Introduction

Cytological data within a taxon (e.g. family, genus or species) may vary with respect to the number of chromosomes (including ploidy level and aneuploidy), but may also show variation in absolute and relative size, in morphology, and in staining properties of the respective chromosomes (Sharma & Sen 2002). The position of the centromere is the most important feature to characterize morphological differences of chromosomes (Levan & al. 1964). Information on chromosome morphology is a powerful tool to characterize genomes. For any organism, karyological data represent essential information and provide important characters for plant systematics and evolutionary analysis (Baltisberger & Widmer 2009; Baltisberger & Hörandl 2016).

The genus Achillea L. comprises 110–140 species and has a rather wide geographic distribution spanning Europe, Asia and N Africa (Ehrendorfer & Guo 2006; Oberprieler & al. 2007). Its centre of diversity is located in SE Europe and SW Asia, and the taxa vary broadly in morphology, life cycle and ecology (Guo & al. 2004). Polyploidy is rather frequent, and evidence for natural hybridization has been reported (Guo & al. 2005). Recent investigations including molecular methods show that Achillea has to be newly circumscribed and the two related and monotypic genera Leucocyclus Boiss. and Otanthus Hoffmanns. & Link have to be included in Achillea (Guo & al. 2004; Ehrendorfer & Guo 2005; 2006). Based on available molecular data, four sections are currently accepted within Achillea, viz. A. sect. Achillea s.l., A. sect. Anthemoideae (DC.) Heimerl s.l., A. sect. Babounya (DC.) O. Hoffm. and A. sect. Ptarmica (Mill.) W. Koch (Guo & al. 2004).

Similar to Achillea, the genus Ranunculus L. (Ranunculaceae) is highly diverse and also varies broadly in morphology, life cycle and ecology. Ranunculus shows pronounced cytological variation that supports the phylogeny of the genus (Baltisberger & Hörandl 2016). To investigate whether cytology is also variable in Achillea and reflects phylogenetic relationships, we evaluate novel cytological information (chromosome numbers and karyotypes) within Achillea using our own data, as well as data from literature, and we discuss the phylogenetic relevance of such data.

Material and methods

Living plants were sampled at 85 natural populations (32 from Switzerland, 22 from Greece, 15 from Italy, four each from Austria and Slovenia, three from France, two each from Bulgaria and Macedonia, and one from Kosovo). The plants were transferred to Zurich and cultivated in the greenhouse.

The taxa are arranged alphabetically. Geographic indications are given for each site. The sites are arranged according the country and then the year of sampling; indications of Greek sites have been adapted to the new organization of Greece that was part of the “Kallikratis” administrative reform starting on 1 Jan 2011 (for information and maps see  https://en.wikipedia.org/wiki/Regional_units_of_Greece). The identification numbers refer to the herbarium specimens in the collection of Matthias Baltisberger, deposited in the herbarium Z+ZT (herbarium codes follow Thiers [continuously updated]).

The cytological investigations were carried out on root tips (for method see Baltisberger & Widmer 2009). Five to ten metaphases were counted for each investigated individual to determine chromosome numbers. Numbers of investigated individuals (N) are indicated in Table 1. Chromosome numbers in literature were checked with Goldblatt & Johnson (1979+). To investigate karyotypes, good metaphases were drawn with a camera lucida. The number of analysed metaphases per population is given in Table 1. The nomenclature for chromosome morphology follows Levan & al. (1964). The chromosomes are named according to the position of the centromeres. This is expressed with the ratio “long arm to short arm”. We used the following terms: metacentric (arm ratio 1–1.7), submetacentric (ratio 1.7–3), subtelocentric (ratio 3–7); further chromosome types have not been observed in Achillea. As there are several polyploids, we indicate the haploid karyotype to more easily compare the indications of the respective taxa. In Fig. 1 metaphases are shown for taxa of which the chromosome number has not been previously reported, for taxa with B-chromosomes and for the deviating karyotype found in some plants of one population of A. holosericea. Searching in literature for karyotype information, it turned out that papers with figures of good metaphases were very helpful. We therefore offer illustrations of one metaphase of each investigated taxon for which the chromosome number was known but no karyotype information was found in literature (Fig. 2).

Table 1.

