Does the health condition of the common ash tree affect its pollen viability?

Abstract

Pollen viability plays a crucial role in reproduction. Given the enormous threat posed to the common ash ( Fraxinus excelsior L.) by ash dieback, it is important to investigate the disease's effects on pollen viability and germination. Thus, we conducted an analysis of these pollen characteristics across three distinct forest stands in southern Bavaria, with a maximum of 23 ash trees per study site. These ash trees exhibited varying degrees of ash dieback-related damage symptoms, enabling us to assess differences between mildly and severely affected trees ( via Mann-Whitney-U/Wilcoxon test). Pollen viability was assessed using the TTC test, while pollen germination capacity was evaluated using a sucrose agar solution. Our findings revealed no significant differences in pollen viability between healthy and diseased trees, as indicated by both the TTC test and pollen germination assay. However, a tendency towards higher pollen viability was observed in healthier, more robust ash trees across both methods. Non-significant differences, however, suggest that ash trees can produce viable pollen necessary for successful fertilisation irrespective of their health status. Nonetheless, it was observed that severely diseased trees were linked to less inflorescences, as the severely diseased or dead shoots produced few to no flowers. Consequently, the likelihood of pollen from severely diseased trees fertilising other ash trees is substantially diminished. In conclusion, it is evident that flower and pollen production are most important in the reproductive ecology of ash trees.

Full text

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Does the health condition of the common ash tree
affect its pollen viability?
Georgia Kahlenberg
Katholische Universität Eichstätt-Ingolstadt: Katholische Universitat Eichstatt-Ingolstadt
https://orcid.org/0009-0007-0922-4958
Lisa Buchner
Susanne Jochner-Oette
Anna-Katharina Eisen
Research Article
Keywords: Fraxinus excelsior L., pollen germination, TTC-test, ash dieback
Posted Date: May 27th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-4411385/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

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Abstract
Pollen viability plays a crucial role in reproduction. Given the enormous threat posed to the common ash
(
Fraxinus excelsior
L.) by ash dieback, it is important to investigate the disease's effects on pollen
viability and germination. Thus, we conducted an analysis of these pollen characteristics across three
distinct forest stands in southern Bavaria, with a maximum of 23 ash trees per study site. These ash
trees exhibited varying degrees of ash dieback-related damage symptoms, enabling us to assess
differences between mildly and severely affected trees (
via
Mann-Whitney-U/Wilcoxon test). Pollen
viability was assessed using the TTC test, while pollen germination capacity was evaluated using a
sucrose agar solution. Our ndings revealed no signicant differences in pollen viability between healthy
and diseased trees, as indicated by both the TTC test and pollen germination assay. However, a tendency
towards higher pollen viability was observed in healthier, more robust ash trees across both methods.
Non-signicant differences, however, suggest that ash trees can produce viable pollen necessary for
successful fertilisation irrespective of their health status. Nonetheless, it was observed that severely
diseased trees were linked to less inorescences, as the severely diseased or dead shoots produced few
to no owers. Consequently, the likelihood of pollen from severely diseased trees fertilising other ash
trees is substantially diminished. In conclusion, it is evident that ower and pollen production are most
important in the reproductive ecology of ash trees.
1. Introduction
Pollen play a crucial role in the life cycle of forest trees as they present a key element in reproduction
(Smith 1981; LaDeau and Clark 2006; Larue et al. 2021). They act as central vectors for gene ow and
the transfer of genetic material to the next generation, which is essential for maintaining the diversity
and vitality of tree populations (Smith 1981; Ladeau & Clark 2006; Jump et al. 2009; Isabel et al. 2020;
Wang et al. 2022). To increase the probability of fertilisation of female owers by male pollen,
anemophilous plants, which include most forest trees, produce a large number of owers and pollen
(Holsinger and Steinbachs 1997; Dellinger 2020; Timerman and Barrett 2020). Regarding the long
generation times in natural forest landscapes, exploring pollen production is crucial as it provides
valuable insights into phenomena such as the alternating patterns in mast and non-mast years (Dahl et
al. 2013). Additionally, it provides insights into the effects of air pollution or climate change (Ziska et al.
2003; Darbah et al. 2008; Talwar et al. 2022) or the inuence of diseases (Kozlowski 1971; Eisen et al.
2024). For example, CO, SO2, NO2 and O3 were often associated with negative effects on pollen
production (Darbah et al. 2008), while CO2 repeatedly showed a positive effect (LaDeau and Clark 2006;
Darbah et al. 2008). Both Kozlowski (1971) and Eisen et al. (2024) found a link between the vigour of
forest trees and their ability to produce owers.
However, pollen production alone is not sucient to conclude on the effective fertilisation of owers in
the plant kingdom. Pollen viability, which is dened as "having the capacity to live, grow, germinate or
develop" (Lincoln et al. 1982), is also of utmost importance for pollination success and thus for
reproduction (Dafni and Firmage 2000; Burkhardt et al. 2009; Bochenek and Eriksen 2011). Pollen

