A Morphometric and Karyological Study of the Anthemis macedonica Group (Asteraceae, Anthemideae) Reveals a New Species from Greece

A recent study of the Anthemis collections in the Balkans indicated that the taxa of the Anthemis macedonica group (A. macedonica subsp. macedonica, A. macedonica subsp. thracica, A. meteorica, A. orbelica) exhibit noteworthy morphological patterns not evaluated before. We applied morphometric approaches (principal components analysis, PCA; factor analysis on mixed data, FAMD) by considering 19 qualitative and 20 quantitative morphological characters, together with three ratios, in 26 populations of this group. Furthermore, the chromosome numbers and karyotype morphology were investigated in eight populations of the group, covering the taxa participating in the study. Our results revealed that the southernmost populations of the group represent a hitherto unknown species confined to serpentine: it is described here as Anthemis serpentinica Goula & Constantinidis. The morphological evidence supports the proximity of A. macedonica and A. orbelica, which would be better considered as subspecific entities of the same species. On the contrary, A. meteorica and A. thracica are retained as independent entities at species level. All taxa share the same diploid chromosome number of 2n = 2x = 18 with similar but not identical karyotypes. A brief description of all taxa, based on recent new collections, and a dichotomous key are presented. Lectotypes are designated for Anthemis macedonica and A. meteorica.


Introduction
Anthemis L., the second largest genus of Asteraceae tribe Anthemideae, comprises about 175 species in its narrow circumscription [1], and has a rather complex taxonomic and phylogenetic history. According to Lo Presti et al. [2], the pronounced variability of micro-morphological characters in Anthemis and the associated difficulty in recognising unique morphological features that consistently discriminate independent taxa, result in problems of its infrageneric classification. Several characters that were once used to define sections or even species within the genus, are proved to be unsatisfactory and of limited use. Oberprieler [3,4], for example, indicated that the distinction of sections composed of either perennials or annuals within Anthemis is problematic. Likewise, the presence or absence of scales on the receptacle disc does not necessarily characterise different species [1], whereas the appendages on the corolla lobes of disc florets "are too variable to characterise species or species groups" [5].
The group of taxa around Anthemis macedonica Boiss. & Orph. is not an exception to the above-cited rule. According to Dimopoulos et al. [6], there are three representatives of

Morphometric Analyses
The morphometric analyses that we used (principal components analysis, PCA; factor analysis on mixed data, FAMD; Figures 1 and 2, respectively) indicated a clear distinction of Anthemis macedonica subsp. thracica and Anthemis 1 from the rest of the examined material, i.e., A. macedonica subsp. macedonica, A. meteorica and A. orbelica. This distinction was more evident in the FAMD, i.e., when evaluating both the qualitative and quantitative characters ( Figure 2). In the same analysis, the group of A. meteorica was also defined, albeit remaining close to the macedonica subsp. macedonica-A. orbelica complex, which appeared coherent.
In both the PCA and FAMD, the quantitative characters that had the most significant relative contribution to the ordination of individuals (Figures 3 and 4a) were mainly those referring to the achene size and the corona length (OACL, IACL, see Table 1). The number of stem leaves divisions (DSLs), as well as the involucre width (IW) and the size of both the ligules and disc florets (LL, DFLL, DFLTPLR), also contributed significantly to the analysis. Regarding qualitative characters, the surface of the disc floret achenes (OAT, IAT), the pubescence of the involucre and the leaves (IP, LP), together with the shape of the involucre and the receptacle were among those that participated the most to the ordination of the FAMD (Figure 4b). Anthemis 1, emerging as the most distinct group of individuals in both analyses, split off from the rest of the groups because of its larger disc floret achenes (OAL, IAL, OAW, IAW), the more dissected leaves (DSLs), the tomentose leaves and involucre (LP, IP), the hemispherical to obconical shape of the involucre (IS) and the elongated-conical, sharply acute receptacle (RS) (Figures 3 and 4). A. macedonica subsp. thracica was defined as another distinct group, separated by the longer coronas in both the outer and inner disc floret achenes (OACL, IACL), the dense, sericeous pubescence on the leaves and involucre (LP, IP) and the shorter, hemispherical to conical receptacle (RS) (Figures 3 and 4).
Plants 2022, 11, x FOR PEER REVIEW 3 of 25 and Mn [24]. The ultramafic substrates quite often include endemic species adapted to their geoedaphics [25].