Alphabetical list of investigated Achillea taxa with the year of sampling. — Voucher: number associated with vouchers deposited in the herbarium Z+ZT; N: number of individuals investigated for chromosome numbers; 2n: chromosome number in somatic cells; KT: number of metaphases investigated for karyotype analysis.

Continued

Based on indications in Richardson (1976), Hess & al. (1980), Pignatti & Ehrendorfer (1982), Franzén (1991) and Aeschimann & al. (2004), geographic distributions and interesting systematic aspects are discussed within the respective taxon including further literature if needed. A general discussion concerning Achillea and its cytological data (chromosome numbers, ploidy levels and karyotypes) follows the treatment of the taxa.

Results and Discussion

The rather variable Achillea ageratifolia occurs in the S parts of the Balkan Peninsula and comprises three subspecies (Franzén 1986, 1988). We investigated the two mountain subspecies, viz. subsp. ageratifolia (growing throughout the range of the species) and subsp. aizoon (occurring in N Greece). The lowland subspecies A. ageratifolia subsp. serbica (Nyman) Heimerl was not investigated here.

All three taxa are known to be diploid with 2n = 2x =18 chromosomes (Franzén 1988), as we found in one population of subsp. ageratifolia as well as in subsp. aizoon. But in subsp. ageratifolia one population (Macedonia, 12251) proved to be tetraploid with 2n = 4x = 36 chromosomes, which was not known up to now. Both subspecies showed the same haploid karyotype with seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites as it was indicated also by Baltisberger (2006) for subsp. ageratifolia as well as by Međedović (1976, and repeated 1984) and Baltisberger (1994) for subsp. aizoon.

See comments under Achillea ageratifolia subsp. ageratifolia.

Achillea alexandri-regis is a narrow-endemic taxon known only from Mt Ošljak in Kosovo (Gaijic 1975). Mt Ošljak is the northernmost part of Šar Planina, which spans the joint borders of NE Albania, NW Macedonia and S Kosovo. Achillea alexandri-regis grows in alpine meadows on limestone. It is characterized by yellow flowers and undivided (rarely shallowly pinnate), dentate and sericeous leaves. Despite its rarity and restricted geographic range, it was subject of several pharmacobotanical investigations (Kovačević & al. 2005; Kundakovi´ & al. 2000, 2004, 2005a, 2005b).

The chromosome number of this species was not known until now. With 2n = 2x = 18 chromosomes (Fig. 1A) it is diploid. The haploid karyotype consists of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites.

Achillea alexandri-regis combines some taxonomically relevant morphological characters of two other mountain Achillea species: A. holosericea with pinnatifid, sericeous leaves and yellow flowers is also calcicole and grows on gravel and stony meadows in the S part of the Balkan Peninsula (Albania, Kosovo, Macedonia and Greece). Achillea lingulata Waldst. & Kit. with undivided, glabrous to pubescent leaves and white flowers is calcifuge and occurs on meadows and rocky slopes in the E and C Carpathians as well as scattered in mountains of the Balkan Peninsula southwards to N Greece. Based on the somewhat intermediate morphology and the fact that these two species grow not far away from Mt Ošljak, Diklić & al. (1991) suggested that A. alexandri-regis is of hybrid origin with these two species as putative parents. They also proposed to investigate the karyology of these three species to support or reject this hybrid hypothesis. All three species are fully or at least mostly diploid and show the same haploid karyotype (for A. alexandri-regis see above; for A. holosericea see below; for A. lingulata see Međedović & Siljak 1978, Međedović 1984 and Baltisberger & Widmer 2009). Consequently, caryological data do not allow to support or reject the hybrid origin hypothesis for A. alexandri-regis.

Fig. 1.

Somatic metaphases of Achillea species, all with 2n = 2x = 18 chromosomes. — A: A. alexandri-regis (12278); B: A. fraasii (12522) with three additional B-chromosomes (marked with B); C: A. holosericea (12520) with one additional B-chromosome (marked with “B”); D: A. holosericea (12596b), one metacentric chromosome with deviating morphology, changed into a submeta- to subtelocentric chromosome (marked with “st”), the long arm distinctly longer than the arms of the metacentric chromosomes; E: A. lucana (13826).

