Cytotype and morphological study of two widespread taxa within Pulmonaria officinalis agg. (Boraginaceae) in Central Europe: the natural and anthropogenic contact zones with confirmation of a new hybrid

Abstract The distribution of cytotypes and their contact zones provides insights into the spatial, morphological, and ecological differentiation of individual lineages, as well as their rates of reproductive isolation, gene flow, and/or selection. In this study, we focused on the Pulmonaria officinalis group, which comprises two widely distributed and morphologically similar species, P . obscura and P . officinalis , that differ in chromosome number. Intermediate plants, often of questionable identity, are rarely reported from mixed populations. In addition, several ornamental cultivars exhibit morphological similarities to P . officinalis . We employed flow cytometric relative genome size estimation, chromosome counting, morphometric analysis, and pollen staining methods to evaluate cytotype distribution, morphological variation, and to confirm the hybrid origin of intermediate plants and their frequency. We identified two main cytotypes, each associated with specific taxa, i.e. P . obscura (2 n = 2 x = 14), predominant in the northern regions, and P . officinalis s. str. (2 n = 2 x = 16), more common in southern areas of Central Europe. We also occasionally detected DNA-triploids in natural populations of both taxa and confirmed an intermediate cytotype (2 n = 15), mainly in mixed populations of P . obscura and P . officinalis in natural contact zone, although such populations were rare. The intermediate cytotype displayed differences in relative genome size and chromosome number, as well as several morphological characters (e.g. calyx length and calyx indumentum) compared to the parental taxa. These plants also have lower fertility, with pollen viability ranging from 2 to 33%, compared to their parents, which had pollen viability 70–98%. On the basis of these results, including the confirmation of the hybrid status and the ability to distinguish these plants from both parental taxa, we describe a new hybrid, Pulmonaria × superflua Vejvodová and Kobrlová, as a hybrid between P . obscura and P . officinalis s. str. We also revised the frequency of non-native populations (garden escapes) and explored their potential coexistence with native populations of the P . officinalis complex and forming of the anthropogenic contact zone.