The Balkan endemic Centaurea grbavacensis ( Asteraceae ) ─ New evidence on variation, typification, and distribution, with karyological studies

Phitos, D., Niketić, M., Kipopoulos, E. & Kamari, G.: The Balkan endemic Centaurea grbava-censis ( Asteraceae ) ─ New evidence on variation, typification, and distribution, with karyolog-ical studies. — Fl. Medit. 33: 111-129. 2023. — ISSN: 1120-4052 printed, 2240-4538 online. Centaurea grbavacensis , a Balkan endemic species of North Macedonia and N and C Greece, has been thoroughly studied throughout its distribution, based on living, photographic and herbarium material as well as literature. Ιt was found that all forms of the species grow in both Greece and North Macedonia. We studied the variation of the main morphological features, especially for the Mt. Olympos (Greece) subpopulations. It was found that spine length of the involucral phyllaries varies significantly within individuals and subpopulations, irrespective of flower colour (the typical colour is dark brownish purple, whereas in C. grbavacensis f. lutea it is yellow). Therefore, C. grbavacensis f. spinescens cannot be maintained as a separate taxon. A karyological study of the two colour forms was performed, and karyotype photos are provided for the first time. The chromosome number, 2 n = 2x = 22, was confirmed and the karyotype morphology is mentioned.


Introduction
was first to recognize Centaurea grbavacensis (Rohlena) Stoj.& Acht.as a new taxon, initially described it as a variety of Centaurea immanuelis-loewii Degen, which also had been based on plants from southern former Yugoslavia, known today as North Macedonia.
Centaurea grbavacensis belongs to C. sect.Acrocentron (Cass.)DC., a section of c. 100 species mainly found in the Mediterranean region (Font & al. 2002(Font & al. , 2009)).Micevski (1975), in his relevant publication on some species of the section, treated the variation of C. grbavacensis by formally recognising three formae: f. grbavacensis (Fig. 1), f. spinescens Rohlena (Fig. 2) and f. lutea Micevski (Figs 3 and 4).The said variation is not surprising in view of the relatively wide distribution of the species.
Centaurea grbavacensis is a characteristic and attractive Balkan endemic.The most noticeable and interesting feature of its variation is flower colour.C. grbavacensis was considered endemic to North Macedonia until 1976 (Dostál 1976).It has been well studied throughout its scattered distribution in North Macedonia and all its forms (as recognized by Micevski 1975) have often been found to coexist in the same local population (Map 1, Appendix 1).The yellow flower colour, first informally referred to as Centaurea immanuelis-loewii "fl[ore] luteo" by Soška (1938: 230), was known, so far, only in the northernmost distribution area of the species (Map 1, Figs 3 and 4), where it grows side by side with the typical colour form (Map 1, Fig. 5).
In 2015 Ms Themis Nasopoulou, member of the Olympus National Park Management Agency, together with Assist.Prof. G. Fotiadis, found plants of C. grbavacensis with yellow flowers on Mt.Olympos, at the place named Skandaliara, Agios Ioannis -Τrochalo, (Figs 6a & 6b); this was the first time such plants were observed in Greece.In the same locality, Ms Nasopoulou also found individuals with the typical flower colour (Figs 6c & 6d).Photographs of flower heads from Mt. Olympos, showing both colours together, were posted on the Internet in May 2016 by Vasileiadis (2016).Several more recent photo posts of yellow-flowered C. grbavacensis plants from the same locality on Mt.Olympos followed, as Eleftherios Κipopoulos (co-author of this paper) found and studied C. grbavacensis in several localities on that mountain (Map 2).