Mt Ošljak is situated on the border of the geographic areas of both potential parent species (Diklić & al. 1991): the mountain is located on the N border of the geographic range of Achillea holosericea, the northernmost sites of A. holosericea being in Šar Planina S and SE of Mt Ošljak. Concerning the geographic distribution of A. lingulata, Mt Ošljak is in the range of the S and SW border of the respective geographic area. This restricted and peripheral distribution of A. alexandri-regis concerning the geographic areas of both widespread species suggests another scenario of speciation than the hybrid origin of A. alexandri-regis: It may have originated from one of these species by quantum speciation. Quantum speciation is defined as the occurrence of a new daughter species from a semi-isolated (mostly peripheral) population of the ancestral species (Grant 1971). As A. alexandri-regis is closer to A. holosericea (shape and hairiness of leaves, colour of flowers, ecology) it seems more likely that it was separated by quantum speciation from A. holosericea.

Fig. 2.

Somatic metaphases of Achillea taxa (all with 2n = 2x = 18 chromosomes) for which no karyotype information was found in literature. — A: A. atrata (12623); B: A. erba-rotta (14446); C: A. grandifolia (12611); D: A. macrophylla (1840); E: A. moschata (11630); F: A. nana (11833); G: A. pindicola subsp. integrifolia (11337); H: A. ptarmica (12709); I: A. tomentosa (11454).

Achillea atrata is a widespread species on calcarous soils in the Alps. We investigated plants from twelve sites from four different countries (Austria, Italy, Slovenia and Switzerland). All plants proved to be diploid with 2n = 2x = 18 chromosomes and with the same haploid karyotype of seven metacentric and two submeta- to subtelocentric chromosomes, the latter chromosomes with mostly visible satellites. As no karyotype information was found in literature, we present a metaphase of this species (Fig. 2A).

Achillea barrelieri is a diploid endemic mountain species with 2n = 2x = 18 chromosomes occurring only in Italy in C and S Appennini. The species is known to be diploid, which was confirmed by the plants from Mt Vettore (14408; the chromosome number of the other site [11533] was published earlier, see Baltisberger 1990). Plants from both sites showed a haploid karyotype of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. The same karyotype was indicated by Baltisberger & al. (1996) for plants from three different mountains.

Achillea chrysocoma is closely related to the diploid lowland species A. tomentosa (see below). Achillea chrysocoma is a species of mountain grasslands in the Balkan Peninsula growing in Albania, Macedonia, Bulgaria and N Greece. Plants from E Albania, Macedonia and Bulgaria are hexaploid with 2n = 6x = 54 chromosomes (Baltisberger 1992, 2006), whereas plants from mountains in S Albania are octoploid with 2n = 8x = 72 chromosomes (Baltisberger & Baltisberger 1995; Baltisberger 2002). The plants from N Greece investigated here proved to be hexaploid with 2n = 6x = 54 chromosomes. This fits well with the separation of the geographic areas of the respective cytotypes, with hexaploid plants occurring in more eastern areas than the octoploid ones (Baltisberger 2002).

The haploid karyotype shows seven metacentric and two submeta- to subtelocentric chromosomes. The 12 submeta- to subtelocentric chromosomes probably all have satellites, but these are not always visible. This has been observed earlier in several Achillea species, especially in plants with higher ploidy levels (e.g. Baltisberger & Baltisberger 1995; Baltisberger 2002; Baltisberger & Widmer 2009). The haploid karyotype found in plants from Mt Vourinos (12309a) is the same as that indicated for plants from other localities (Baltisberger 1992, 2002; Baltisberger & Baltisberger 1995).

The group of Achillea clavennae consists of five species growing in stony meadows, screes and ledges on limestone mostly on mountains (Franzén 1986). Two species are rather widespread, viz. A. clavennae in the Alps and the Balkan Peninsula (southwards to Mt Korab on the border between Albania and Macedonia) and A. fraasii (Balkan Peninsula from Montenegro to Greece and W Anatolia, see below). Three species have rather narrow distribution areas, viz. A. ambrosiaca (Boiss. & Heldr.) Boiss. (endemic on Mt Olympus), A. pindicola (endemic to C and S Pindhos in Greece, see below), and A. umbellata Sm. (endemic to C and S Greece).

