Litter leachate as a potential selector of woody species germination at alpine treeline

ABSTRACT The advancement of upper forest limits is driven by environmental conditions, but our current understanding overlooks the attributes of habitats and germination ability of woody species. Habitats, through plant litter, impact the competitive relation of germination and seedling growth. The aims of this study were to identify the selective effect of six litter leachates on the germination of indigenous Norway spruce (Picea abies) and nonindigenous dwarf pine (Pinus mugo) and compare the germination rates of the species. We collected plant litter and seeds from the (sub)alpine belt of the Hrubý Jeseník Mts. (the Eastern Sudetes Mts.; the Czech Republic). We evaluated the effect of plant litter leachates from alpine heathlands, wind-swept alpine grasslands, subalpine tall-herb plants, Pinus mugo scrub, subalpine Vaccinium vegetation, and Norway spruce clonal groups on germination process under standard light and temperature conditions. The germination of Norway spruce was inhibited by the litter leachate from subalpine tall-herb vegetation mainly dominated by Calamagrostis villosa, whereas that of dwarf pine was not. The other five litter leachates had no significant effect on the both. Under standard conditions, the germination time of dwarf pine is on average one day faster. These results suggest that most of the litter leachates examined may have small impacts on the germinability and germination time of Norway spruce and dwarf pine, but litter from the subalpine tall-herb vegetation can act as a filter that influences the seedling composition of the woody species.


Introduction
The advancement of upper forest limits as a consequence of climate change is commonly related to abiotic environmental conditions (Körner 2003;Krajick 2004) but also to attributes of particular habitat (Nagy and Grabherr 2009), and the quality of ground conditions has been associated with conifer germination and recruitment in (sub)alpine belts (Treml et al. 2016;Crofts and Brown 2020).
The ongoing tree encroachment depends on seedling recruitment that is controlled by both abiotic conditions and by biotic characteristics of the community (Holtmeier and Broll 2007;Marsman et al. 2020;Šenfeldr and Treml 2020;Šenfeldr et al. 2021). An essential determinant of vegetation dynamics and community structures in this sense is surface for germination formed by plant litter (George and Bazzaz 1999;Hovstad and Ohlson 2008). Plant litter can exert profound positive or negative effect on the success of particular plant species by affecting probability of germination and hence successful establishment. Through litter leachates, plant communities may have a selective impact on a particular woody species in their recruitment, advancement of the upper forest limit, and dominance. However, the effect of leachates from distinct communities on the germination of treeline-forming species has been inadequately studied in mountain areas.
In Central Europe, Norway spruce (Picea abies L., Karst) and dwarf pine (Pinus mugo Turra) are apparent examples of anemochorous woody species that play specific ecological roles in treeline ecotone (Zeidler et al. 2012;Šenfeldr et al. 2014;Zeidler and Banaš 2020). Norway spruce is considered the most abundant treeline-forming tree species (Treml and Banaš 2008) and dominates montane forests up to the timberline, forming clonal groups or solitary trees above the limit of the closed forest in Central Europe (Šenfeldr and Maděra 2011). Prostrate dwarf pine is limited to alpine areas from the Pyrenees to the Balkan Peninsula and is common in the altitudinal belt above the upper limit of closed forests in the eastern Alps, Sudetes, and Carpathians (Hamerník and Musil 2007). However, it expands into surrounding communities in many areas of the Carpathians (Švajda et al. 2011), Apennines (Calabrese et al. 2018), Alps (Dullinger et al. 2003), and Sudetes (Treml et al. 2010), which has a strong impact on biodiversity (Zeidler et al. 2021). Studies comparing spruce and pine germination are still missing. The ability to germinate both of these tree species in different habitats can have serious consequences for their spread and impact on communities in protected (sub)alpine areas.
The main aims of the study were to (i) identify the selective effect of six litter leachates on the germination of Norway spruce (Picea abies) and dwarf pine (Pinus mugo) and (ii) compare the germination rates of the species.

