First report on adverse effects of cyanobacterial anabaenopeptins, aeruginosins, microginin and their mixtures with microcystin and cylindrospermopsin on aquatic plant physiology: An experimental approach.

Cyanobacteria produce a variety of oligopeptides beyond microcystins and other metabolites. Their biological activities are not fully recognized especially to aquatic plants. Acute toxicity tests on Spirodela polyrhiza and Lemna minor exposed to a range of concentrations of cyanobacterial metabolites: anabaenopeptins (ANA-A, ANA-B), aeruginosins 98 (Aer-A, Aer-B), microginin-FR1 (MG-FR1), microcystin-LR (MC-LR) and cylindrospermopsin (Cyl) were carried out to compare their influence on plant physiology. Effects of their binary mixtures were determined by isobole approach and calculation of the combination index (CI) that indicates a type of metabolites' interaction. Cyclic oligopeptides microcystin-LR and anabaenopeptin-A revealed the strongest inhibition of S. polyrhiza growth while other metabolites appeared less toxic. Oxygen evolution was inhibited by Cyl, MC-LR, ANA-A, ANA-B, while both variants of aeruginosins and MG-FR1 did not affect this process. Photosynthetic pigments' contents decreased in S. polyrhiza exposed to ANA-A and Cyl, while MC-LR and Aer-A caused their slight increase. 96 h-EC50 values showed that the growth of L. minor was more sensitive to MC-LR, ANA-A, MG-FR1 and Cyl than the growth of S. polyrhiza. In S. polyrhiza synergistic effects of all the binary mixtures of peptides with MC-LR on oxygen evolution were observed, while antagonistic one on the growth of S. polyrhiza exposed to the mixtures with aeruginosins and ANA-A. The mixtures of MC-LR and MG-FR1 with cylindrospermopsin revealed synergistic effects on the growth but antagonistic one to the O2 evolution. Quadruple mixtures (ANA-A + MC-LR + MG-FR1+Cyl) did not reveal any inhibitive effect on the plant growth and very slight on the oxygen evolution, irrespectively of their total concentrations. Various effects caused by ANA-A and ANA-B suggest the importance of molecule structures of metabolites for toxicity. Composition of the mixtures of cyanobacterial metabolites was essential for the observed effects.

The Influence of Shallow Peatland Water Quality on Characteristics of the Occurrence of Selected Herb Species in the Peatlands of Eastern Poland.

The aim of the analysis was to compare the physicochemical variables of the quality of shallow groundwater in the peatlands of Eastern Poland in the context of the occurrence of selected herb species with similar habitat requirements: bogbean (Menyanthes trifoliata), small cranberry (Oxycoccus palustris), and purple marshlocks (Comarum palustre). The analysis of the quality variables of the shallow groundwater included the following physicochemical variables: reaction (pH), electrolytic conductivity (EC), dissolved organic carbon (DOC), total nitrogen (Ntot.), ammonium nitrogen (N-NH4), nitrite nitrogen (N-NO2), nitrate nitrogen (N-NO3), total phosphorus (Ptot.), phosphates (P-PO4), sulphates (SO2), sodium (Na), potassium (K), calcium (Ca), and magnesium (Mg). Internal metabolism was shown to influence the hydro-chemical status of peatland water, free of substantial human impact. The variables tested were within the range of the habitat preferences of the herb species and indicated that they have a wide ecological tolerance. However, their identical habitat preferences were not reflected in identical values for the physicochemical variables of the water essential for building populations of these species. The occurrence of these plant species was also shown to be determined by the hydro-chemical characteristics of the habitat, but the characteristics of their occurrence did not indicate the hydro-chemical aspect of the habitat.

Micropropagation Protocol and Genetic Stability of the Salix myrtilloides Plants Cultivated In Vitro.

