Introduction

Potentillo albae-Quercetum Libb. 1933 (Quercetalia pubescenti-petraeae order) is a type of thermophilous oak forest occurring farthest north and covering within its range southern Germany, Austria, the Czech Republic, Slovakia, Hungary, south-western Russia and Poland1,2. This type of forest occurs on flatland, gentle sunny slopes or in shallow depressions. The habitat is considered a priority type (9110*)3.

The Potentillo albae-Quercetum communities include forests with tall canopy. The shrub layer is usually developed, although its cover is low2. Abundant light reaches the ground as a result of both the spacing of the oaks and the high transmission by their canopy. The light-demanding species that dominate the herb layer are found in dry grasslands (from Festuco-Brometea), brushwood (from Rhamno-Prunetea) and in wet meadows (from Molinio-Arrhenatheretea). The lowest level of this herb layer is composed of species with low light requirements (from Fagetalia)1,4.

Dynamic tendencies of phytocoenoses observed in thermophilous oak forests indicate urgent need to continue active protection in order to stop the process of transformation of thermophilous oak forests to closed oak-hornbeam forest1. According to previous studies5, it is well established that understanding of vegetation dynamics requires a combination of intensive field studies (phenology, mortality, seed bank characteristics) and experimental approaches (germination characteristics of the seeds). Knowledge of the factors regulating germination of helio- and thermophilous species of oaks is fragmentary.

Within the oak forest the encroachment of Coryllus avellana L., Carpinus betulus L. and Robinia pseudoacacia L. undergrowth in forest patch margins has been a widespread phenomenon in Poland. The invasion of them caused considerable deterioration in light conditions of the ground layer, as deduced from a decrease in Ellenberg’s light index values. This results in progressing elimination of heliophilous species1,4,6.

The amount of light affecting undergrowth is one the principal conditions controling plant survival and growth. Under open conditions, red (R) light (650–700 nm) predominates over far-red (FR) wavelengths (700–750 nm) with the ratio of R to FR (R/FR) light energy averaging between 0.2 and 1.2 on a clear day7,8. As sunlight filters through the canopy, its spectral distribution is changed because of selective spectral absorption of leaves. It is considered that R light radiation is almost fully absorbed by leaves and green light and FR are largely transmitted9. Hence, the canopy-filtered light is rich in FR and poor in R. Unfiltered daylight has a typical R/FR ratio of about 1.2 and leaves of canopy may reduce this value to 0.2–0.3, depending on the leaf area index10. As a consequence, R/FR ratio increases with gap size and decreases with foliage or litter density11. The inhibitory effect of light filtered through leaves on germination is a result of a decreased R/FR ratio which effects the phytochrome system of imbibed seeds7,10,12. Most positively photoblastic seeds are inhibited by low R/FR ratio of the canopy-filtered light.

Seed germination is a process controlled by both internal properties of seeds and by an array of environmental factors including light quality, soil moisture availability, alternating temperature and nitrate ions10,13,14. It is also known that germination is under control of two phytohormones which act antagonistically: gibberellins and abscisic acid. Production of gibberellins induces germination, while accumulation of abscisic acid negatively regulates this process and is responsible for dormancy15,16. Recently it has been found that reduction of the abscisic acid level is controlled in a nitrate-dependent manner by proteins which are transcription factors binding to a promoter of a gene coding an abscisic acid catabolic enzyme17. Therefore, the optimal form and dose of nitrogen seem to promote germination by lowering the abscisic acid/gibberellins ratio. This proportion can be also altered by induction and/or inhibition of gibberellins biosynthesis. Metabolism of gibberellins is sensitive to changes of environmental factors, including light quantity and quality (e.g. changing R/FR ratio)18. Then, alternations are detected by a phytochrome19, converted to an internal signal and transducted to a nucleus, which triggers biosynthesis of gibberellins. Therefore, germination that can be observed in field is the result of the cross-talk between endogenous factors and environmental-triggered stimuli and shows high complexity.

