Achillea setacea and A. asplenifolia are two diploid species of the monophyletic A. millefolium agg. [11, 19]. They represent two extremes of morphological and ecological differentiation within this species aggregate, the former hairy, small, and adapted to xeric steppe environments, the latter tall, glabrous, and adapted to undisturbed wet environments. Achillea setacea-2x is sporadically distributed from NE Anatolia and SE Europe to the Balkans, Hungary, Slovakia, Moravia, Austria and interior valleys of the Alps, and in the north to S Poland, E Germany and the N Czech Rep; whereas, A. asplenifolia-2x occurs locally from Bulgaria and Hungary to E Austria and the southern Czech Republic [10, 11, 27]. In the ncpGS gene tree, haplotype sequences of A. setacea-2x and A. asplenifolia-2x group well into two clades corresponding to the two species (Fig. 1a), the PgiC gene tree, however, does not completely correspond to the divergence of the diploid species (the subclade IIa of Fig. 2a inclues both A. asplenifolia-2x and A. setacea-2x) (Fig. 2a). Our data clearly show that both the ncpGS and PgiC genes are single-copy in Achillea millefolium agg.. To explain the partial incongruence of the PgiC gene tree with the divergence of the diploid species (Fig. 2a), two interpretations can be put forward: i) incomplete sorting of ancestrally polymorphic alleles, or ii) of introgression during secondary contact of the two diploid species. Considering the current allelic distribution, the former interpretation is more likely as shown below.
Assuming incomplete lineage sorting (Fig. 2c) , allele A2 might have been retained from an ancestor of A. millefolium agg. in some populations of the extant A. setacea (the Greek and Anatolia populations, GR and SeAA) and in A. asplenifolia, but was apparently lost during the migration of A. setacea to the north and the west, e.g., in the Ukrainean and Austrian populations (K4 and NS1). Allele A3, which appears in A. asplenifolia, could have arisen from A2 after the divergence of this species in the Pannonian area, where it has survived locally in lowland areas in Hungary, Bulgaria, Austria, and Moravia (Figs. 2a &2c).
Alternatively, one could also assume subclade IIa of Fig. 2a (A2) to be the result of hybrid introgression from A. asplenifolia-2x into A. setacea-2x. This is unlikely considering the current geographic distribution of the two diploid species and the occurrence of allele A2 among populations of A. setacea-2x (only in its south-eastern populations, SeAA and GR, that grow outside the distribution area of A. asplenifolia-2x). However, the refugia of the two species may have been in closer proximity in SE Europe during the ice-ages, and they may have hybridized there. If so, allele A2 must have been lost from A. setacea-2x during its northward migration. But this scenario is again unlikely because there are no signs of hybrid introgression between A. asplenifolia-2x and A. setacea-2x throughout the Pannonian area, where they often occur in close proximity. A clear separation of the two diploid species is also strongly suggested by the ncpGS gene tree (Fig. 1a). Thus, we assume that two PgiC alleles A1 and A2 existed already in the ancestral lineage and may have been sorted incompletely after the divergence of A. asplenifolia and A. setacea, while allele A3 has arisen within A. asplenifolia after its species separation (Fig. 2c).
In contrast to the clear genetic and morphological separation of Achillea setacea-2x and A. asplenifolia-2x, A. collina-4x is morphologically intermediate between these two diploid species and also linked by intermediates to other 4x-taxa of A. millefolium agg.. Unlike the two relic diploid species, A. collina-4x has widely expanded in various mesic and open vegetation types from SE and E to C Europe and is much more aggressive in disturbed habitats. From experimental crosses between A. asplenifolia-2x and A. setacea-2x, synthetic allotetraploid and A. collina-like plants were produced and successfully backcrossed to natural A. collina-4x . These early results were supported by AFLP analyses which showed that species-specific bands of the two diploids are combined in A. collina-4x .
The present sequence data from single-copy nuclear genes ncpGS and PgiC (Figs. 1, 2) demonstrate that all the haplotype sequences of the diploid individuals or populations are grouped corresponding to the two species, Achillea setacea-2x and A. asplenifolia-2x respectively. In contrast, sequences of nearly all populations and many individuals of A. collina-4x (and its suspected 4x-hybrids) are placed among both the diploid Achillea setacea and A. asplenifolia clades. Therefore, homeologs of the nuclear single-copy genes in A. collina-4x demonstrate its allotetraploid origin. Additional evidence for this conclusion comes from the AFLP Neighbor Net (Fig. 3). That many of the A. collina-4x individuals (in the PgiC gene tree, most individuals) harbor homoeologous gene copies (Additional file 1 &2: Figs. S1 & S2) suggests at least partly disomic inheritance of this tetraploid species. Its diploid progenitors must have been closely related to the extant A. setacea-2x and A. asplenifolia-2x, and probably have differentiated in SE Europe. Their hybridization and the origin of an allotetraploid progeny may have taken place in the Pannonian region, where their distribution areas still overlap.
With the establishment of A. collina-4x, a first cycle of hybridization and differentiation was completed. But was the further expansion of this young allotetraploid species accompanied by complete isolation from or by continued backcrossing with its diploid progenitor lineages? Earlier experiments of crossing 2x- and 4x-taxa of the A. millefolium agg. never have produced 3x-hybrids but can occasionally gave rise to 4x-progeny via unreduced egg cells from the 2x side . Such unreduced gametes occur frequently in A. millefolium agg. . In Burgenland, Austria, populations of A. setacea-2x, A. asplenifolia-2x and A. collina-4x grow in two areas about 4 km apart: southeast of Rust and St. Margarethen (see Additional file 3, Table S1 for population sampling information). Ongoing gene flow may exist among their populations: Polymorphic populations M1 and M2 with mixed ploidal levels of 2x and 4x were found in disturbed grassland surrounding the morphologically more typical A. setacea-2x population NS1 on natural steppe islands near St. Margarethen, whilst NS1 itself also contains a few phenotypically intermediate 4x-plants. Similarly, at the outer border zone of lake Neusiedlersee near Rust, in contact zones between A. asplenifolia-2x in natural humid meadows and A. collina-4x from adjacent disturbed grassland, 4x-plants with intermediate phenotype were found in populations R1, R2 and NS2 (see Additional file 3, Table S1 for population sampling information). Our study, especially the AFLP network (Fig. 3), suggests these 4x-plants result from backcrosses of the 2x-taxa to A. collina-4x via unreduced female gametes. The possibility of reverse gene flow from 4x to 2x will need a further critical study.
There are several other examples for ongoing hybridization between taxa on different ploidy levels in Achillea: A contact zone between A. asplenifolia-2x and A. collina-4x, comparable to the one in Austria, was studied in W Hungary . A. virescens is an allo-4x-species, which has arisen from hybridization between A. collina-4x and A. nobilis-2x. Its backcrossing with A. collina-4x has been demonstrated in NE Italy . The yellow flowering SE-European A. clypeolata-2x has formed an extensive 4x-hybrid swarm with A. collina-4x in Bulgaria [19, 31]. In addition, natural and experimental crosses between A. collina-4x and A. millefolium-6x are quite successful; via semifertile 5x-F1, aneuploid-F2 and backcrosses they rapidly produce normal euploid 4x or 6x progeny and support gene flow between the two ploidy levels .