Genetic Variation versus Morphological Variability in European Peatland Violets (Viola epipsila-V. palustris Group).

In Europe, the V. epipsila-V. palustris group comprises V. epipsila Ledeb., V. palustris L., V. pubifolia (Kuta) G. H. Loos (=V. palustris subsp. pubifolia Kuta), interspecific hybrids, and putative introgressants. The genetic affinity of V. pubifolia to V. palustris, and their shared origin via hybridization followed by polyploidization, were confirmed using inter simple sequence repeat (ISSR) markers, restriction site-associated DNA sequencing (RAD-Seq), and a low-copy nuclear gene, GPI, which encodes glucose-6-phosphate isomerase. The other taxa of subsect. Stolonosae were not identified as putative parents of V. pubifolia by GPI. Our analyses indicated that V. pubifolia can be included in the morphological and genetic variation of V. palustris. The ISSR, RAD-Seq, and genome size value separated well V. palustris from V. epipsila and hybrids. The results also reopen the discussion on intraspecific variation in the context of taxa ranks and species concepts. The reduced tolerance of V. epipsila in Europe to changing environmental conditions might result from low genetic differentiation and heterozygosity, as well as the increased number of interspecific hybrids (V. epipsila × V. palustris), and eventually can possibly lead to its extinction. The disappearance of populations/individuals of this species may indicate anthropogenic changes occurring in peatlands.

Re-introduction of an extinct population of Pulsatilla patens using different propagation techniques.

The study focuses on the propagation of a rare and endangered plant species (Pulsatilla patens) to re-introduce an extinct population from calamine area in Southern Poland. The plants were propagated from seeds, rhizome cuttings, or regenerated in vitro from shoot tips, hypocotyls with roots or cotyledons of seedlings on Murashige & Skoog (MS) medium supplemented with 0.25 or 0.50 mg L-1 BAP (Benzylaminopurine) via direct and indirect organogenesis or somatic embryogenesis (SE). The most efficient micropropagation method was with shoot tips as an explant on MS + 0.25 mg L-1 BAP where 97% of the explants produced multiple shoots, mass SE was observed after transfer on ½ MS with 2% saccharose; 267 (35%) shoots rooted on ½ MS + 2% saccharose were acclimatized to ex vitro conditions. Flow cytometry revealed genome size stability of propagated plantlets. Low genetic differentiation between micropropagated plantlets and initial material was indicated by ISSR (Inter Simple Sequence Repeat) markers. Totally, 132 vigorous plantlets obtained on various pathways were introduced to the field plots in 2020; 30.33% survived the winter, and several reached the generative stage and flowered in the spring 2021. In next season (March/April 2022) the number of introduced plants decreased to 25% while the number of flowering and fruiting shoots in different clumps increased in some plots. This is the first report of successful re-introduction of the endangered P. patens based on micropropagation, rhizome cuttings, and seed germination.

Seasonal and Simultaneous Cleistogamy in Rostrate Violets (Viola, subsect. Rostratae, Violaceae).

The special mixed reproductive system, i.e., the ability of an individual plant to develop both open, chasmogamous (CH) flowers adapted to cross-pollination and closed, cleistogamous (CL) flowers with obligate self-pollinating, is a common phenomenon in Viola L. In most sections of Northern Hemisphere violets, cleistogamy is seasonal, and CH and CL flowers develop sequentially in the season. Non-seasonal cleistogamy (simultaneous) is a rare phenomenon in rostrate violets. In the current study, we focused on modification of the CH/CL mating system in V. caspia by environmental conditions, resulting in a gradual switch from temporal cleistogamy, occurring in nature, to simultaneous cleistogamy under greenhouse conditions. V. reichenbachiana with seasonal cleistogamy was the control for V. caspia with the labile seasonal/simultaneous cleistogamy system. In simultaneous cleistogamy, the CH and CL flowers, fruits and seeds developed on an individual plant at the same time on the same branch. The typical difference between CH and CL flowers' pistils is a straight style ending with a head-like stigma in CH and a curved style in CL adapted to self-pollination. This trait persists in the fruit and seed stages, allowing for easy recognition of fruit of CL and CH flowers in simultaneous cleistogamy. Floral meristems of CH flowers of V. reichenbachiana developed on the rhizome at the end of the growing season under short-day conditions and remained dormant until the following season. The CL floral meristems formed under long-day conditions on elongating lateral branches in the upper leaf axils. The daily temperature influenced the variable CH/CL ratio of V. caspia in nature and greenhouse conditions. Regulation of the CL/CH flower ratio by modifying environmental factors is important for basic research on genetic/epigenetic regulation of cleistogamy and for practical use to produce genetically stable lines of economically important species via CL seeds.