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Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non-metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore-bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non-polluted sites, high levels of intra- and inter-population variability, and the occurrence of inter-specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter-specific hybrids from non-polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal-polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.

Morphological characters that identify and describe living organisms have been a major practical criterion used in plant and animal systematics since morphological data form the basis of virtually all systematic descriptions [1]. However, results of the combined molecular and morphological research techniques, give insight into speciation processes and are fundamental to species-level taxonomy. They are successfully used in phylogeny reconstruction, to examine the causes of variability, to construct natural classification systems, and to define taxonomic borders [2]. Genetic studies of species complexes, within which taxonomic borders based on morphology are usually blurred, often permit to assess the level of inter-species distinction and, on the other hand, their relationships [3,4].Phylogenetic and taxonomical relationships between Melica ciliata L. and M. transsilvanica Schur (Poaceae) have not been exhaustively explained and established [5][6][7]. M. ciliata was described by Linnaeus [8] as a species that occurs in rocky and infertile hills of Europe but the exact place of its collection is unknown. M. transsilvanica, distinguishes by very unequal glumes and pubescent lower leaf-sheaths, was described by Schur [9] from the vicinity of the Sibiu town in the Transylvanian Plateau in Romania. An intricate infraspecific variability and some morphological overlap between species makes them taxonomically problematic [5,6]. M. transsilvanica has for a long time been regarded either as a subspecies or as a variety of M. ciliata in many European floras [10][11][12][13][14][15]. However, Papp [16] pointed out that M. transsilvanica is a separate species distinguished from M. ciliata by several characters, including a dense inflorescence, flatter leaves and details of leaf-sheath pubescence.Melica ciliata L. is a sub-Mediterranean species whose main continuous geographical range covers the area from the Atlantic and Mediterranean region, Central Europe, to southern Ukraine and the Crimea (Fig. 1). It also occurs in the southern part of the Scandinavian Peninsula and in north Africa. Single scattered records have been reported from the Middle East [17]. M. transsilvanica Schur is a sub-Mediterraneancontinental, mostly steppe and steppe-forest species. Its main Abstract A good knowledge of species delimitation is crucial for the biodiversity protection and the conservation of wild species. We studied the efficiency of AFLP markers and morphological characters to assist species determination for Melica ciliata L. and M. transsilvanica Schur within European range of distribution, including isolated and range-limit populations of "M. ciliata" (i.e. M. cf. ciliata) from the Polish Sudetes, where it is regarded as critically endangered. AFLP markers were found to be more effective then morphological characters (more or less continuous) in distinguishing the both studied species. AMOVA revealed very low genetic diversity within populations and high differentiation among populations of M. ciliata and M. transsilvanica (F ST ...

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Hybrid speciation within the genus Elymus is analyzed and discussed. The supposed hybrid origin of Elymus ×mucronatus between Elymus repens and Elymus hispidus was tested. In this aim, pollen viability and AFLP variation of putative progenitors and progenies from two natural populations were studied. Pollen fertility of hybrids ranging from 0 to 91.95% (average 59.50%) indicates the occurrence of F1 hybrids and backcross hybrids. Seven EcoRI/MseI AFLP primer combinations generated a total of 477 AFLP fragments (300 polymorphic) for 48 parental and hybrid individuals. UPGMA and principal coordinates analysis of the AFLP data set show that hybrids are closer to E. hispidus than to E. repens and suggest unidirectional introgression towards E. hispidus. AFLP fragments of the hybrid taxon were additively inherited from the parental species and no hybrid-specific bands were identified. AFLP analysis validates the hybrid status of E. ×mucronatus. Populations in which E. repens, E. hispidus and their hybrids co-occur still progress from early hybridization stages of F1 hybrids to an advanced hybridization stage of a well-established hybrid swarm. E. ×mucronatus is potentially self-sustaining by predominant vegetative reproduction by rhizomes and partial fertility leading to the development of mature seeds. We propose to consider Elymus ×mucronatus as a new species of hybrid origin in the Polish flora.

To determine the relative importance of clonal growth and sexual reproduction, the Randomly Amplified Polymorphic DNA (RAPD) method was used to study genetic diversity and clonal structure of six populations of Elymus repens and four populations of Elymus hispidus from Poland. These outbreeding species are virtually self-sterile and form widely spreading and long-lived rhizomes. Using 12 primers, a total of 150 unambiguous RAPD fragments were amplified and scored. Results of AMOVA showed no significant genetic distinction between morphologically distinguished varieties of E. repens and E. hispidus. E. repens had slightly higher intra-specific genetic polymorphism than E. hispidus; the percentage of polymorphic bands per population ranged from 38 to 49 and from 19 to 38 respectively. Clonal diversity measured using the Simpson diversity index (D) indicated different contributions of clonal reproduction in particular populations of E. repens (D: 0.20-0.72). Populations of E. hispidus were dominated by one or a few clones, which were generally restricted to a single population (D: 0.00-0.22). RAPD revealed that most genetic diversity resided within populations of the two studied species, suggesting that, despite their clonal character, propagation by seeds contributes considerably to reproduction of E. repens and E. hispidus.

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