliptic in outline, one- to two-pinnatisect, 1.0–6.5 ¥ 0.5–2.5 cm; leaf-lobes oblanceolate to obovate, four to nine on each side, 3.0–8.0 ¥ 1.0–2.5 mm, terminal one often longer and wider than laterals, all or most distinctly mucronate at apex; mucro 0.4–1.0 mm long, dark-brown at base, whitish at the apex. CAULINE LEAVES like basal ones but gradually decreasing in size and number of lobes; uppermost 0.6–1.2 ¥ 0.2–0.4 cm, with one to two lobes on each side or entire. CAPITULA radiant, involucre narrowly ovoid to semiglobose, 1.0–1.2 ¥ 0.5–1.0 cm; involucral bracts straw-coloured, pale-brown at central part, lanulose to glabrous, three- or fiveveined, middle ones 6.0–9.5 ¥ 5.0–7.5 mm (including the appendage); appendages of middle bracts broadly triangular to almost rhombic, decurrent, pectinate, 2.8–3.2 mm ¥ 5.0–7.5 mm (including fimbriae), comprising c. one-third of the bract, straw-coloured to pale-brown at central and upper parts, whitish membranous at lateral edges, with transparent membranous lateral wings 0.3–0.6 mm wide completely covering bracts to base; lateral fimbriae up to 3 mm long, always longer than apical mucro, straw-coloured or white; apical mucro 0.3–1.2 mm long; outer bracts like middle ones but smaller; inner bracts

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Figure 1. Centaurea carystea Trigas & Constantin. sp. nov.: A, habit; B, rosette leaf; C, involucral bracts (C1 outer, C2 middle, C3 inner); D, achene. Drawn from Trigas 3117 and cultivated material. Scale bars: A, 2 cm; B, 1 cm; C, 1.5 mm; D, 1 mm.

linear-lanceolate, 10.5–12.5 ¥ 2.0–4.0 mm. Florets c. 25–36 per capitulum, corolla pale-yellow. Outer florets 8–14, radiant, sterile, c. 15 mm long, lobes 5, unequal, 3.5–5.5 mm long; inner florets cylindrical and somewhat expanded at limb, 12–16 mm long, with four to five lobes c. one-third as long as tube; stamens with anthers connate into a tube, filaments hairy papillose, anther appendages c. 0.5 mm, glabrous. Style c. 1.5 mm long, with a ring of hairs below bifurcation, branches with minute hairs dorsally. ACHENES sparsely hairy to glabrous, c. 3 mm long; pappus up to 1.5 mm long, one-third to one-half as long as achenes, composed of a few (usually three) rows of white, unequal, scabrid to barbellulate bristles, innermost row short.

Etymology: The name of the new species refers to the town of Carystos (Karistos) and the prefecture of the same name in southern Evvia. This region includes

Mt. Ochi and Cape Kafireas, areas which constitute an important centre of endemism on Evvia (Trigas, 2003).

Habitat and ecology: Centaurea carystea grows in open, steppe-like, rocky habitats with extremely low vegetation cover (< 10%). This is not an unusual habitat for the whole of section Acrolophus as many of its species are found in xeric grasslands and steppelike vegetation, whereas others live in more specialized habitats, such as those provided by the steep rocky slopes and crevices (Georgiadis, 1980; Blanca Lopez, 1981). The plants on Mt. Ochi form small tufts of rosette leaves and grow on almost plane ground on a small plateau, west of the Profitis Ilias summit area. The geological units of the area consist of muscovite, epidotite, chlorite and quartz schist and micaceous marbles with chloritic partings. The soil is thin and infertile and usually limited to small patches

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Figure 2. A flowering plant of Centaurea carystea in its natural habitat.