Achillea clavennae is highly variable (Heimerl 1926), but, as the characters show weak correlations and many intermediate plants can be found, no infraspecific subdivision seems useful (Franzén 1986). We counted the chromosome number of plants from eight sites from four countries (Austria, Italy, Slovenia and Switzerland; the chromosome number of one Italian site [10992] was published earlier, see Baltisberger 1990). All plants were diploid with 2n = 2x = 18 chromosomes. Plants from all nine sites investigated showed the same haploid karyotype of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. The same karyotype was indicated by Mededović (1976, 1984) and Baltisberger & al. (1996).

Achillea clypeolata shares several important characters and is therefore rather similar to A. coarctata (see below). Both grow in dry, rocky meadows in the Balkan Peninsula. They mainly differ in the density of the indumentum and the dissection of the leaves. Achillea clypeolata is less densely hairy, and the leaves are less dissected (1- to 2-pinnatisect) and mostly flat. A. clypeolata is known to be a diploid species with 2n = 2x = 18 chromosomes, as was found in the plants investigated here. The haploid karyotype consists of seven metacentric and two submetato subtelocentric chromosomes, the latter with satellites. The same karyotype was indicated in Kuzmanov & Kozuharov (1973), Baltisberger (1993, 2006), and Nedelcheva (1995).

Achillea coarctata is rather similar to A. clypeolata (see above), but in contrast to A. clypeolata the indumentum of A. coarctata is denser and the leaves are more dissected (2- to 3-pinnatisect) and subterete. Two ploidy levels are known, viz. diploid with 2n = 2x = 18 and tetraploid with 2n = 4x = 36 chromosomes. With 2n = 2x = 18 chromosomes all plants from both Greek sites proved to be diploid (the chromosome number of the Bulgarian site was published earlier, see Baltisberger 2006). The haploid karyotype of plants of all three sites shows seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. The same karyotype was given in Kuzmanov & Kozuharov (1973) and Baltisberger & Baltisberger (1995).

Achillea crithmifolia grows in meadows and shrubby areas mostly on mountains in the Balkan Peninsula. Diploid plants with 2n = 2x = 18 as well as tetraploid plants with 2n = 4x = 36 chromosomes are known. The plants from Macedonia investigated here were diploid with 2n = 2x = 18 chromosomes and showed a haploid karyotype with seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites, which corroborates the indications in Kuzmanov & al. (1989), Baltisberger (1994, 2002, 2006) and Baltisberger & Baltisberger (1995).

Achillea distans occurs in rocky meadows in the Alps, the Carpathians, as well as in the mountains of the Balkans. It is a member of the highly polymorphic and difficult A. millefolium group (see below), with diploid to highly polyploid taxa, or is at least closely related to that group (Danihelka & Rotreklova 2001). Achillea distans is known to be mostly hexaploid with 2n = 6x = 54 chromosomes, as were the plants from Greece investigated here. No suitable metaphases for an analysis of the karyotype were obtained from the material investigated here. Nevertheless the haploid karyotype of seven metacentric and two submeta- to subtelocentric chromosomes was indicated in Baltisberger (1992) and Baltisberger & Widmer (2009).

Achillea erba-rotta belongs to a group of taxa growing on rocks and screes and in stony meadows in the Alps, the Appennini, as well as on Mt Olymbos in Greece. The taxa are treated at species or subspecies levels. We investigated three taxa of the group, viz. A. erba-rotta (here) as well as A. moschata and A. lucana (see below).

In the Alps three taxa of this group are indicated, all being calcifuge (Richardson 1976; Aeschimann & al. 2004): Achillea erba-rotta occurs in the SW Alps (France and Italy), A. moschata (see below) is widespread in the C and E Alps (from France to E Austria), and A. erbarotta subsp. ambigua (Heimerl) Richardson growing in the SW Alps (France and Italy) is morphologically intermediate between the other two species.

Only diploid plants of Achillea erba-rotta are known, and the plants investigated here corroborate this ploidy level with 2n = 2x = 18 chromosomes with the haploid karyotype of seven metacentric and two submetato subtelocentric chromosomes, the latter chromosomes with satellites. As no karyotype information was found in literature, we present a metaphase of this species (Fig. 2B).