Morphometric Analyses
Τhe morphometric analyses that we used (principal components analysis, PCA; factor analysis on mixed data, FAMD; Figure 1 and Figure 2, respectively) indicated a clear distinction of Anthemis macedonica subsp. thracica and Anthemis 1 from the rest of the examined material, i.e., A. macedonica subsp. macedonica, A. meteorica and A. orbelica. This distinction was more evident in the FAMD, i.e., when evaluating both the qualitative and quantitative characters ( Figure 2). In the same analysis, the group of A. meteorica was also defined, albeit remaining close to the macedonica subsp. macedonica-A. orbelica complex, which appeared coherent.   In both the PCA and FAMD, the quantitative characters that had the most significant relative contribution to the ordination of individuals (Figures 3 and 4a) were mainly those referring to the achene size and the corona length (OACL, IACL, see Table 1). The number of stem leaves divisions (DSLs), as well as the involucre width (IW) and the size of both the ligules and disc florets (LL, DFLL, DFLTPLR), also contributed significantly to the analysis. Regarding qualitative characters, the surface of the disc floret achenes (OAT, IAT), the pubescence of the involucre and the leaves (IP, LP), together with the shape of the involucre and the receptacle were among those that participated the most to the ordination of the FAMD (Figure 4b). Anthemis 1, emerging as the most distinct group of individuals in both analyses, split off from the rest of the groups because of its larger disc floret achenes (OAL, IAL, OAW, IAW), the more dissected leaves (DSLs), the tomentose leaves and involucre (LP, IP), the hemispherical to obconical shape of the involucre (IS) and the elongated-conical, sharply acute receptacle (RS) (Figures 3 and 4). A. macedonica subsp. thracica was defined as another distinct group, separated by the longer coronas in both the outer and inner disc floret achenes (OACL, IACL), the dense, sericeous pubescence on the leaves and involucre (LP, IP) and the shorter, hemispherical to conical receptacle (RS) (Figures 3 and 4).       A second FAMD was attempted after excluding the well-circumscribed Anthemis 1 and A. macedonica subsp. thracica from the group. The results, as well as the contribution of both the qualitative and quantitative characters to this analysis are shown in Figures 5 and 6a,b. A. meteorica formed a well-defined group, compared to the remaining two taxa, a conclusion that strengthens the results shown in Figure 2. The variables that have the most significant contribution to this FAMD are the leaf pubescence (LP), the characters of the bracts likemargin colour and shape (BMC, IBS, IBA), the ratio of the total disc floret length to the length of the swollen part (DFLTPLR), and the leaf mucro length (LML) ( Figure 6). A. meteorica is detectable from the rest of the individuals, mainly by its more acuminate leaf-lobes (LMLs), its pubescent leaves (LP), the more acute apex of its inner bracts, compared to the obtuse to sometimes subacute apex of the two remaining taxa (IBA), and by the characters of the receptacle, reflected both in the quantitative ratio of the receptacle length to width (RLRWR) and the qualitative receptacle shape (RS) ( Figure 6). The slightly longer and wider disc floret achenes (IAW, IAL, OAW, OAL) and the darker bracts (BMC), as well as the regularly cupuliform involucre (IS) and the conical receptacle furnished with scales, often trifid at the apex (RS, SCS) that characterise A. orbelica, are the most reliable characters that support its distinction from A. macedonica subsp. macedonica ( Figure 6).