Material and Methods
Herbarium material from the whole distribution range in North Macedonia and north and central Greece was used (Maps 1 & 2), kept in the herbaria ATH, ATHU, B, BEO, BEOU, C, EGE, G, GOET, HUTH, K, LD, M, MCF, P, PRC, S, SKO, UPA, WU (abbreviations according to Thiers 2022+) and in the private herbarium "Phitos & Kamari" (deposited at UPA).The cited records from living and herbarium material or from literature are listed in Appendix 1. Distribution is mapped using a 10 × 10 km squares grid, based on the Military Grid Reference System (MGRS) of Greece (Lampinen 2001).
Living plants kept in cultivation at the University of Patras, originating from Mt. Olympos, were used for karyological study, which followed is the classical squash technique by Östergren & Heneen (1962), with some small modifications (especially in the pretreatment: 00.03% v/w aqueous 8-hydroxyquinoline, in 5 °C, for 3h).Details of the squash technique and the karyotype morphology have been reported in previous papers (Kamari 1992; Samaropoulou & al. 2013; Bareka & al. 2015).
Flora Mediterranea 33 -2023 115 Fig. 5. Specimen of Centaurea grbavacensis (in Flora Exsiccata Macedonica) with two individuals belonging to the two colour forms of the species, collected by Teofilovski (30.5.1999) in Suva Gora, North Macedonia.

Variation
The most noticeable variable features in Centaurea grbavacensis are flower colour, size of the apical spines of the involucral bracts (phyllaries) of the capitula (flower heads), especially of the outer middle phyllaries, as well as the segment shape of the basal leaves (simple-intact or sickle-shaped).In particular, the colour of the flowers varies significantly.The typical flowers have dark brownish-purple colour and appear to occur in almost all species subpopulations or populations (Maps 1 & 2, Figs 1, 2, 5, 6c & 6d, 7, 8).
Early literature (Soška 1938: 230) and herbarium material from North Macedonia show that individuals of Centaurea grbavacensis with yellow flowers are scattered in three very close localities or subpopulations in the northern part of the species distribution (Map 1).For example, according to Teofilovski (2011) both forms abound in the lower parts of the Treska gorge (Fig. 5), while upstream from the village Zdunje f. lutea is prevalent and on the peak Visoka Čuka (Zdunje) that variant no longer exists.On the other hand, in most of the populations or subpopulations scattered in North Macedonia, individuals of otherwise typical C. grbavacensis vary in the length of their phyllary spines (Fig. 2, Map 1).
In Greece, the most populations consist of typical plants with dark purplish-brown flowers only.Additionally, on Mt.Olympos, where several scattered subpopulations of Centaurea grbavacensis occur (Map 2), we also observed predominance of the typical flower colour (f.grbavacensis), but considerable variation in phyllary spine length (Fig. 8).However, on Mt.Olympos, in two subpopulations in the SE part of its range plants with yellow flowers (f.lutea) are prevalent (Figs 6a & 6b & 9); in subpop.no 8 (Map 2), not individuals with dark brownish-purple flowers have so far been observed.
It is noteworthy that from the abundant living and photographic material of Centaurea grbavacensis studied from Mt. Olympos, we observed that plants with spiny phyllaries (with spines up to 10 mm long, corresponding to "f. spinescens") coexist with spineless ones in several localities, irrespective of flower colour (Fig. 6a-b, Fig. 8a-d).
We conclude that, as the length of the phyllary spines varies significantly among the individuals of both colour forms of the species, "f.spinescens" cannot be considered as a separate taxon.
With regard to the variation of the segment shape of the basal leaves, our observation on living plants from Mt. Olympos, and of photographs of herbarium specimens (notably the type material of C. grbavacensis) and can be summarised as follows: individuals with flowers of brownish-purple colour tend to have basal leaves with simple (entire), falcate or straight segments spreading at a right angle from the leaf rachis.Conversely, individuals with yellow flowers have basal leaves with toothed or deeply lobed segments, but sometimes a few with an undivided lamina (Figs 1 & 2).However, as the mentioned differences in basal leaf shape are not significantly diagnostic for the two different flower colour types.
On Mt.Tzena, Centaurea grbavacensis coexists with C. kotschyana Heuff, with which it shares the dark brownish-purple flower colour, but which is easily distinguished by a combination of stem and leaf characters: However, C. kotschyana has leafy stems and undivided basal leaves, whereas in C. grbavacensis the stems are consistently leafless stem and the basally crowded leaves are pinnatisect to bipinnatisect, with numerous linear or linear-lanceolate ultimate segments arranged in different planes.According to Micevski (1975), Centaurea grbavacensis is also similar to C. immanuelis-lοewii Degen (both having dark brownish-purple flowers); he regards all Yugoslav (North Macedonian) records of the latter as belonging to the former.
In Greece, the two species Centaurea grbavacensis and C. immanuelis-lοewii never grow together and can be distinguished easily by their stems and habit.C. immanuelislοewii has numerous branched stems with leaves, while C. grbavacensis, consistently, has unbranched, leafless stems.
-Flowers yellowish to yellow.
Flora Mediterranea 33 -2023Micevski (1975: 183) described Centaurea grbavacensis f. lutea referring to "C. immanuelis-loewii fl.luteo" of Soška (1938: 230), which is a descriptive designation, not a validly published name.Even though Micevski failed to use the term "type" referring to the single specimen he cited for his f.lutea, thus apparently failing to meet the requirements of valid publication of that time (Turland & al. 2018: Art. 7.11 & 40.1), the name is nevertheless validly published under Art.40.2.The fact that, in the German summary, two localities are mentioned for f. lutea is not tantamount to the inclusion of more than one specimen in the new taxon (see Turland & al. 2018: Art. 40 Note 2).
Plants with clearly spiny phyllaries, named "f.spinescens" or corresponding to the relevant description, have not been reported from Greece, so far.However, our study of the photographic material collected over the years on Mt.Olympos by E. Kipopoulos, showed that in the locality Rema Arapi (subpop.no. 2 in Map 2) a majority of otherwise typical plants of C. grbavacensis have up to 10 mm long phyllary spines (Fig. 9) and according to Rohlena (1935: 4) correspond to his "f.spinescens".
The same kind of phyllary variation was also observed in several subpopulations of Mt.Olympos.
Additionally, we can here report, for the first time, that the same kind of variation in phyllary spines also exists in yellow-flowered plants (f.lutea) in material from subpop no.7 (Map 2) on Mt.Olympos (Fig. 6), some of which, based on spine length, would correspond to "f. spinescens".Probably, further study of all subpopulations of C. grbavacensis will show that throughout the species distribution the appearance of spiny phyllaries is a variable character, and that spiny plants appear in both colour forms recognized here.