between the rocks. The whole area is exposed to strong north winds, especially during the summer. Several endemic and noteworthy taxa grow together with C. carystea. These include Allium cupani Raf. s.l., Anthemis wiedemanniana Fisch. & Mey., Centaurea raphanina Sm. subsp. mixta (DC.) Runem., Cerastium comatum Desv., Crocus cancellatus Herb. subsp. mazziaricus B.Mathew, Dianthus monadelphus Vent. subsp. pallens (Sm.) Greuter & Burdet, Hieracium hoppeanum Schult. subsp. testimoniale Nägeli ex Peter, Moenchia graeca Boiss. & Heldr., Myosotis incrassata Guss., Ononis spinosa L. subsp. antiquorum (L.) Arcang., Petrorhagia dubia (Rafin.) G.Lopez & A.M.Romo, Phleum exaratum Hochst. ex Boiss. subsp. exaratum, Poa bulbosa L. subsp. bulbosa, Scabiosa argentea L., Scleranthus perennis L. subsp. marginatus (Guss.) Nyman, Sedum amplexicaule DC. subsp. tenuifolium (Sm.) Greuter, Sedum eriocarpum Sm. subsp. apertiflorum t’Hart, Thlaspi bulbosum Boiss., Trifolium uniflorum L., Veronica glauca Sm. subsp. peloponnesiaca (Boiss. & Orph.) Maire & Petitm., etc. Centaurea carystea grows at an altitude of c. 1200 m and flowers from the middle of June to July. Mature achenes are formed at the end of July. The pappus is reduced and the heavy achenes are not fit for long-distance dispersal. The achenes are probably dispersed by ants (many species of Centaurea are myrmecochorous (Wagenitz & Hellwig, 1996; Hellwig, 2004) and often have capitula closely adpressed to the ground). This may be a reason why the new species occupies a very restricted distribution area.

Distribution: Centaurea carystea is known only from the type locality on Mt. Ochi in southern Evvia. Although the steppe-like rocky habitats in which it grows are common in the area, no other populations have been observed so far. Conservation: Centaurea carystea forms small groups, each consisting of 5–20 individuals and growing in an area of a few square metres. The total known population extends over c. 5 ha and comprises less than 500 individuals. Genetic erosion is presumably a threat for the only known population of the species. The vegetation cover of Mt. Ochi is heavily grazed during the summer by domestic herbivores, mostly goats. The animals consume the flowering stems and leaves of C. carystea, usually leaving the lower plant parts intact. The prostrate habit of the species offers some protection from further damage, but the easily visible capitula are extensively grazed. We have estimated that more than 95% of the annual seed production is consumed by goats; therefore, overgrazing appears to be a serious threat to the long-term survival of the species. The area in which the population of C. carystea grows is relatively easily accessible, being only 30 min walking time from the Mt. Ochi shelter. The track that leads to the entrance of Dimosari gorge, one of the most famous attractions of the area, passes close to the population, thus creating additional risks. Southern Evvia, located close to the city of Athens, has seen intensive building activity and tourist development in the last decade. The construction of wind generators in the area is another serious threat. More

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NEW HEXAPLOID SPECIES OF CENTAUREA FROM GREECE than 800 wind generators have already been placed in southern Evvia in recent years, particularly in areas exposed to the strong north winds. The habitat of C. carystea is an ideal place for a wind park development because of the ground morphology. In the case of any such construction, the species will face the immediate threat of extinction. Centaurea carystea is a rare and localized species, which should be characterized as Critically Endangered, fulfilling the criterion B2ab(v) of the World Conservation Union (IUCN) Red List Categories and Criteria (IUCN, 2001). Monitoring the unique small population is imperative in order to prevent the species from going extinct. Fortunately, Mt. Ochi and its surroundings (Cape Kafireas and the Dimosari gorge) have been selected for inclusion in the ‘Natura 2000’ network of ecologically important areas in Greece. The protection of the area is expected to have a beneficial effect on the population of C. carystea. However, any protection or management scheme requires strict implementation, something that is currently lacking.

TAXONOMIC DISCUSSION Centaurea carystea belongs to C. section Acrolophus because of its small capitula bearing triangular, decurrent, regularly ciliate appendages on the involucral bracts. The members of this section in Greece have been revised by Georgiadis (1980), who recognized 29 species in the country, together with several infraspecific taxa. To these, C. incompleta Halácsy and the recently described C. johnseniana Kit Tan & Strid should be added, whereas a few species, such as C. prespana Rech. f., need critical re-evaluation. The colour of the corollas is considered to be an important character for the classification of taxa within C. section Acrolophus (Georgiadis, 1980). The paleyellow corollas of C. carystea group it with a small number of Greek Centaurea species which have yellow, cream or white corollas. This group includes nine species distributed mainly in the eastern parts of continental Greece and the Aegean Islands (Georgiadis, 1980). On the basis of the morphological features, the closest yellow-flowered allies of C. carystea are C. pelia DC., C. mantoudii T.Georgiadis and C. laureotica Halácsy. Centaurea pelia is distributed mainly in eastcentral continental Greece at low and moderate altitudes (Fig. 3), and differs from C. carystea in a number of morphological features, including habit, leaves, involucral bracts and pappus length (Table 3). Centaurea mantoudii, a local endemic of the serpentine areas of northern Evvia (Fig. 3), is also a