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dispersal is mostly stochastic, which implies that the order in which pollen are released and reach the
female ower has an inuence on pollination success (Burkhardt et al. 2009; Bochenek and Eriksen
2011). However, if several pollen grains reach the female ower at the same time, competition between
the male gametophytes can occur, whereby the speed of germination of the pollen grains and sperm
transport is inuenced by the tness of the pollen (Bochenek and Eriksen 2011). It is therefore assumed
that increased competition between pollen grains and high pollen viability can lead to improved progeny
quality (Marshall et al. 2007; Lankinen et al. 2009).
Questions on reproduction especially emerge in terms of
Fraxinus excelsior
L. (common ash), as this
tree species is acutely threatened by ash dieback, a disease caused by the fungus
Hymenoscyphus
fraxineus
(T. Kowalski) Baral, Queloz, Hosoya (Baral et al. 2014), and is becoming increasingly
fragmented in its populations (Metzler et al. 2012; McKinney et al. 2014; Enderle 2019). The ascomycete
rst attacks the leaf spindles and leaves of the trees and then gradually spreads to the shoots and wood
of the ash trees. This can lead to the death of branches and twigs and within a few years to the death of
the entire tree due to the disruption of nutrient and water transport (Timmermann et al. 2011; Pautasso
et al. 2013; Cleary et al. 2016; Enderle 2019; Hultberga et al. 2020; Gašparović et al. 2023). The
proportion of ash trees that are less susceptible to ash dieback is very low and is estimated at 10–20%
(Westergren et al. 2020) or even 1–5% (McKinney et al. 2014; Rigling et al. 2016; Enderle 2019). In
addition, the ongoing ash dieback could lead to limited gene ow and pollination success and have a
substantial impact on the viability of pollen and thus on the reproductive ecology of
Fraxinus excelsior
L.
(Semizer-Cuming et al. 2021; Buchner et al. 2022; Eisen et al. 2023, 2024).
Natural regeneration is considered crucial in controlling the disease, as several studies indicate that
resistance to ash dieback is inherited and not associated with the population (McKinney et al. 2011;
McKinney et al. 2014; Lobo et al. 2015; Semizer-Cuming et al. 2019). In numerous studies, this high
degree of genetic variation in ash tree susceptibility was observed at the individual level (McKinney et al.
2014; Semizer-Cuming et al. 2017, 2019; Fussi 2020), with heritability estimated to be high at around 50%
(McKinney et al. 2014; Enderle et al. 2015). This implies that seeds are necessary for natural
regeneration, representing the successful fertilisation outcome that combines genetic information from
both parent trees
via
gamete fusion (Bochenek and Eriksen 2011; Fussi et al. 2014). Although ash trees
are considered hermaphroditic, Saumitou-Laprade et al. (2018) suggested that self-fertilising seeds do
not develop due to inbreeding depression, implying that the species is functionally sub-dioecious
(dioecious). In general, ash trees can achieve regeneration rates of up to 150,000 individuals per hectare
under favourable site conditions and due to their high seed production (Tabari and Lust 1999;
Dobrowolska et al. 2011); although, it is assumed that the actual germination rate is only 58–65% (Roloff
and Pietzarka 1997; Schirmer 2002). It is also speculated that resistance development might be
associated with epigenetic effects in fungus-infected individuals (Sollars and Buggs 2018).
Consequently, the future of ash primarily depends on effective gene ow among partially resistant adult
trees to establish a healthier next generation (Lobo et al. 2015; Semizer-Cuming et al. 2019; Semizer-
Cuming et al. 2021).