Leachates preparation
Plant litter samples were taken in the (sub)alpine belt of the Hrubý Jeseník Mts. (the Czech Republic) on October 2017. We collected plant litter from six distinct alpine habitats (Chytrý et al. 2010): Alpine heathlands (H) -dominated by Calluna vulgaris and species of families Vacciniaceae, Ericaceae, and Empetraceae; Wind-swept alpine grasslands (G) -dominated by Festuca supina and Avenella flexuosa; Subalpine tall-herb vegetation (T) -dominated by Calamagrostis villosa; Pinus mugo scrub (D) -dominated by shrubs of dwarf pine; Subalpine Vaccinium vegetation (V) -dominated by Vaccinium myrtillus; and Norway spruce clonal groups (N; Šenfeldr and Maděra 2011) -dominated by Norway spruce. At each habitat, 10 samples of litter were haphazardly taken and mixed before next use. The litter samples were dried in the shade at room temperature (Loydi et al. 2015). The dried matter was stored at room temperature until use. Leachate from powdered and homogenized dried litter was prepared in January 2018. A 100 g portion of each sample was mixed with 1 l of deionized water and allowed to leach for 72 h on a shaker at room temperature (Loydi et al. 2015). Then, the mixture was filtered and immediately used for germination experiments.

Germination conditions
Seeds of Norway spruce and dwarf pine from the area of interest (the Hrubý Jeseník Mts.) were stored in an airtight, cold room at 4°C (Leinonen 1998) for 3 months before testing. Sowing was performed over 3 layers of standard filtration paper (80 g.m −2 ) in germination boxes (8 × 6 cm) with lids to prevent drying. For each type of litter (H, G, T, D, N, and V), we prepared 10 boxes with 40 seeds in each box (10 × 40 = 400 seeds in total). In each box, seeds were evenly distributed, and filtration paper was saturated with the leachate. One extra set of 10 boxes with filtration paper soaked in deionized water was added as a control (C). The same design was used for both Norway spruce and dwarf pine. All germination boxes were placed into a growth room under day (light 16 h, 30 ± 2°C) and night (8 h, 20 ± 2°C) regimes for 22 days following Czech standard No. 481,211 (from the Czech office for standards, metrology and testing). Seeds were counted once a day and were considered to have germinated when the radicle first emerged.

Data analysis
To evaluate the germination process of both species, we used the measurements of Ranal et al. (2009): the emergence of the germination process, germinability; mean germination time; coefficient of variation of the germination time. The effects of six leachates and deionized water (control) on Picea and Pinus seed germination were analysed using two-way ANOVA model with the litter treatment (leachates and control) and species as explanatory variables. A post hoc test of differences among the combinations of experimental factors by using the Tukey HSD method was performed. The effects of experimental treatments on seed germination were evaluated using repeated measures ANOVA. The multiple paired t-tests with a Bonferroni correction were also performed. All data analyses were performed using STATISTICA 13 software.

Results
The two-way ANOVA indicated the significant interaction between species and treatment for germinability (Table 1).
The only significant contrast was between the germinability of Norway spruce and dwarf pine in the subalpine tall-herb vegetation (T) leachate (Figure 1). The germinability of Norway spruce was lower in the leachate than that of dwarf pine (p < 0.001). There was no significant difference between the germinability of dwarf pine seeds in deionized water (C) and all the leachates used.
Significant differences in the mean germination time were found between Norway spruce and dwarf pine ( Table 1). The mean germination time of Norway spruce was approximately 1 day longer than that of dwarf pine on average. Although variability in germination time appeared under the influence of plant litter leachate (Figure 2), the effect was nonsignificant in all cases within particular species.
Significant differences in the coefficient of variation of the germination time (CV) were found only between Norway spruce and dwarf pine (Table 1, Appendix S1). Multiple comparisons did not reveal a significant contrast in CV among plant litter leachates.