Karyological Analyses
All populations of the Anthemis macedonica group examined, share the same diploid chromosome number of 2n = 2x = 18 ( Table 2). Diploid populations of Anthemis macedonica subsp. macedonica, A. macedonica subsp. stribrnyi and A. orbelica are known to grow in Bulgaria [26]. Aneuploidies have also been reported in A. orbelica and A. macedonica

Karyological Analyses
All populations of the Anthemis macedonica group examined, share the same diploid chromosome number of 2n = 2x = 18 ( Table 2). Diploid populations of Anthemis macedonica subsp. macedonica, A. macedonica subsp. stribrnyi and A. orbelica are known to grow in

Karyological Analyses
All populations of the Anthemis macedonica group examined, share the same diploid chromosome number of 2n = 2x = 18 (Table 2). Diploid populations of Anthemis macedonica subsp. macedonica, A. macedonica subsp. stribrnyi and A. orbelica are known to grow in Bulgaria [26]. Aneuploidies have also been reported in A. orbelica and A. macedonica subsp. stribrnyi (2n = 18 + 4 and 2n = 18 + 3, respectively) together with a triploid population (2n = 3x = 27) of A. orbelica from Mt Rila [26]. The chromosome numbers, metaphase plates and idiograms of A. macedonica subsp. thracica, A. meteorica and the population from the serpentine area of Central Greece (Anthemis 1) are presented here for the first time ( Figure 7). With respect to the karyotype formula, all populations had 12 metacentric (m) chromosomes (Table 2), which appeared very similar amongst the taxa. However, there was an interesting differentiation concerning the remaining submetacentric (sm) and subtelocentric (st) chromosomes: two sm and four st chromosomes were present in all metaphase plates of A. macedonica subsp. macedonica, A. orbelica and the populations from the serpentine area of Central Greece, whereas four sm and two st chromosomes were present in all metaphase plates of A. meteorica and A. thracica. Small satellites were ob-  With respect to the karyotype formula, all populations had 12 metacentric (m) chromosomes (Table 2), which appeared very similar amongst the taxa. However, there was an interesting differentiation concerning the remaining submetacentric (sm) and subtelocentric (st) chromosomes: two sm and four st chromosomes were present in all metaphase plates of A. macedonica subsp. macedonica, A. orbelica and the populations from the serpentine area of Central Greece, whereas four sm and two st chromosomes were present in all metaphase plates of A. meteorica and A. thracica. Small satellites were observed on the short arm of the st chromosomes in all cases, whilst an additional satellited sm chromosome pair was observed in a metaphase plate of A. meteorica. The lack of satellites on the sm chromosomes in the rest of the A. meteorica metaphase plates, as well as the observed inequality of certain chromosome pairs in the ideogram reconstructions, may partly be an artifact of image processing. THL varies from 31.19 to 49.16 µm. A. orbelica tend to have a longer THL, compared to the rest of the taxa (Figure 8), although there are not enough data to test the statistical significance. A scatter plot of the asymmetry indices M CA [27] and CV CL [28] constructed by 21 metaphase plates did not contribute any further to the distinction of the different taxa, based on the chromosome features ( Figure 9). The same result is reached with different asymmetry indices (CV CI -CV CL [28]; A1-A2 [29]).
Plants 2022, 11, x FOR PEER REVIEW 10 of 25 metry indices MCA [27] and CVCL [28] constructed by 21 metaphase plates did not contribute any further to the distinction of the different taxa, based on the chromosome features ( Figure 9). The same result is reached with different asymmetry indices (CVCI-CVCL [28]; A1-A2 [29]).

Evaluation of Taxa within the Anthemis macedonica Group
Our investigation aimed to shed light on the variable morphological complex of Anthemis macedonica, a group of taxa that have been treated in different ways and at various taxonomic levels in the past. Although closely related, from a phenetic and karyological point of view, some of these taxa had been allocated to different Anthemis sections, thus hampering the assessment of their actual relationships. Our study was particularly concentrated in the southern parts of the complex distribution, where certain populations clearly did not fit the known morphological patterns of the group.
The morphometric and chromosome data support the placement of Anthemis macedonica s.str. and A. orbelica under a single taxonomic entity. Greuter et al. [21] expressed the same opinion, based on morphological grounds and Lo Presti et al. [2] provided some evidence of phylogenetic proximity. Kuzmanov et al. [26] corroborated, based on their karyotype similarity, and argued that these taxa, at subspecific level, namely A. macedonica subsp. macedonica and A. macedonica subsp. orbelica, should be members of sect. Hiorthia, thus contradicting Fernandes [20]. Although these two taxa appear to have predominatelybut not always-a different life cycle and some fine morphological differences (Table 3, Key to taxa), they are otherwise difficult to distinguish. Their habit, and in particular their lifespan and overall size, seem to be dependent also on environmental factors. In our results (Figure 6b) longevity (or otherwise, lifespan) has an insignificant contribution to the ordination of individuals that belong either to A. macedonica subsp. macedonica, or A. macedonica subsp. orbelica and A. meteorica. Field observations indicated that plants from high montane habitats that mostly grow in moist forests (Mt Rodopi, Mt Vitsi) keep quite often a biennial or short-lived perennial life-form, with a few well-developed leaf rosettes at flowering time. On the other hand, plants from drier habitats at lower altitudes are usually annuals, without additional leaf-rosettes at flowering time. This observation comes in agreement with the life-form shifts deduced in Anthemis, where the annual habit appeared to have evolved several times and independently in various groups during the past, following a progressive aridification in the Mediterranean area [1]. A. macedonica subsp. macedonica and subsp. orbelica meet in Greece, close to the borders with Bulgaria, but show a parapatric distribution around the Mt. Rodopi area ( Figure 10).