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lowland species, closely related to C. pelia, which it replaces on Evvia. It is easily distinguished from C. carystea by its tall, many-headed stems, the glabrous or scabrous rosette leaves, the glabrous and narrow involucral bracts and the longer achenes and pappus (Table 3). It has recently been treated as a synonym of C. laureotica for the needs of the Euro + Med Plantbase (Greuter, 2008) but we still consider it to be sufficiently distinct and prefer to keep it as an independent entity. Centaurea laureotica was considered by Georgiadis (1980) to be a species of hybrid origin, which originated by introgression between C. mantoudii and C. attica Nyman. It is locally restricted to south-east Attiki, close to the small town of Lavrio (Fig. 3). This remarkably polymorphic species may have pink, reddish, yellow or white corollas and a variable habit. Its morphological similarities to C. carystea are rather weak, as it is a robust, usually tall plant, with up to 100 capitula per stem, scabrous leaves, narrow bracts, a long mucro on bract appendages and achenes with a longer pappus. The pink- or purple-flowered C. attica is a polymorphic species distributed in east Sterea Ellas and Evvia. Four subspecies have been recognized: subsp. attica, on Mts. Imittos, Parnitha, Penteli and central Evvia; subsp. pentelica (Hausskn.) Dostál, on the same mountains of Attiki and also in northern Evvia; subsp. megarensis (Halácsy & Hayek) Dostál, endemic to the serpentine parts of Mt. Gerania; and subsp. pateraea (Halácsy) T.Georgiadis, on Mts. Gerania, Pateras, Kitheronas, Pastra and Elikonas (Fig. 4). The latter subspecies has recently been considered as a synonym of C. subsericans for the needs of the Euro + Med Plantbase (Greuter, 2008), and even Georgiadis (1980) places C. subsericans under the synonyms of subsp. pateraea. However, having seen both taxa in the field, we prefer to keep them independent, at least for the time being. Within the group of C. attica, individuals with yellowish flowers have been reported only exceptionally (Strid & Andersson, 1985; Constantinidis, 1997), and have been attributed to hybridization with yellow-flowered taxa growing at the same locality or adjacent places. Centaurea carystea shares significant morphological similarities with C. attica, although the latter has pink or purple corollas. Some populations of C. attica consist of decumbent plants, an important feature also found in C. carystea. Moreover, the densely whitish tomentose indumentum is a common feature of C. carystea and certain populations of C. attica. Despite these morphological similarities, however, the two species are well differentiated by their habit, number of capitula and features of the involucral bracts (Fig. 5). When, one by one, the subspecies of C. attica are taken into consideration, their

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Figure 3. Total known distribution of Centaurea carystea (circle), C. pelia (square), C. mantoudii (triangle) and C. laureotica (diamond).

morphological differences from C. carystea are even greater (Table 3). To conclude, a set of morphological characters that includes the decumbent habit, the whitish indumentum, the leaves (at least some of which are clearly

two-pinnatisect with oblanceolate to obovate lobes), the wide appendages of the involucral bracts which bear a short mucro, and the pale-yellow florets distinguish C. carystea from all other members of its section.