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A few prior studies already focused on ash pollen characteristics. Castiñeiras et al. (2019) investigated
the production and viability of
Fraxinus
pollen in Spain independently of ash dieback and Buchner et al.
(2022) tested different methods to assess pollen viability. In addition, Buchner et al. (2022) investigated
factors such as temperature and UV radiation that may have an inuence on the viability of the pollen,
e.g., during their long-distance transport. The authors conrmed an accelerated decrease in pollen
viability with increased or prolonged UV radiation and under warmer conditions.
This methodological background was utilised in the eld study by Eisen et al. (2024). The authors
investigated whether ash dieback affects pollen production and the viability of pollen and seeds in two
seed orchards and a riparian forest. Regarding viability of pollen, a slight tendency but no signicant
difference was detected between mildly affected and heavily diseased ash trees of the two seed
orchards. However, a signicantly higher pollen viability was found for less affected ash trees in the
riparian forest. Due to these disparate results, the rationale of this study was to conduct a more detailed
investigation focusing on three different forest stands in Southern Germany, Bavaria, which was
replicated in two study years. The main aim was to ascertain whether ash dieback and thus the health
status of ash trees have an impact on pollen quality in different forest stands.
2. Material and methods
Study areas
Our investigations were conducted in three Bavarian forest stands (Fig.1a): BY1 Monheim (48°48'23.4"N,
10°47'36.7"E, 470 m a.s.l.; Fig.1b), BY2 Bruckberg (48°28'59.9"N, 11°57'8.4"E, 419 m a.s.l; Fig.1c), and
BY3 Isen (48°11'47.0"N, 12°5'2.9"E, 519 m a.s.l.; Fig.1d). The sites BY1 and BY3 represent typical
deciduous, broad-leaved mixed forests, while the study site BY2 is a riparian forest. The average annual
temperature for BY1 (German Weather Service (DWD) station Harburg, 1991–2020) is 8.9°C; for BY2
(DWD station Munich Airport, 1991–2020) and for BY3 (DWD station Ebersberg, 1991–2020) it is both
8.7°C. The average annual precipitation sum is 824 mm (BY1), 757 mm (BY2) and 1,005 mm (BY3),
respectively. For each study site, we selected 12 to 23 ash trees of varying health status (Table1), which
were easy to reach with a lifting platform. The health status of the trees was classied according to a
scoring system for ash trees affected by ash dieback (Peters et al. 2021). Class 0 represents a healthy
tree without any infection symptoms and no disease-associated leaf loss. In class 1, the rst infection
symptoms, such as reduced foliage, are visible, but no dead twigs are identied yet. In class 2, the
progressive loss of leaves is more visible, and the rst dead twigs can be seen. With class 3 and 4, the
proportion of dead wood continues to increase, and the leaf biomass is reduced drastically. The last two
stages represent the dead (class 5) and fallen (class 6) trees. The vitality assessments for both 2021
and 2022 were conducted at the end of July, as trees typically reach their maximum leaf mass during
this period (Lenz et al. 2012). Table1 shows the different numbers of trees investigated at each site per
year and their associated health statuses. The number of trees varies through the years and for the study
sites, due to the different availability of inorescences and the accessibility of the study sites and trees.
For further statistical analyses, the trees were summarised in two groups: mildly affected (health statues

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1 and 2) and severely affected (health statues 3 and 4). Class 0 was omitted due to the absence of
healthy trees. Trees classied as vitality class 4 were the least analysed group in both years. This was
primarily due to our observation that many trees in this health state no longer produce owers.
Table 1
Observed number of trees with different health statuses for the study sites BY1 (Monheim), BY2
(Bruckberg) and BY3 (Isen) in the years 2021 and 2022
2021 2022
Class
1Class
2Class
3Class
4Sum Class
1Class
2Class
3Class
4Sum
BY1 4 11 4 2 21 3 8 8 2 21
BY2 3 8 5 2 18 3 9 10 1 23
BY3 4 2 5 1 12 1 5 9 2 17
Pollen viability
The TTC test (2,3,5-triphenyltetrazolium chloride) can be used to determine cell activity as
triphenyltetrazolium chloride causes a colour change in living cells, based on the presence of active
enzymes. The TTC test is used to examine the respiratory activity of tissue and thus the activity of pollen
(Iannotti et al. 2000). The red colour change of the redox reaction to formazan does not occur in non-
viable pollen. In this manner, viable pollen can be easily distinguished from non-viable pollen under the
microscope. Some studies have already conrmed that this approach is suitable for ash pollen
(Castiñeiras et al. 2019, Buchner et al. 2022, Eisen et al. 2024). The 1% TTC solution is made from 1 g
2,3,5-triphenyltetrazolium chloride and 60 g sucrose in 100 ml of distilled water (Buchner et al. 2022).
A few days prior to the beginning of the pollen seasons, we selected seven branches with closed owers
that were cut from up to 23 ash trees (see Table1) at different heights and in as many different
orientations as possible. The twigs were placed in vases lled with 100 ml tap water and stored at room
temperature in the laboratory. After 24 to 30 hours, when the anthers began to dehisce, pollen were
harvested by shaking and collecting them on microscope slides. Five slides with pollen from all branches
of one tree were prepared for each tree. Two drops of the TTC solution were added on the pollen, the
microscope slides were subsequently sealed with cover slips and placed on moist lter paper in petri
dishes (90 mm diameter) at room temperature in the dark. After 24 hours, the viable pollen were
quantied under the light microscope (Olympus CX23, magnication X40). We assessed the percentage
of viable pollen according to the pollen’s colour (red = viable) from in total 400 pollen per slide.
Pollen germination
Pollen germination experiments were carried out only in 2022 on the same trees and twigs used for
assessing pollen viability. The pollen were collected from opened owers into petri dishes and stored in
the freezer (-80°C) until further analyses were conducted in mid-May. We followed the approach outlined