Although various aspect of the autecology of species from Potentillo albae-Quercetum have been studied in considerable detail1,4, until recently not much attention was given to seed germination. For example, it is not known whether the seeds germinate readily in the dark or if their emergence from the seed bank is restricted to disturbances that expose them to adequate light. Basic information on germination of the presented taxa should add to our understanding its mechanisms in Potentillo albae-Quercetum species and assist restoration as well as conservation efforts in thermophilous oak forest.

The aim of this study was to test the hypothesis that seeds of the dominating species occuring in herb layer (Table 1) require light to germinate. The following questions were addressed; (1) how do plants respond to different quality of light; (2) can GA3 and nitrogen source (KNO3, NH4Cl or NH4NO3) promote seed germination; (3) is seed germination photoreversible by FR light after exposure to white or R light.

Table 1 List of the studied taxa, their life-form, centre of abundance and environmental conditions preference.
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Results

Effect of Different Quality of Light

Seed germination was greatly affected by light quality (Table 2). Exposure to white light significantly (Tukey’s test, P < 0.05) increased germination compared to the dark control for all species (Table 3). The white light effect was greatest for the seeds of Serratula tinctoria and Stachys officinalis which germinated poorly (<16%) in the dark but high germination rate (>96%) was observed in the light. The seeds of other six taxa germinated between 23–43% in dark. R light fully replaced the effects of white light in seed germination for four (Aquilera vulgaris, Calamintha acinos, Digitalis grandiflora, Festuca amethystina) taxa. For Calamintha acinos, Digitalis grandiflora and Lychnis viscaria no difference in germination between light and darkness conditions were observed. Seeds of Calamintha acinos, Digitalis grandiflora and Festuca amethystina were highly responsive to R light. Seed germination depended upon the kind of treatment applied at the end. The effect of R light was reversed by FR light and vice versa. Seeds of all taxa imbibed in FR light and then exposed to R light had increased germination compared to those not exposed to R light, but this increase was much smaller than that for seeds imbibed in darkness followed by R light. This indicates insensitivity of the seeds to FR light following an initial exposure to R light.

Table 2 Germination percentages of seeds exposed to different light treatments.
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Table 3 The effect of light (12-h daily photoperiod) or dark in relation to the presence of nitrate and/or ammonium ions (10 mM) on the final germination.
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The PGI values varied between 0.44 and 0.88 (Table 4). Six of the eight studied taxa (rated 6 or 7 for light requirements) occured as adults in large gaps, had a seed mass <1.5 mg, and required light for germination (PGI > 0.56). The most shade-tolerant species tested, Melica nutans (Ellenberg’s value of 4), had a seed mass 1.59 mg and germinated better in light than in dark (PGI = 0.44).

Table 4 Photo-requirement germination index (PGI) and average seed mass for taxa included in the study.
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Effect of Different Type and Concentrations of Nitrogen Source and Chilling

Germination was generally very low in the seeds incubated immediately after harvesting. Maximum promotion of germination of all taxa was obtained with the concentration of 10 mM NH4NO3 after 16 wk chilling. Calamintha acinos, Festuca amethystina, Lychnis viscaria and Melica nutans presented the highest germination. A considerable enhancement of germination was also obtained at 1 mM. Germination of all taxa was again shown to be promoted by KNO3 (NH4NO3 being slightly more effective than KNO3). NH4Cl had less effect than both NH4NO3 and KNO3 (Table 5).

Table 5 Effect of different concentrations of KNO3, NH4Cl and NH4NO3 combined with chilling on total germination of the examined taxa.
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In the absence of exogenous N or chilling, only 0–8% of seed germinated. Chilling and addition of either nitrate (NO3 −) and ammonium (NH4 +) significantly enhanced germination. There was also significant interaction between the influence of NO3 − or NH4+ and chilling on germination. In the absence of chilling and after 4 weeks of chilling, germination increased with growing NO3 - and NH4 + concentration. Overall, chilled seeds germinated better than non-chilled ones. Chilling stimulated germination in seven of the eight taxa. N level, chilling period and their interaction significantly affected germination (Table 6).