Achillea fraasii belongs to the A. clavennae group (see above). It is highly variable (Heimerl 1926), but, as no subunit merits recognition, no infraspecific taxa are recognized (Franzén 1986). Achillea fraasii occurs in the W part of the Balkan Peninsula (Montenegro, Albania, Kosovo, Macedonia and Greece) as well at one site in W Anatolia (Mt Ida, Franzén 1991). Most plants are diploid with 2n = 2x = 18 chromosomes, but tetraploid plants with 2n = 4x = 36 chromosomes also exist. All plants investigated here showed 2n = 2x = 18 chromosomes and proved to be diploid, but in one of the two populations (Mt Loupata, 12522) additionally two to four B-chromosomes were observed in every plant investigated (Fig. 1B). Neglecting the B-chromosomes, the haploid karyotype of plants from both sites consists of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. The same haploid karyotype was indicated in Baltisberger (1993 [on tetraploid plants], 2002) and Baltisberger & Baltisberger (1995). Additional B -chromosomes have also been indicated for other species of the A. clavennae group: for A. pindicola see below; for A. umbellata, Contandriopoulos & Martin (1967) indicated B -chromosomes in plants from Mt Parnassos, and Tzanoudakis & Iatrou (1981) found one to three B -chromosomes in all plants of their five investigated Greek sites.

Achillea grandifolia grows in mountain woods in the Balkan Peninsula and in Turkey. Only diploid plants with 2n = 2x = 18 chromosomes are known, as we found in the plants investigated here. The haploid karyotype shows seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. As no karyotype information was found in literature, we present a metaphase of this species (Fig. 2C).

Achillea holosericea grows on gravel and stony meadows from lowlands to the mountains in the S part of the Balkan Peninsula (Albania, Kosovo, Macedonia and Greece). It is mostly diploid with 2n = 2x = 18 chromosomes, but tetraploid plants with 2n = 4x = 36 chromosomes are also known. We investigated plants from five Greek sites. Four of them proved to be diploid with 2n = 2x = 18 chromosomes. From one of these sites with diploids (Mt Kamvounia, 12520) all four investigated plants constantly showed one additional B-chromosome (Fig. 1C) . Plants with one to three B-chromosomes were also reported by Persson (in Franzén 1991) from two mountains on Peloponnisos (S Greece). All plants from one site (Mt Karava, 12307) were tetraploid with 2n = 4x = 36 chromosomes.

Neglecting the B -chromosomes, the haploid karyotype found in the plants from four sites always proved to consist of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. The same haploid karyotype was indicated in Baltisberger (1984, 1991, 1993, 1994). Five plants were investigated from the fifth site (Vikos gorge, 13596b) and all proved to be diploid with 2n = 2x = 18 chromosomes. One of these plants showed the typical haploid karyotype, as do the plants from the other sites. But four plants had a deviating karyotype with one of the 14 metacentric chromosomes changed into a submetato subtelocentric chromosome. The short arm of this chromosome was similar to the short arm of the other four satellited submeta- to subtelocentric chromosomes, but the long arm of this chromosome was distinctly longer than the arms of the metacentric chromosomes. The diploid karyotype of these four plants therefore consisted of 13 metacentric chromosomes, one submeta- to subtelocentric chromosome without satellites and four submeta- to subtelocentric chromosomes with satellites. These findings suggest that a change within one metacentric chromosome took place and a substantial part of one arm has been translocated to the other arm of the same chromosome (Fig. 1D).

In 1992 we sampled six living rosettes of young plants in the Vikos gorge (from where the plants with the deviating karyotype originate). Five individuals survived, four of them showed the deviating karyotype, and only one plant proved to have the “regular” karyotype. As we sampled six rosettes by chance and got at least four with the deviating karyotype, those plants seem to be rather frequent and viable within that natural population and their survival is not just the result of protection in culture. We checked important morphological characters comparing the five plants from the Vikos gorge all flowering in the greenhouse (shape and indumentum of the leaves, hairs on the stems, number of capitula per corymb, size and hairs of the involucres). No significant morphological difference could be detected. We stained pollen grains of the respective plants with carmine acetic acid, and all five plants proved to have well-developed and well-staining pollen. It seems that the deviating karyotype has no negative impact on the fitness of the individuals. Nevertheless we do not currently know whether plants with the deviating karyotype are able to successfully reproduce and produce viable offspring.