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1–3, decumbent, (4–)8–24 cm long, tomentose 1–4 Densely whitish lanatetomentose; leaf-lobes oblanceolate to elliptic Lanulose to glabrous, middle ones 6.0–9.5 ¥ 5.0–7.5 mm

Stems

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Straw-coloured to palebrown, broadly triangular, 2.8–3.2 ¥ 5.0–7.5 mm 0.3–1.2 Pale-yellow

c. 3 Up to 1.5 54

Apical spine (mm) Corollas

Achene length (mm) Pappus length (mm) 2n

Appendages

Involucral bracts

Capitula per stem Mature rosette leaves

C. carystea

Character 1–5(–10), erect to procumbent, 5–50 cm long (1–)3–45 Glabrous to tomentose; leaf-lobes oblong-lanceolate to linear Pubescent to glabrous, middle ones 5.0–10.5 ¥ 2.5–5.1 mm Brown to dark-brown, narrowly triangular, 2.5–8.0 ¥ 2.5–5.1 mm 1–4(–6) Pink to purple, rarely whitish or bicoloured 3–4 1–3 18, 36

C. attica s.l.

3.5–4.5 1.5–2.5 36

0.5–1 Yellow or ochre

Brown, triangular, 1.8–3.5 ¥ 2.5–4.5 mm

(2–)4–46 Glabrous or scabrid; leaf-lobes lanceolate to ovate Glabrous, middle ones 5.5–7.5 ¥ 2.5–4.5 mm

Erect, 20–50 cm long

C. mantoudii

2.5–3.4 1.5–3 18

0.5–3 Pale-yellow to bright yellow

Straw-coloured, triangular, 2.0–4.0 ¥ 3.0–4.0 mm

Glabrous, middle ones 4.2–7.8 ¥ 3.0–4.0 mm

Erect or ascending, 20–60 cm long 3–16 Sparsely hairy to tomentose; leaf-lobes linear

C. pelia

Table 3. A comparison of selected characters useful in distinguishing Centaurea carystea from its relatives C. attica s.l., C. mantoudii and C. pelia

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Figure 4. Total known distribution of the subspecies of Centaurea attica: C. attica subsp. attica (square), C. attica subsp. pentelica (circle), C. attica subsp. pateraea (diamond) and C. attica subsp. megarensis (triangle).

PHYTOGEOGRAPHY On the basis of the morphological, palynological and cytological characters, Georgiadis (1980) distinguished tendencies of differentiation and evolution in the Greek members of C. section Acrolophus, which he related to geography. The species distributed to the

western and southern parts of the mainland are considered to be the most advanced, judging from the morphological characters and ploidy level. Likewise, taxa growing on the mountains appear to have a more recent origin, compared with those of the lowland, especially in central and southern Greece. Consequently, evolutionarily advanced species are generally

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Figure 5. Middle involucral bracts of Centaurea carystea and its relatives: A, C. pelia; B, C. mantoudii; C, C. carystea, D, C. laureotica; E, C. attica subsp. attica; F, C. attica subsp. megarensis; G, C. attica subsp. pateraea; H, C. attica subsp. pentelica. Scale bars: A–E, H, 1.5 mm; F, G, 2 mm.

expected to occur in western and southern Greece at high altitudes. Therefore, the discovery of the hexaploid C. carystea at the summit area of Mt. Ochi in southern Evvia comes as no surprise. Its yellowflowered allies, C. pelia (diploid) and C. mantoudii (tetraploid), are mainly distributed in east-central Greece (Fig. 3). Centaurea attica (diploid and tetraploid cytotypes), presumably a species of recent origin, replaces the Balkan endemic C. affinis Friv. in eastern Sterea Ellas (Attiki and Evvia). The discovery of C. carystea in southern Evvia may constitute an interesting link in the evolutionary processes responsible for the differentiation of C. section Acrolophus in southern Greece.

KARYOLOGY Two plants of C. carystea were used for chromosome counts and estimation of the ploidy. All metaphase plates examined revealed the same chromosome number of 2n = 54. The species is therefore hexaploid, based on the base number of x = 9, one of the most common base numbers in Centaurea. To our knowledge, this is the second time that a hexaploid Centaurea species has been reported in Greece, the first being the equally rare C. cithaeronea (section Phalolepis), known from Mts Kitheronas and Elikonas (Phitos & Constantinidis, 1993). The relatively small size of the chromosomes (c. 2–5 mm) did not

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Figure 6. A chromosome metaphase plate of the hexaploid Centaurea carystea with 2n = 54. Scale bar, 8 mm.

permit detailed morphological analysis; however, metacentric, submetacentric and acrocentric types exist in the karyotype (Fig. 6). Small satellites were observed on at least two chromosomes, situated on the short arm of metacentric or acrocentric chromosomes. The hexaploidy of C. carystea poses the question of its origin. It is possible that the species is a stabilized hybrid of allopolyploid origin. Nevertheless, no related species were found growing in the same locality of C. carystea or in the wider vicinity of Mt. Ochi, and thus the parental taxa are rather obscure. Unfortunately, the appearance and small size of the chromosomes in the metaphase plates did not permit any grouping of homologous chromosomes in sets of six, four or two that would provide evidence of allopolyploidy or autopolyploidy. Meiosis was not studied, mostly because of the scarcity of the material available.