Evaluation of Taxa within the Anthemis macedonica Group
Our investigation aimed to shed light on the variable morphological complex of Anthemis macedonica, a group of taxa that have been treated in different ways and at various taxonomic levels in the past. Although closely related, from a phenetic and karyological point of view, some of these taxa had been allocated to different Anthemis sections, thus hampering the assessment of their actual relationships. Our study was particularly concentrated in the southern parts of the complex distribution, where certain populations clearly did not fit the known morphological patterns of the group.
The morphometric and chromosome data support the placement of Anthemis macedonica s.str. and A. orbelica under a single taxonomic entity. Greuter et al. [21] expressed the same opinion, based on morphological grounds and Lo Presti et al. [2] provided some evidence of phylogenetic proximity. Kuzmanov et al. [26] corroborated, based on their karyotype similarity, and argued that these taxa, at subspecific level, namely A. macedonica subsp. macedonica and A. macedonica subsp. orbelica, should be members of sect. Hiorthia, thus contradicting Fernandes [20]. Although these two taxa appear to have predominately-but not always-a different life cycle and some fine morphological differences (Table 3, Key to taxa), they are otherwise difficult to distinguish. Their habit, and in particular their lifespan and overall size, seem to be dependent also on environmental factors. In our results (Figure 6b) longevity (or otherwise, lifespan) has an insignificant contribution to the ordination of individuals that belong either to A. macedonica subsp. macedonica, or A. macedonica subsp. orbelica and A. meteorica. Field observations indicated that plants from high montane habitats that mostly grow in moist forests (Mt Rodopi, Mt Vitsi) keep quite often a biennial or short-lived perennial life-form, with a few well-developed leaf rosettes at flowering time. On the other hand, plants from drier habitats at lower altitudes are usually annuals, without additional leaf-rosettes at flowering time. This observation comes in agreement with the life-form shifts deduced in Anthemis, where the annual habit appeared to have evolved several times and independently in various groups during the past, following a progressive aridification in the Mediterranean area [1]. A. macedonica subsp. macedonica and subsp. orbelica meet in Greece, close to the borders with Bulgaria, but show a parapatric distribution around the Mt. Rodopi area ( Figure 10). Field observations and cultivation experiments regarding Anthemis meteorica, revealed that this species has a variable lifespan shared with the members of the A. macedonica group, rather than those of the A. cretica group. In its habitat, it mostly behaves as a biennial or a short-lived perennial, but some annual plants were observed as well. Its close affinity to A. macedonica became obvious in our morphometric analyses: the representatives of A. meteorica form a coherent group together with A. macedonica subsp. macedonica and subsp. orbelica (Figures 1 and 2). However, when samples of all three taxa were analysed as a single entity, A. meteorica appeared to stand out ( Figure 5). Therefore, we treat A. meteorica as an independent species with affinities to the A. macedonica group. The shape and colour of the involucral bracts are useful discriminating features between these two taxa. In A. meteorica, all involucral bracts have a very thin margin, concolorous to the remaining bract part, and a prominent, light green midvein, whereas A. macedonica s.l. usually has a brown margin and a green midvein, very distinct from the rest of the bract. Furthermore, the inner involucral bracts in A. meteorica are more lanceolate and acute than those of A. macedonica (Table 3). Some significant differences were also found in the karyotypes of these two species (see Results and Table 2) indicating that, despite their overall morphological similarity, A. meteorica would better be treated as a separate species. Most of the Anthemis meteorica populations occur south of the A. macedonica distribution area ( Figure 10) and we presume that new populations can be found to the south and to the west or north-west of the latter species.