Table 6 Results of a three-way ANOVA applied to the germination percentages of seeds incubated at different nitrogen level (0, 1, 10, 25, 50 mM), forms (KNO3, NH4Cl and NH4NO3) and chilling periods (0 vs 16 weeks).
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Effect of Nitrogen Source Type and Light

Nitrogen added as nitrate ions (KNO3), was statistically more effective than that from ammonium ions (added as NH4Cl). In addition, germination in the combined presence of the two ions, added as NH4NO3 solution, was statistically higher than with either one of them (Table 3).

Although both light and exogenous nitrogen alone resulted in statistically significant promotion of germination, the combined presence of these factors was the most inductive. Two-way interactions of N form and light were significant (Table 7).

Table 7 Results of a two-way ANOVA applied to the germination percentages of seeds incubated at different nitrogen forms (KNO3, NH4Cl and NH4NO3) (each 10 mM) under 12-h daily photoperiod or in continuous dark.
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Effect of GA3

GA3 promoted dark germination of all species. Seeds of all examined taxa given R light irradiation and then imbibed in 100 ppm GA3 germinated just as well as dark-treated seeds kept in water. Exposure of imbibed seeds to 48 h FR before the application of GA3 prevented germination (Table 8).

Table 8 Effect of GA3 on germination of red or far red-treated seeds.
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Discussion

Seeds of all studies species sown soon after harvest showed very low germination which indicates presence of primary dormancy. Furthermore, germination rates of all taxa increased after wet-stratification treatment. In nature such a mechanism effectively delays germination until spring. It is consistent with the previous study on the forest species in a temperate region20.

It was shown in this study that germination can be induced by short R irradiation applied after 24 h of RF light inhibition. Promotion of seed germination caused by R light was reported for a great number of plant species and is a phenomenon well established in literature21,22,23. For example, germination of Ruellia tuberose L.24 and Asteracantha longifolia (L.) Nees25 seeds can be promoted by R light treatment. This promotion can be reversed by a subsequent exposure to FR. Described inhibitory effect of FR expired after 17–20 h of R light treatment. All physiological actions that can be altered by changing R/FR ratio (including germination) are considered as processes controled by phytochrome. Our study showed that Lv and Dg were weakly inhibited by FR while level of inhibition for Av, Ca, Fa, Mn, St and So was moderate. Similar results were obtained for two of these species (Lv and Av) after field studies23. It may indicate that phytochrome-mediated regulation of Av, Ca, Fa, Mn, St and So seed germination is tuned to maximalize chance for fast population establishment by germination of banked seeds. It is worth mentioning that germination of some forest woody species (e.g. Abies alba L., Betula pubescens Ehrh., P. strobus L. and P. sylvestris L.) is also sensitive to light quality: R light stimulates germination while FR light inhibits this process26,27. Elimination of specimens with canopy that strongly affects light spectrum may therefore lead to competition beetwen herbaceous and woody species. It also suggests that many herbs occuring in Potentillo albae-Quercetum are able to win competition for resouces due to efficient phytohormone regulation of germination.

Phytochromes are well known to mediate light-promoted germination; they are also known to increase the amount of bioactive gibberellins in seeds28. Our results showed that exogenously applied gibberellins promoted dark germination of the studied species. Prolonged FR light pretreatment caused permanent loss of sensitivity to GA3. However, it was stated that continuous FR light irradiation delayed gibberellin mediated promotion of germination but did not prevent germination permanently29. It was also indicated that different kinds of processes are involved in the biochemical control of germination30. This was consistent with studies on Lactuca sativa L.31.