Salix myrtilloides L. is a relict species, threatened with extinction in many European countries. To prevent the loss of the species, tissue culture was established to produce plant material for reintroduction in natural habitats. Micropropagation was chosen as a method to obtain new plants. S. myrtilloides shoots were disinfected with NaOCl, AgNO3, or with a two-step disinfection with NaOCl, and then placed on MS medium supplemented with BA at 1 mg·dm-3 and IBA at 0.1 mg·dm-3. Regenerated shoots were cultivated in presence of BA, KIN, and 2iP to select the treatment with the highest multiplication rate. The obtained plants were acclimatized to ex vitro conditions. Inter-simple sequence repeat (ISSR) and flow cytometric analyses were conducted on in vitro regenerated plants to check their genetic stability. The best disinfection results were obtained when explants were treated with 1.5% NaOCl for 20 min. The highest multiplication rate and good quality plants were noted in the control media, without growth regualtors and in presence of kinetin at 0.5 mg·dm-3. Flow cytometry and ISSR analyses confirmed genetic stability in plantlets, which indicated the possibility to use the in vitro obtained plants for reintroduction.

The Search for Suitable Habitats for Endangered Species at Their Historical Sites-Conditions for the Success of Salix lapponum and Salix myrtilloides Reintroduction.

Restoring endangered plant species to their historical sites is not always possible due to constantly changing habitat conditions. The aim of this study was to test the effects of reintroduction of two relict willow species in eastern Poland. The experiment consisted of planting 48 individuals of Salix lapponum and S. myrtilloides, obtained by micropropagation, at each of the two selected sites and observing their survival after one year. At the same time, selected physicochemical and biocenotic factors of the environment were monitored. About 70% of S. lapponum individuals and 50% of S. myrtilloides plants survived the one-year period. This result can be considered satisfactory and confirms the effectiveness of this means of active protection. The results of measurements of selected abiotic factors of the environment and the observations and ecological analysis of the flora indicated that the habitat conditions of both historical sites have changed, resulting in accelerated succession of vegetation. However, complete habitat degradation did not occur, although the development of a multi-story structure of one of the phytocenoses intensified competition for light and other environmental resources, which narrowed the potential ecological niche of the reintroduced species.

Optimization of Propagation of the Polish Strain of Aldrovanda vesiculosa in Tissue Culture.

Aldrovanda vesiculosa is a rare and critically endangered carnivorous plant species. Its populations have declined worldwide, so there is a need to protect the species from extinction. The research was conducted to establish an effective method of in vitro propagation of the species in order to obtain plants for reintroduction in the wild. The procedures included disinfection, multiplication, and acclimatization of plants. Contamination-free in vitro cultures were established using shoots and turions, which were disinfected with 0.25% sodium hypochlorite. The shoots were first defoliated. The explants regenerated better in liquid 1/5 MS medium than in solidified one. The optimum medium for the multiplication phase contained MS macro- and microelements diluted to 1/10. Plants cultivated in that medium were of good quality, long, and branched. The advantageous effect of medium was also confirmed by the content of photosynthetic pigments in the plant material. The content of chlorophyll a was highest in plants cultivated in 1/5 or 1/10 MS medium. The plants obtained were acclimatized to ex vitro conditions and reintroduced in the wild.

Searching for Optimal Substitute Habitats for Plants by Biological Experiments-A Case Study of the Endangered Species Aldrovanda vesiculosa L. (Droseraceae).

The selection of appropriate locations for the reintroduction of endangered plant species is an important process, because it usually influences the success of the conservation. The aim of this study was to select the optimal substitute habitats for Aldrovanda vesiculosa, taking into account the influence of physical-chemical factors (light intensity, temperature, pH, concentration of dissolved forms of nitrogen and cyanobacterial toxin microcystin-LR) on the efficiency of plant growth. Water analysis and field observations of the habitats of six lakes in Eastern Poland typified as potential substitute habitats for aldrovanda were carried out. The results of the experiments showed that both the concentration and the form in which nitrogen compounds are present in the environment were the factors limiting the growth rate and condition of plants. The second factor that caused the inhibition of aldrovanda growth was microcystin-LR. It was found that the habitat conditions in Lake Brzeziczno were within the ecological tolerance of the species. Particularly important was the low content of mineral compounds and the available forms of nitrogen and phosphorus in the water. Therefore, the probability of development of toxic cyanobacteria, the metabolites of which may affect the growth of A. vesiculosa, is also minimal.