The calcicole Achillea lucana belongs to the group of A. erba-rotta (see above). It is a taxon of a cluster of plant populations from screes and mountains scattered in C and S Italy (Richardson 1976; Pignatti 1979; Pignatti & Ehrendorfer 1982). Several names on various taxonomic levels have been assigned to the populations of this cluster [e.g. A. erba-rotta var. ambigua Heimerl, A. erba-rotta subsp. ambigua (Heimerl) Richardson, A. erba-rotta subsp. calcarea (Porta) Richardson, A. moschata var. calcarea Porta, A. lucana Pignatti, A. rupestris Huter vel Porta, A. erba-rotta subsp. rupestris (Porta) Richardson]. The delimitations of the respective entities is unresolved, and further investigations (including molecular methods) studying plants from as many origins as possible are needed.

Mt Pollino is mentioned in Pignatti & Ehrendorfer (1982) as the southernmost site with plants of this group. These plants from Mt Pollino are named as Achillea rupestris Huter, a taxon with undivided leaves and bracts with narrow margins (less than 0.2 mm). The plants investigated here originate from Saracena, which is situated about 20 km S of Mt Pollino. As the plants from Saracena show pinnatifid leaves and bracts with rather broad margins (more than 0.3 mm), they best fit with A. lucana even though this species has only been reported from sites N of Mt Pollino (Pignatti 1979). The chromosome number of A. lucana was not known until now. With 2n = 2x = 18 (Fig. 1E) the chromosome number is the same as that indicated for plants from Mt Pollino [Cesca & Morrone 1981, named as A. erba-rotta subsp. calcarea (Porta) Richardson]. The plants investigated here show a haploid karyotype of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites.

Achillea macrophylla grows in the Alps and in the N Appennini. We investigated plants from four Swiss sites. All plants proved to be diploid with 2n = 2x = 18 chromosomes, corroborating the indications in literature. The haploid karyotype shows seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. As no karyotype information was found in literature, we present a metaphase of this species (Fig. 2D).

The polyploid complex of Achillea millefolium occurs in Europe, W Asia and North America, and diploid to octoploid plants are known. Hybridization and introgressions seem to be frequent, and identification of a given plant is therefore often difficult. Plants of this complex that could be determined are presented under the respective name (viz. A. distans, A. setacea or A. stricta). Plants summarized here under A. millefolium s.l. could not be assigned to a specific taxon within the complex.

The plants from Greece proved to be diploid with 2n = 2x = 18 chromosomes, and the plants from France and Norway were hexaploid with 2n = 6x = 54 (the chromosome number of the Swiss site was published earlier, see Huber & Baltisberger 1992). All plants from all four sites showed the same haploid karyotype with seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites (in hexaploid plants not always with all satellites visible).

The calcifuge Achillea moschata belongs to the group of A. erba-rotta (see above) and is sometimes treated on the subspecies level as A. erba-rotta subsp. moschata (Wulfen) Richardson. It occurs in the C and E Alps (from France to E Austria). It is diploid with 2n = 2x = 18 chromosomes, which was confirmed by plants from five sites from Switzerland (the chromosome numbers of two sites [11881, 12545] were published earlier, see Huber & Baltisberger 1992). The plants from all seven sites showed the same haploid karyotype consisting of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. As no karyotype information was found in literature, we present a metaphase of this species (Fig. 2E).

Achillea multifida occurs in the S parts of the Balkan Peninsula (Albania, Macedonia, Bulgaria and Greece) and on Uludağg in NW Turkey. It is closely related or even conspecific with A. clusiana Tausch, which has a distribution area restricted to the E Alps. The separation of plants from the Balkans and Turkey as A. multifida is therefore not generally accepted, but it seems that the two entities are not conspecific and might be treated as distinct taxa (Ehrendorfer 2005, in litt.).

Diploid with 2n = 2x = 18 chromosomes (Turkey: Martin-Noguet 1969; Bulgaria: Kuzmanov & Kozuharov 1970; Baltisberger 2006) and decaploid plants with 2n = 10x = 90 chromosomes (Mt Korab and Sar Planina in Albania and Macedonia; Baltisberger 1992) are known. The plants from Bulgaria and Greece investigated here showed 2n = 2x = 18 chromosomes and proved to be diploid. The haploid karyotype consists of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. The same or at least a rather similar karyotype was indicated for diploid (Kuzmanov & Kozuharov 1973; Baltisberger 2006) as well as for decaploid plants (Baltisberger 1992).