Discussion
Although a positive effect of plant litter on germination was often registered at the treeline (Dullinger et al. 2003;Lett and Dorrepaal 2018;Šenfeldr and Treml 2020), we documented only both neutral and a negative impact without any positive effects. The impact of leachate from subalpine tall-herb vegetation lowered the germinability of Norway spruce by approximately 15%, but the leachate had no effect on the germination of dwarf pine (Figure 1). On semi-natural grasslands, Hovstad and Ohlson (2008) demonstrated the inhibitory effect of litter leachates on seven plant species. The more concentrated leachates can reduce germinability of forbs by more than 10%. As documented Jäderlund et al. (1996), the more concentrated leachates of bilberry (Vaccinium myrtillus) can reduce germinability of Scots pine (Pinus sylvestris) and Norway spruce by up to 7% and 17%, respectively. In both studies, low germinability was attributed especially to chemical features of litter leachate. The authors also pointed out the species composition of litter determining intensity of the germination inhibitory effect. In the Eastern Sudetes Mts., the dominant species of subalpine tall-herb vegetation is Calamagrostis villosa; its litter was documented to have a significant inhibitory effect on other plant species (Pyšek 1990(Pyšek , 1993. The F column represents the values of the test statistic for each model term, which differs in degrees of freedom among germination characteristics and the course of germination. The p-value represents the test significance for this term. Not only graminoids but also other (sub)alpine species have the ability to influence germination processes. Inhibiting effects have been demonstrated for Calluna vulgaris in heathlands (Jalal and Read 1983) and bilberry litter (Jäderlund et al. 1996). However, we did not confirm the effect of leachates from other habitats ( Figure 1). As demonstrated by Hovstad and Ohlson (2008), the effect of litter increases as a function of litter amount. Hence, the small effects of leachates in this study could be partly attributed to the low concentration.
As shown by Halvorson et al. (2017), woody plants are able to germinate under weakly acidic to neutral pH values, but they are negatively affected by highly conductive soil solutions. The Sudetes Mts. were exposed to strong soil acidification due to atmospheric deposition in the 1980s (Reininger et al. 2011). The effect still persists, the soil has  a pH of approximately 3-4, and the concentration of minerals in the soil is low (Štýbnarová et al. 2014). In our case, litter leachates reached approximately pH 4-5, and their conductivity ranged from 300-800 μS (data not shown). The effect of leachates we do not attributed to pH, osmotic effects, and salinity on the study area.
Despite the low effect of most leachates, germination time course of Norway spruce and dwarf pine differed (Figure 2). The germination time of Norway spruce is in congruence with germination time of seeds originating from close natural forest regions (Houšková et al. 2021). The study also documented difference in mean germination time about 1 day between Norway spruce and Scots pine. Much more, germination treatment had only limited impact on germination time of both species. After water soaking, most of seeds of both species were capable germinating within about 10 days which is in line with our findings (Appendix S1). Early and fast germination is an important feature of successful invaders that support their establishing in new habitats (Milbau et al. 2003), but the slight difference in germination time between Norway spruce and dwarf pine will not contribute to creating distinctions in encroachment between the two species.

Conclusions
The aqueous leachate of the graminoid litter evokes different effects on Norway spruce and dwarf pine germination and thus may select among them in the forest limit ecotone. Leachates from other (sub)alpine habitats had no significant effect that might be attributed to the quality and quantity of inhibitory substances. The proved difference in germination time between Norway spruce and dwarf pine is not considered decisive for distinguishing the strategies between the two species in the encroachment process. Litter attributes should be considered only a part of the environmental filters applying on tree seeds and germination processes in real conditions of the forest limit ecotone; hence, there is necessity of next studies focusing on interplay among the environmental filters, leachate attributes, germination, and woody species Leinonen (1998) recruitment in real conditions. Acknowledgement I am grateful to Kateřina Jurníková and Jan Černohorský for help with laboratory work. The two anonymous reviewers are acknowledged for their advises and comments on this paper.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by Palacky University in Olomouc under grant IGA_PrF_2021_014.

Geolocation information
Plant litter samples, seeds of Norway spruce and dwarf pine were taken in the (sub)alpine belt of the Eastern Sudetes Mts., the Hrubý Jeseník Mts. (the Czech Republic); GPS 50.13N, 17.11E.

Supporting Information
Further information on germination characteristics of both species are available in Appendix S1.