Nitrates which naturally occurr in soil, can substitute for the light requirement in some cases32,33. In this study, four taxa: Ca, Mn, St and So are connected with poor-soil environments and their Ellenberg’s nitrogen index is 3 (Table 1). Up to now little was known about the effects of nitrate concentration combined with light quality on germination. Our results show that germination of these species was enhanced by the lowest concentration (in the range 1–10 mM) of any of the nitrogenous solutions. High concentrations of nitrogen compounds (25–50 mM) resulted in lower germination percentage than controls. This could be related to their ability to colonize soils with low nitrogen concentration. Similar results for seeds soaked in nitrogen solutions as those used in our study have been shown for 10 species from shrubby woodlands in central-western Spain34. However, the optimal conditions for seedling growth might not correspond to those for seed germination and seedling survival10,35. It is interesting to note that the critical nitrate concentration range for germination induction, observed in laboratory experiment, is spectacularly close to that encountered in natural ecosystems36.

Seeds of all examined species need light exposure to complete germination. Only Ca, Mn and Lv germinated >30% in the dark. Based on the white light germination characteristics, the data indicate that many of these species may be able to form a large persistent seed bank. This response, previously shown for many small-seeded temperate species37,38, can be seen as an evolutionary adaptive mechanism that prevents seed germination under shaded conditions10, as well as under excessively deep soil layers39.

The distinct light requirements for seed germination of the studied taxa could be a major factor hampering its natural regenerations. Strong requirements of light suggest germination preference for large vegetation gaps. For forest canopy of temperate forests, areas under canopy openings were found to have higher light intensities as well as higher air and soil temperatures than the surrounding closed forest40,41. Therefore, to improve the natural regeneration of Potentillo albae-Quercetum, disturbance (e.g., thinning) should be applied to allow more light reaching the understory layer.

A general correlation between seed weight and light requirement for germination has been suggested37,38,42. It was found that in temperate forest seed dry mass of 1.5 mg was an approximate cutoff between herbaceous species that are light-dependent and light-independent39. Our experiments indicated that seed germination of all 8 studied taxa of Potentillo albae-Quercetum was promoted by light. A light requirement for germination was stronger in smaller than in larger seeded species of Potentillo albae-Quercetum. PGI decreased with increasing seed mass. Such relationship was reported previously for example for Campanulaceae42 and for herbaceous species of northern temperate deciduous forests43.

In recent years fast advancing changes in some types of heliophilous oak and oak-pine forests, where there are the best habitat conditions for the studied species, have been observed. The gradual invasion of C. avellana and C. betulus shrubs is connected with a considerable deterioration of light conditions in the ground layer which results from the closure of the canopy1. This variation in vegetation density creates new conditions with altered R/RF ratios of irradiance. Thus, germination of any seed falling under plant canopy (e.g. cover shrubs) could be predominantly inhibited by R/RF ratio which would also contribute to the formation of persistent seed banks of these species7,8,11. Germination of any seed falling under a plant canopy may be inhibited by exposure to FR and by lack of R light. Removal of C. avellana and C. betulus results in greater irradiance of R light than under intact canopies. It could be hypothesized that seedling emergence of the studied taxa may be increased after canopy removal as a result of increased germination of seeds exposed to white and R light. Moreover, the germination of most seeds of the studied taxa occurs in early spring before leaf expansion. The subsequent reduction in light transmittance after leaf expansion would be disadvantageous to growth of seedlings of helio- and thermophilous species.