Anthemis macedonica subsp. thracica was attributed to section Hiorthia since its very beginning. Grisebach [8] described it as a variety of Anthemis montana L., with the latter accepted nowadays as a synonym of A. cretica. A more recent work [19] considered it a separate species and placed it close to the A. macedonica group. This group was re-arranged by distinguishing three varieties under A. thracica: var. orbelica (Panč.) Stoj. et Acht., var. macedonica (Boiss. et Orph.) Stoj. et Acht., and var. stribrnyi (Vel.) Stoj. et Acht., which correspond to three out of the four currently [7] accepted subspecies of A. macedonica. In this work [19] A. thracica was regarded as a biennial species, thus adding a second life-form to its perennial habit provided by Grisebach [8]. According to our morphometrical and karyological results, A. thracica is well-distinguished from the A. macedonica complex and should be treated as a separate species (Figures 1 and 2). A. thracica shares several features in common with A. rumelica [18,20]. However, they present obvious differences in the morphology of their disc floret achenes: the latter has achenes with a very short auricle, while the former has a relatively long corona, measuring 0.4-0.7 mm and reaching 1/3 of the achene's body in the outer achenes, whereas the corona further increases to the 1/2 of the achene's body in the inner achenes. The morphological features useful in distinguishing A. thracica from the rest of the group are given in Table 3 and in the Key to Taxa.
Anthemis thracica is distributed in East Macedonia and Thrace ( Figure 10) and shared between Greece and Bulgaria. It appears to be allopatric with respect to the rest of the taxa in its group; however, its border area with A. macedonica should be better investigated for the possible discovery of new, neighbouring populations.
The Anthemis 1 populations from the serpentine parts of Central Greece stands out in our results as a distinct Anthemis group well separated from the rest of the A. macedonica taxa (Figures 1 and 2). They seem to have a stabilised biennial lifespan, documented by field observations carried out during different months of the year. In winter, particularly, we noticed no sign of last season's living flowering stems on any plant, only leaf rosettes. Several plants with well-developed leaf rosettes but without flowering stems were also found intermixed with flowering individuals during late spring. The karyotype of this species is similar to that of A. macedonica, but several morphological differences, i.e., type of pubescence, bracts and achene shape and size, disc floret features and receptacle shape, allow for safe distinction between the two species ( Table 3). The achenes of Anthemis 1 resemble those of the A. cretica group; however, the strictly biennial lifespan and the sharp, elongated conical receptacle differentiate the species from A. cretica and bring it closer to A. macedonica. The geological substrate of Anthemis 1 is also unusual: it appears to be the only member of the A. macedonica group confined to ultramafic rock types, also known as ophiolites or serpentines, in Greece. The wider serpentine area of East Sterea Ellas is also home to a few local Greek endemics (Onosma stridii Teppner, Polygonum papillosum Hartvig) and hosts several Greek serpentine endemics with a wider distribution.
Distribution and habitat: Balkan endemic growing in Greece, Bulgaria and North Macedonia [7]. In Greece, it is apparently restricted to the Rodopi mountain range, at the northeastern part of the country. More widespread but rare and threatened in Bulgaria [31,32]. It grows in semi-shaded woodland edges and sometimes in rock fissures, at an elevation of 1000-1850 m. Young individuals consisting of rosette leaves usually present at flowering time.
Distribution and habitat: Balkan endemic, distributed in Greece, Albania and North Macedonia [7]. In Greece, it is known from its locus classicus in Meteora and a few additional localities in the east-central and north-central parts of the mainland ( Figure 10). It grows in rather dry, open places, in meadows or scrub, at an elevation of 500-1500 m. Anthemis serpentinica Goula & Constantinidis,sp. nova (Figures 11 and 12) Type: Greece. Nomos Fthiotidos, ca. 17 km NNW of Lamia town, road embankment with sparse Juniperus shrub, serpentine, 38 • 59 N/22 • 22 E, 763 m, 14 May 2018, K. Goula 2464isotype, B).
Distribution and habitat: Endemic to Central Greece, where it has so far been located only in a few localities on the ultramafic substrate that forms a continuum from the western parts of Mt Othris area (eastern border) to the low hills around Mt Tamasio (Mt Katachloro) to the west. It grows on bare slopes, scree, and road embankments, together with the local serpentine endemic Silene fabaria subsp. domokina Greuter. Other plant species that were found growing at its locus classicus are Convolvulus cantabrica L., Crucianella graeca Boiss., Echium italicum L., Melilotus neapolitanus Ten., Minuartia attica (Boiss. & Spruner) Vierh. subsp. attica, Onobrychis caput-galli (L.) Lam. and Thymus teucrioides subsp. candilicus (Beauverd) Hartvig. Young individuals consisting of rosette leaves present at flowering time.