Distribution
Centaurea grbavacensis is a Balkan endemic that occurs in North Macedonia, north and central Greece (Map 1, Appendix 1), where it forms small, scattered subpopulations.
In three subpopulations in the northern part of North Macedonia (Map 1), both colour forms of C. grbavacensis coexist, and several subpopulations show strong variation in length (up to 10 mm) of the phyllary spines, even on single plants.
In Greece (Map 1) the typical colour form of C. grbavacensis predominates.The exception is Mt.Olympοs (Map 2), where several scattered subpopulations of typical, dark-purple flowered C. grbavacensis, grow on the NE slopes (Figs 7 & 8), but in the two only subpopulations of the SE slopes, also f. lutea appears.In subpop.no.7 (Map 2) some typical, dark-purple flowered plants also exist (Fig. 6), but in the subpop.no. 8 (Map 2), only yellow-flowered plants have been observed, so far (Fig. 9).
Even though the flora of Mt.Tzena and Mt.Voras of N Greece has been well studied, no yellow-flowered plants of C. grbavacensis have been reported from there, in spite of the shorter distance separating these mountains from northern North Macedonia.
We conclude that yellow-flowered plants (f.lutea) only occur in few localities in the northern and southern the periphery of the total species range (Map.1).
We counted 2n = 2x = 22 chromosomes in both colour forms of Centaurea grbavacensis, on material from Mt. Olympos (see above).The chromosome number in both forms is diploid, the karyotypes are symmetrical and include metacentric (m) and submetacentric (sm) chromosomes.Two chromosome pairs in the karyotypes of both forms bear satellites.These two pairs of sm-SAT chromosomes bear spherical satellites on their shorter arms and are usually well visible.In some cases, we observed a third pair of small spherical satellites, but these are not always visible (in Figs 10a, 10c & 10d we observed five, and in Fig. 10b, six satellites).The karyotype formula is 2n = 2x = 8m+8sm + 6sm-SAT = 22 chromosomes, varying in size from 3.0 to 5.7 μm.