Microhabitat Conditions and Inter-Species Competition Predict the Successful Restoration of Declining Relict Species Populations.

The local populations of relict plant taxa living near the limits of their geographical range are particularly vulnerable to extinction. For example, Salix lapponum is one of the rarest and most endangered glacial relicts in Western and Central Europe. In Poland, the number of its sites has dramatically decreased over the past few decades, prompting us to take conservation measures focused on saving endangered populations. During a field experiment aimed at the reconstruction of the downy willow population in the Knyszyn Forest (NE Poland), 730 individuals of the species were planted in four different natural sites. The seedlings were obtained by micropropagation from parts of vegetative individuals taken from the most abundant population of this species in eastern Poland (Lake Bikcze). The success of the reintroduction, measured by the number of individuals that survived 2-3 years in the wild and took up growth, was about 67%, however, with low flowering efficiency (7.5%). Additionally, monitoring showed significant differences in plant survival and growth rates under different habitat conditions prevailing at the site and with different cover from competing species, especially tall grasses. However, the restoration projects for relict shrub species should include periodic removal of competing plants and protection of plants from trampling and browsing by herbivorous mammals to increase reintroduction success.

Genetic Stability of the Endangered Species Salix lapponum L. Regenerated In Vitro during the Reintroduction Process.

Salix lapponum L. is a boreal relict species, threatened with extinction in Poland. An 80% decrease in the number of its stands was confirmed in the last half-century, so that to prevent the loss of downy willow, attempts were made to reintroduce this species in natural habitats. Micropropagation was chosen as a first stage of its active conservation. S. lapponum shoots were collected and disinfected with NaOCl, AgNO3, or HgCl2 or with a two-step disinfection with NaOCl and then placed on MS medium with BA 1 mg·dm-3 and IBA 0.1 mg·dm-3. Regenerated shoots were cultivated with addition of BA, KIN, or 2iP, alone or in combination with auxins, to find the highest multiplication rate. Inter-simple sequence repeat (ISSR) analysis and flow cytometric analyses were conducted on in vitro regenerated plants to check their genetic stability. Disinfection was quite difficult and the use of HgCl2 was the most efficient. The highest multiplication rate was obtained in presence of KIN at 0.5 mg·dm-3 + IAA at 0.5 mg·dm-3. The analysis confirmed the genome size stability, which is in agreement with the results obtained by ISSR, revealing no somaclonal variation in plantlets and therefore allowing the use of the obtained plants for reintroduction.

Ciliates as restoration indicators in peatbogs - 10 years of study.

There is almost no information on how restoration of Sphagnum-dominated peatbogs, and in particular removal of reeds, affects the functioning of ciliate communities. Changes in vegetation patterns caused by restoration procedures may take years to be observed, while microbial processes already display effects after short-term exposure to changes in environmental conditions due to restoration. Based on the results of a long-term study, we assumed that mowing down of reeds causes changes in the physicochemical properties of peatbog water and sought to answer the question of how ciliate communities react to these changes and whether these microorganisms can play a significant role as bioindicators in evaluating the restoration process. Removal of reeds clearly modified the taxonomic composition and abundance of ciliates. This was reflected in an increase in the abundance and diversity of these protozoa and in a significant increase in the proportion of sphagnophilous species. Our results suggest an indicator species approach based on functional groups may be appropriate for biomonitoring peatbog restoration. A better understanding of what regulates microbial populations and activity in peatbogs and unravelling of these fundamental mechanisms are particularly critical in order to more accurately predict how peatbogs will respond to anthropogenic disturbances.