Genetic diversity, reproductive capacity and alkaloids content in three endemic Alkanna species

Abstract

The Balkans endemic species Alkanna primuliflora Griseb., A. stribrnyi Velen., and A. graeca Boiss. & Spruner have limited distribution in the Balkan Peninsula and a large variation in the morphological characteristics. The populations of the three Alkanna species in the Bulgarian flora are small and fragmented. There are no previous reports on the chemical profile or on the embryology of these species. The hypothesis was that the limited distribution of A. primuliflora, A. stribrnyi, and A. graeca was due to their reproductive capacity and genetic diversity. Furthermore, we hypothesized that the three species will contain pyrrolizidine alkaloids (PAs), as other species of the genus Alkanna (Boraginaceae), but they would have differential alkaloids composition. The population genetic structure and differentiation showed a clear distinction between species and revealed average levels of genetic diversity among the natural populations of the three Alkanna species. The embryological investigation observed stability of the processes in the male and female generative spheres and high viability of mature pollen and embryo of the three species. The normal formation of male and female gametophytes without deviations or degenerative processes, and observed levels of genetic diversity between Alkanna individuals are important in maintaining the size and resilience of the Alkanna populations. Eight alkaloids were identified by GC-MS in A. primuliflora and A. graeca and six alkaloids in A. stribrnyi. The main pyrrolizidine alkaloids (PAs) in all investigated species was triangularine. A. primuliflora and A. graeca showed similar chemical composition that comprised 9-angeloylretronecine, 7-tigloylretronecine, 9-tigloylretronecine, triangularicine, dihydroxytriangularine, dihydroxytriangularicine, whereas, in A. stribrnyi 9-tigloylretronecine, triangularicine and dihydroxytriangularicine were not found. This is the first report on the presence of PAs in A. primuliflora, A. stribrnyi and A. graeca. Besides, this is the first report on the embryology of these endemic species. The results contribute to the knowledge of the three endemic Alkanna species and will facilitate policy-making and defining new strategies for their conservation.

Fig 1. A two-dimensional PCoA plot of the all analyzed individuals of Alkanna primuliflora, A. graeca and A. stribrnyi in Bulgaria.

The first and second principal coordinates account for 32.2% and 18.7% of the total variation, respectively.

Fig 2

(A) Plot of K clusters versus mean (±SD) likelihoods (L[K]): (B). K plotted against the second order rate of change of the likelihoods (ΔK); (C). Genetic relationships among the populations of Alkanna estimated using STRUCTURE program based on ISSR data. STRUCTURE clustering results for K = 3 to as suggested in (B). Estimated genetic clustering obtained with the STRUCTURE for all investigated Alkanna individuals.

Fig 3. Graphical representation of hierarchical cluster analysis by UPGMA method based on Nei’s genetic distances among Alkanna individuals obtained by ISSR genetic markers.

Fig 4. Pecularities of the reproductive sphere of studied Alkanna species.

(A) tetrasporangite anthers in A. primuliflora; (B) two-celled mature pollen grains in A. primuliflora; (C) two-loculate ovary in A.stribrnyi; (D) anatropous ovule with one-celled archesporium in A. primuliflora; (E) mature embryo sac with postament in the chalazal part in A graeca; (F) globular embryo in A.sribrnyi; (G) mature embryo with part of endosperm and seed coat in A. primuliflora; (H) proterandry in A. primuliflora (x40 for Fig H); (x100 fpr Figs A,B,C,G), (x400 for Figs D,E,F); ac- archesporium cell, it-integumentary tapetum, pt-postament, em-embryo, en-endosperm, ov-ovule, al-anther locule.