In many habitats the establishment of seedlings depends on the presence of sites that are clear of vegetation, namely vegetation gaps12. Environmental conditions in vegetation gaps often differ considerably, depending on type and size of the gaps, as compared to those of the intact forest44. For example, it was found that vine maple gaps, compared with closed canopy of conifer forest, had significantly higher pH values and higher concentrations of Ca, Mg and K in the forest floor45. A tendency for lower C/N ratios and higher total N concentrations in the surface mineral soil was also observed. Germination in response to elevated NO3 − concentrtion in our study can be interpreted as gap formation or as gap-detection mechanisms10,37. Some authors stated that nitrates could be a useful indicator of small scale disturbances in forests, since rise in the soil nitrate level can even be observed in single tree-fall gaps46. Seedling that rapidly establish in gaps have an advantage over plants that germinate later, when there is a greater competition for resources10. The results of this study have important implications for Potentillo albae-Quercetum restoration programmes. The distinct light requirements for seed germination of the species of Potentillo albae-Quercetum phytocoenoses could be the most important factor hampering its natural regeneration. In the phytocoenoses of Potentillo albae-Quercetum, emergence of light-demanding woody species from the seed bank is triggered by disturbance when gaps in litter or plant canopy expose seeds to light or higher R/FR ratio. Therefore, to improve the natural regeneration of phytocoenoses in thermophilous oak forests, disturbances (e.g., thinning) should be applied to allow more light reaching the understorey layer. Moreover, cold stratification breaks dormancy and promotes germination of selected species occurng in temperate forest and this suggests that the examined taxa are specialized to germinate in spring before leaf expansion of canopy. It leads to the conclusion that for many of these species revegetation is strictly connected with seed bank establishment and efficient detection of light condition change.

Materials and Methods

Study Site and Seed Collection

Seeds of eight taxa from termophilous oak forest, Potentillo albae-Quercetum, located in the vicinity of Poddębice, central Poland (51°91′72″N, 18°89′42″E) (Table 1), were collected for all the experiments. The taxa were as follows: Aquilegia vulgaris L., Calamintha acinos (L.) Clairv., Digitalis grandiflora Miller, Festuca amethystina L. subsp. ritschli (Hack.) Lemke ex Markgr.-Dann., Lychnis viscaria L. subsp. viscaria, Melica nutans L., Serratula tinctoria L. and Stachys officinalis (L.) Trevisan. The selected species belong to different plant families and are important components of the plant communitiy in the study area. The forest stand is primarily (84%) Quercus robur L. (Fagaceae), some of which are 150–200 yr old, with 12% Pinus sylvestris L. (Pinaceae) and 4% Carpinus betulus L. (Betulaceae). Common species of the shrub layer include C. betulus, Frangula alnus Mill. (Rhamnaceae), C. avellana L. (Betulaceae) and Q. robur seedlings (J. Kołodziejek, personal observation).

The study site is located in the transition zone between the temperate oceanic (Atlantic) climate in the west and the moderate continental climate in the east. Mean annual temperature (2000–2010) is 8.8 °C; mean annual precipitation is 570.1 mm, with a maximum in June–July; the average length of the growing season is 210–220 days47.

Seed material was collected in the study region between May and October 2015 depending on the time of ripening. In order to obtain representative seed samples of the local populations, mature seeds of each taxa were collected from at least 15 individuals of one single large population and mixed before use. In the laboratory, the seeds were removed from fruits, dried at room temperature and stored in paper bags with relative humidity 40–60% in the dark at laboratory temperature (23 °C) for a maximum of two weeks. By selecting eight species we intended to include typical species of termophilous oak forest occuring under oak tree stand with different seed weights and dispersal strategies, representing a broad variety of families and life forms.