Achillea nana is calcifuge and grows in screes and on rocks in the W and C Alps. The chromosome number was investigated on plants from five Swiss sites. They all proved to be diploid with 2n = 2x = 18 chromosomes (the chromosome number of one site [12546] was published earlier, see Huber & Baltisberger 1992). The haploid karyotype proved to be with seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. As no karyotype information was found in literature, we present a metaphase of this species (Fig. 2F).

Achillea nobilis is a variable species and belongs to a difficult group of species (Bässler 1963). It grows in dry habitats in C and S Europe, in Turkey and further eastwards. It is a mostly diploid species with 2n = 2x = 18 chromosomes, as confirmed here by our investigations on Greek plants (the chromosome number of the Swiss site was published earlier, see Huber & Baltisberger 1992). The haploid karyotype in plants from both sites investigated consists of seven metacentric and two submetato subtelocentric chromosomes, the latter with satellites. The same karyotype was indicated by Međedović (1984) in A. nobilis as well as by Kuzmanov & Kozuharov (1973) and Baltisberger & Baltisberger (1995) in A. neilreichii A. Kerner [= A. nobilis subsp. neilreichii (A. Kerner) Velen.], which is closely related and probably even conspecific with A. nobilis.

The Achillea oxyloba group consists of three mountain taxa, which are treated as species or subspecies. The three taxa have clearly disjunct geographic areas, viz. A. mucronulata Bertol. [= A. oxyloba subsp. mucronulata (Bertol.) Richardson] in the C and S Appennini, A. oxyloba in the SE Alps and A. schurii Sch. Bip. [= A. oxyloba subsp. schurii (Sch. Bip.) Heimerl] in the E and S Carpathians. All three taxa are known to be diploid with 2n = 2x = 18 chromosomes, as we found in plants of A. oxyloba from three sites from the Alpi Dolomitiche (the chromosome number of one site [10993] was published earlier, see Baltisberger 1990). The haploid karyotype of plants from all four sites consists of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. All three taxa of the group show the same haploid karyotype, viz. Brullo & al. (1991) for A. mucronulata, Maffei & al. (1986) for A. oxyloba, and Tacina (1979) and Baltisberger & Widmer (2009) for A. schurii.

Achillea pindicola is an endemic species of C and S Pindhos in Greece and belongs to the group of A. clavennae (see above). It is divided into two subspecies differing in shape and indumentum of the leaves (Franzén 1986). Both subspecies are diploid with 2n = 2x = 18 chromosomes (Contandriopoulos & Martin 1967; Franzén 1986), sometimes additional B-chromosomes are indicated (Franzén 1986). All plants investigated here proved to be diploid without any B-chromosomes and with a haploid karyotype of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. As no karyotype information was found in literature, we present a metaphase of this species (Fig. 2G).

Achillea ptarmica is widespread in Europe and grows in damp meadows and ruderal places. The species is known to be diploid with 2n = 2x = 18 chromosomes, which was confirmed by plants from all five sites investigated. The haploid karyotype consists of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. As no karyotype information was found in literature, we present a metaphase of this species (Fig. 2H).

Achillea setacea is a member of the polyploid complex of A. millefolium (see above). It is mostly diploid with 2n = 2x = 18 chromosomes, as are the plants investigated from Greece. The haploid karyotype consists of seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. The same or at least rather similar karyotypes were indicated by Mededović (1984), Kuzmanov & al. (1989) and Danihelka & Rotreklova (2001).

Achillea stricta belongs to the polyploid complex of A. millefolium (see above). It grows in grasslands and ruderal places in the Carpathians, the Alps, as well as in the Appennini. It is a hexaploid species with 2n = 6x = 54 chromosomes. All six plants investigated here proved to be octoploid with 2n = 8x = 72 chromosomes, which is a new ploidy level for this species. As no suitable metaphases have been obtained on the material investigated here, nothing can be said about the karyotype.