Notes: Anthemis serpentinica is the southernmost member of the A. macedonica group in Greece. Compared to its geographically closest relative, A. meteorica, of which the nearest population is located ca. 70 km to the north (Figure 10), the new species is larger in several of its parts, including the involucre width, ligule, disc floret, and achene length, although it has a similar stem height and leaf size with A. meteorica. Regarding the indumentum, it is certainly more densely pubescent than A. macedonica s.l. and A. meteorica. The populations examined (see Appendix A) present a morphological stability that we interpret as the result of speciation on serpentine rather, than serpentinomorphosis (see [33,34]). Seven additional obligate serpentine endemics are found in the same area [35]. Serpentine has long been considered as a driving force in plant evolution and speciation (e.g., [36]). Another interesting example of Anthemis speciation on serpentine is A. rhodensis Boiss.: both its subspecies, subsp. rhodensis and subsp. pulvinalis Rätzel & Ristow grow on the ultramafic substrate of Rodos Island [37]. Description: Predominantly perennial, sometimes flowering the first year, tomentose to tomentose-sericeous, with medifixed hairs, more or less densely glandular-punctate in all parts. Stems usually several, erect, 10-30 cm long, sparingly branched, striate.   Greuter (2003: 40).
Distribution and habitat: Balkan endemic growing in Greece and Bulgaria [7]. Restricted to the north-eastern parts of the countries [31,32]. It grows mainly on dry hills, at an elevation of 90-700 m.

Materials and Methods
Plant specimens of Anthemis macedonica s.l. (including A. meteorica) from ATH, ATHU, TAU, TAUF, UPA were studied in detail. Digital images of plants preserved in B, BEOU, BM, BRNM, G, GOET, JE, K, LD, LY, PRC, W, WU were also studied, with an emphasis on the type material. The herbarium acronyms appearing above follow [38]. The protologues of all the representatives of the group were researched and studied. Moreover, the descriptions, nomenclature, and evaluation of the taxonomic relationships of the taxa were studied in both historic and recent literature [8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Descriptive terminology follows Stearn [39]. Plant material from Northern and Central Greece (the natural distribution of the group) was investigated in the field during excursions in spring and summer of 2017-2020. Several voucher specimens were made for the morphometric work, ripe achenes were collected when possible, sown, and young plants were cultivated experimentally for both chromosome investigations (see below) and phenotypic observations. The morphological variation of the group was studied in recent and intact herbarium specimens prepared from various localities and deposited in ATHU. For A. macedonica subsp. stribrnyi, we checked the type but had no adequate material for further analyses.
The morphometric analysis was carried out based on the detailed measurements of 95 plants, corresponding to 26 populations, and covering the Anthemis macedonica group (A. macedonica subsp. macedonica, A. macedonica subsp. thracica, A. orbelica), A. meteorica from its locus classicus at Meteora (Central Greece) and nearby areas, as well as populations of a particular form growing on the serpentine areas of Central Greece that approached A. macedonica s.l. phenotypically (Anthemis 1). The analysis was carried out mostly on Greek samples. A total of 20 quantitative morphological characters were measured on the herbarium specimens and three ratios were calculated (Table 1). In addition, 19 qualitative characters were evaluated ( Table 1). Many of the selected characters are among those considered as diagnostic for the members of the group and are mentioned as such in the literature. To explore the dominant patterns in the morphological variation of the examined specimens, the principal components analysis [40] was applied on the quantitative morphological characters by using the "princomp" function of the R package "stats" [41]. To explore the variability of both the quantitative and qualitative morphological characters, the factor analysis on mixed data (FAMD [42]) was used, a method that is applicable to a dataset containing both continuous and categorical variables. The FAMD was applied using the "FAMD" function of the "FactoMineR" [43] R package.
The karyological examinations were made in the root tips obtained from cultivated material. Mature achenes from eight populations collected from various localities were sown in pots at the facilities of the Department of Biology, National and Kapodistrian University of Athens. The seedlings were transferred in individual pots and root tips were collected several times. The protocol used for the pretreatment of the roots, the preparation of metaphase plates and the construction of idiograms is described in [44]. The total haploid length (THL, see [45]) was calculated to provide a chromosome size evaluation. The indices M CA [27], CV CL /CV CI [28] and A1/A2 [29] were used to estimate the intrachromosomal and interchromosomal asymmetry. All chromosome measurements and the evaluation of the asymmetry indices were conducted using the KaryoType software, ver. 2.0 [46].