General Germination Procedures

All germination tests were done in sterile plastic Petri dishes (9 cm diameter) lined with two layers of filter paper (Whatman no. 1) moistened with 3 ml of distilled water (pH 6) or the tested solutions. The Petri dishes were sealed with parafilm to minimize the loss of water. Each experiment consisted of four replicate Petri dishes of 25 seeds for each of the eight species and each treatment. Germination was recorded daily for 20 days. Germinated seeds were removed from the Petri dishes. A seed was considered to have germinated when a radicle had emerged. All manipulations in the dark treatments were done under dim green safe light. Germination percentages were calculated on the basis of the number of viable seeds; dead seeds, which were identified based on their softness and brownish embryo colour, were excluded. Number of dead seeds did not differ statistically among groups and treatments (P < 0.05). Then some seeds were stratified in a refrigerator at 5 °C for a 16 weeks to fully break dormancy. This temperature is a standard for testing cold stratification and simulated average winter (5 °C) temperature condition. Non-chilled seeds were stored at 23 °C. The temperature of 23 °C appeared most suitable for optimal germination for herbaceous species from the temperate region14,48,49. In addition, this thermoperiod represents the mean daily maximum and minimum monthly temperatures at the Lodz Weather Station during June and July, when most seeds germinate in natural habitat. Fluorescent and incandescent sources of light were used. Light was filtered through 3 mm thick plexiglas filters (locally manufactured).

Effect of Different Quality of Light

This experiment was performed using the seeds after 16 weeks of stratification at 5 °C prior to germination. Four light treatments plus dark control were used. First, the cold stratified seeds were imbibed in darkness (dishes covered with two layers of aluminium foil) for 12 h on filter paper moistened with 5 ml distilled water. Next, the seeds were exposed to different light treatments: (a) white light provided by a single fluorescent lamp (60 W); (b) R light for 10 min per day obtained by filtering the white light of 100 W incandescent bulb through red plexiglas; (c) FR light for 10 min per day obtained by filtering the white light of 100 W incandescent bulb through blue and red plexiglas; (d) 10 min of R, followed by 10 min of FR per day; (e) 10 min of FR, followed by 10 min of R per day. Dark controls were conducted in absolute darkness. The bulbs were placed 30 cm above the level of the seeds. Germination was observed in a dark chamber at 23 °C for 20 days.

For each taxa, an index of light requirement for germination (photo-requirement germination index; PGI) was derived: PGI = 1 − (FGD/FGL) where FGD is the percentage of germination in the dark and FGL is the percentage of germination in the light. Therefore if all of the seeds germinated in the dark as well as in the light during one day, PGI index would be 0; a value of 1 indicates germination only occurring in the light37.

Effect of Different Type and Concentrations of Nitrogen Source and Chilling

Chilled (16 weeks) and non-chilled seeds were treated with the following nitrogen (N) concentrations: 0, 1, 10, 25, 50 mM N, applied either as KNO3, NH4Cl or NH4NO3. Each seed lot was moistened with the appropriate N solution and placed in the dark for the chilling treatment. For the non-chilling treatment, seeds were treated in a similar manner, but chilling (5 °C) for 24 h was substituted for room temperature (23 °C). Seeds were then returned to Petri dishes and incubated in the dark. Germinated seeds were counted under dim green safe light.

Effect of Nitrogen Source Type and Light

10 mM sources of N were applied as KNO3, NH4Cl or NH4NO3 solutions. All taxa were tested under two light conditions (complete darkness and exposure to daylight). Each seed lot was moistened with the appropriate N solution and placed in the dark for the chilling treatment. Next, seeds were transferred to 23 °C and wrapped in aluminium foil for dark treatment or exposed to light for 12 h. Germinated seeds were counted under dim green safe light or in the daylight, respectively.

Effect of GA3

Seeds were first imbibed in 5 ml of distilled water under R or RF light for 48 h. Then the seeds were imbibed either in distilled water or GA3 solution (100 ppm) in the dark for 24 h. Procedures with imbibed seeds were carried out under a dim green lamp at 23 °C. The seeds were incubated at 23 °C in continuous darkness.

Statistical Analysis

Germination percentages were arcsine transformed in order to stabilize variances. Normality was verified with the Shapiro-Wilk test. The data were analysed using one-, two- and three-way ANOVA. Significance of differences amongst treatment means was assessed using Tukey’s multiple comparisons test with Bonferroni correction. All statistical tests were performed using a statistical software package (Statistica, Statsoft USA).

Data Availability Statement

All data generated or analysed during this study are included in this published article.