MOLECULAR MARKERS We used rapid amplified polymorphic DNA (RAPD) markers to investigate the genetic distances between C. carystea and its morphologically related species, as discussed previously. Centaurea subsericans is included in the study as an independent species which does not comprise C. attica subsp. pateraea. Centaurea pichleri Boiss. [section Cyanus,

or Cyanus pichleri (Boiss.) Holub, when Cyanus is accepted as an independent segregate] and Centaurea raphanina subsp. mixta (section Acrocentron) were also included as reference taxa. The use of 13 selected primers resulted in the production of 173 amplified reproducible bands. We created NJ and UPGMA trees, which both agreed with regard to major dichotomies and topologies. The UPGMA trees produced using either Jaccard or Dice algorithms were practically identical, their only difference being the absolute values of the genetic distance between each pair of species. The cophenetic correlation coefficient between the Dice (1945) similarity matrix and the derived UPGMA cophenetic matrix was 0.976. The UPGMA tree based on the Dice algorithm and following the concepts of Nei & Li (1979) is shown in Figure 7. The position of C. carystea in the dendrogram is discrete. It forms a group distinct from all other taxa used in the analysis. The RAPD markers thus indicate that C. carystea either holds a rather isolated position among both the related pink-flowered C. attica group (including C. laureotica and C. subsericans) and the yellow-flowered group of C. pelia (including C. mantoudii), or its actual allies can only partly be revealed by morphological similarities that guided the selection of taxa included in the molecular analysis. Centaurea laureotica, a possible relative of C. carystea, is clearly clustered in clade B of the dendrogram, together with representatives of C. attica. Georgiadis (1980) has suggested a hybrid origin of C. laureotica, derived from introgression between C. mantoudii and C. attica. Although his hypothesis cannot be ruled out, a clearer placement together with members of the C. attica group is supported by our analysis. The position of the four subspecies of C. attica in the dendrogram of Figure 7 is another interesting point. Although the three subspecies of C. attica cluster in clades B and C, together with either C. laureotica or C. subsericans, all samples of C. attica subsp. attica cluster together with the yellowflowered C. pelia in clade D. Furthermore, the three yellow-flowered centaureas (C. carystea, C. mantoudii and C. pelia) do not form any coherent group of their own, but are found in different parts in the tree. This may indicate that corolla colour should be used with caution as an indication of taxonomic alliance in this particular group. The close genetic relationships between C. attica and C. pelia, indicated in clade D, are further corroborated by the discovery of populations with intermediate morphology between these two species and a wide range of corolla colours (cream, cream with pink tips, yellow, orange, pink, purple and various combinations of these; Constantinidis, 1997).

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Figure 7. Dendrogram showing the relationships of Centaurea carystea and its relatives based on rapid amplified polymorphic DNA (RAPD) data of 28 plants. The similarity matrix was calculated using the Dice coefficient, and the phenogram was generated according to the unweighted pair group method with arithmetic mean (UPGMA). Centaurea pichleri and C. raphanina subsp. mixta were included for comparison.

Hybridization events are not uncommon in Centaurea, even among members of different sections, and may occasionally be revealed directly by high ploidy levels (Garcia-Jacas, 1998). The formation of new species via hybridization and polyploidization, a possible scenario in the creation of C. carystea, may be responsible for the vast number of species found in the genus, the complexity of their relationships and the difficulty of taxonomic and phylogenetic interpretation encountered in many sections.

ACKNOWLEDGEMENTS We thank the curators and directors of the herbaria ATH and UPA for allowing access to their collections. We also thank Professor Emeritus D. Phitos for his assistance with the Latin diagnosis, Associate Professor C. Fasseas for facilities related to the cytological research, Assistant Professor A. Katsiotis for useful advice on the molecular markers, Zoe Tziakou for the illustrations and an anonymous referee for constructive comments.

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