Achillea tomentosa grows in dry habitats in the lowlands of SW Europe (Spain, France, Switzerland and Italy) and is closely related to A. chrysocoma (see above). The plants from Grengiols (11660) are diploid with 2n = 2x = 18 (the chromosome number of the other site [11454] was published earlier, see Huber & Baltisberger 1992). The haploid karyotype of plants from both sites shows seven metacentric and two submeta- to subtelocentric chromosomes, the latter with satellites. As no karyotype information was found in literature, we present a metaphase of this species (Fig. 2I).

General discussion

All taxa of Achillea investigated to date show the basic chromosome number x = 9 on diploid as well as on polyploid levels. The haploid karyotype within Achillea is rather uniform with seven metacentric and two submeta- to subtelocentric chromosomes, the latter with (mostly visible) satellites, as observed in 27 taxa representing three sections, A. sect. Achillea, A. sect. Anthemoideae and A. sect. Ptarmica (we could not investigate any taxon from the fourth section, A. sect. Babounya). Besides the 27 taxa investigated and discussed here, the same karyotype is given in literature for several additional taxa. From A. sect. Achillea: A. collina Rchb. (Baltisberger 2006), A. lingulata Waldst. & Kit. (Baltisberger & Widmer 2009) and A. neilreichii A. Kerner (Baltisberger & Baltisberger 1995); from A. sect. Anthemoideae: A. abrotanoides (Vis.) Vis. (Baltisberger 1993; Baltisberger & Baltisberger 1995), A. ambrosiaca (Boiss. & Heldr.) Boiss. (Baltisberger 1994), A. schurii Sch. Bip. (Baltisberger & Widmer 2009) and A. umbel lata Sm. (Tzanoudakis & Iatrou 1981; Baltisberger 1994); and from A. sect. Babounya: A. fragrantissima (Forssk.) Sch. Bip. (Baltisberger 2012). Thus, taxa from all sections currently recognized in Achillea show the same haploid karyotype. The genus therefore is rather uniform with respect to the chromosome morphology. This might be an important prerequisite to allow even morphologically strongly divergent taxa to hybridize, as is the case in Achillea (Heimerl 1884, 1926; Halacsy 1902; Hayek 1928–1931; Wagenitz 1979; Richardson 1976; Hess & al. 1980; Franzén 1991; Afanas'ev & Bochantsev 1995).

Most Achillea species are diploid with 2n = 2x = 18 chromosomes, but polyploid species up to 2n = 14x = 126 have been reported (Dabrowska 1989). Polyploidy is frequent and occurs throughout the genus (Guo & al. 2004). Despite this extensive variation in ploidy levels, all species show the same haploid karyotype. The chromosomes within Achillea are rather large, but it is interesting that in plants with higher ploidy levels the chromosomes generally are smaller than in diploids, as is also the case in Ranunculus (Baltisberger & Hörandl 2016). As the size of chromosomes varies, we do not consider the size of chromosomes for our analysis.

Ranunculus (Ranunculaceae) shares several interesting aspects with Achillea: with about 600 species, Ranunculus is rather species-rich, has a broad geographic distribution and shows substantial variation in morphology, life cycle and ecology (Tamura 1995; Emadzade & al. 2010; Hörandl & Emadzade 2012). In marked contrast to Achillea, however, Ranunculus shows significant variability in karyotypes, and karyotype evolution mirrors the molecular phylogeny of the genus (Baltisberger & Hörandl 2016).

The splitting of Achillea into four sections is based on molecular data, but the morphology of the chromosomes and therefore the karyotype within Achillea is homogenous. This is in contrast to Ranunculus with eight different karyotypes reflecting the phylogenetic structure of the genus. In general, hybrids between taxa with differing karyotypes are not viable or at least sterile, as pairing of the not or not fully homologous chromosomes in meiosis is problematic or even impossible. This is also reflected by the behaviour of the two genera: in Ranunculus, fertile hybrids have only been reported between species sharing the same karyotype (Baltisberger & Hörandl 2016), but hybrids in Achillea frequently occur (Heimerl 1884), even between morphologically clearly separated taxa and different ploidy levels (e.g. A. clypeolata [2x] and A. collina J. Becker ex Heimerl [4x; member of A. millefolium s.l.]; Sankel & al. 2003). This contrast suggests that karyotype variation has played an important role in the evolution of reproductive barriers in Ranunculus but not in Achillea.