Page 6/20
by Buchner et al. (2022), which is based on a modied concept introduced by Fröhlich (1964): The
nutrient solution was prepared by mixing and boiling 10 g sucrose, 1 g agar, 0.002 g boric acid and 89 ml
of distilled water. Subsequently, 14 mL of the nutrient solution was added to cover the bottom of a petri
dish (90 mm diameter). Once the nutrient medium had cooled and dried, a ne layer of pollen was
sprinkled onto the surface using a brush and the petri dishes were then stored in complete darkness at
25°C and 80% relative humidity in the climate chamber (growth cabinet KBWF, Binder GmbH, Germany).
After 24 hours, the germinated pollen were quantied under the light microscope (Olympus CX23,
magnication X40). The number of germinated pollen was noted from a total of 400 counted pollen per
petri dish. Pollen were considered to have germinated when the pollen tube had grown to at least the
diameter of the pollen (Käpylä 1991).
Statistical Analysis
Since the Shapiro-Wilk normality test indicated that the data were not normally distributed, the Mann-
Whitney-U/Wilcoxon test (2 variables) was employed to calculate the differences between the two
groups, mildly affected (vitality class 1 and 2) and severely affected (vitality class 3 and 4) trees. The
Kruskal–Wallis test (> 2 variables) followed by a post-hoc test (Wilcoxon signed-rank test) determined if
the differences between pollen quality and the years or locations were statistically signicant.
Spearman's correlation coecient was utilized for correlation analyses. All statistical analyses were
performed in R (RStudio Version 4.2.2). Boxplots were generated using the psych package (Revelle
2022).
3. Results
Pollen viability
In 2022, the average percentage of viable pollen (94.4%) was 18% higher than in 2021. In 2021, the study
site BY3 exhibited the highest number of viable pollen, with an average of 73.8%, surpassing BY2 by 1%
and BY1 by 6%. Conversely, in 2022, BY2 presented the highest average of viable pollen with 94%,
compared to BY1 (-5%) and BY3 (-10%). Analysis of variance (Kruskal–Wallis test) between the study
sites and years conrmed a statistically signicant difference (p < 0.001) for both years and all study
sites (Fig.2). The post-hoc test revealed statistically signicant differences for the following
combinations: BY1 2021 – BY1 2022, p > 0.001; BY2 2021 – BY2 2022, p > 0.001; BY3 2021 – BY3 2022,
p = 0.003. In 2021, there were no statistically signicant differences between the study sites, whereas in
2022 there were statistically signicant differences between the study site BY 1 and BY2 (p = 0.041) and
between BY3 and BY2 (p = 0.005).
The comparison between the two groups, mildly (vitality classes 1 and 2) and severely affected (vitality
class 3 and 4) trees, indicated that in most cases less damaged trees tended to exhibit a higher amount
of viable pollen compared to more affected ones, especially in 2022 (Fig.3a). However, this difference
was not statistically signicant (p > 0.050) for any of the analysed cases (2021: BY1 p = 0.697, BY2 p =
0.820, BY3 p = 0.423; 2022: BY1 p = 0.324, BY2 p = 0.578, BY3 p = 0.417; Mann-Whitney-U/Wilcoxon-test).

Page 7/20
A closer examination of the differences within the individual classes (Fig.3b) revealed that the healthiest
trees (vitality class 1) also tend to have the highest percentage of viable pollen, but the Kruskal-Wallis
test indicated no statistically signicant differences.
Pollen germination
As previously mentioned, pollen germination was conducted solely for the year 2022. The percentage of
germinated pollen was comparable between study sites BY2 (11.8%) and BY3 (11.4%), but substantially
lower for BY1 (6.7%).
The pattern of germination mirrored that of pollen viability: pollen from healthier and less affected trees
(vitality classes 1 and 2) tended to germinate more frequently (Fig.4a). However, this was only
marginally statistically signicant for study site BY2 (p = 0.079), according to the Mann-Whitney-
U/Wilcoxon test. There was no statistical signicance for BY1 (p = 0.231) and BY3 (p = 0.688). The
variation in individual scoring levels did not reveal a consistent pattern among the trees (Fig.4b),
although the healthiest trees of study sites BY1 and BY3 showed a higher number of germinated pollen
compared to the severely affected trees. Analysis of variance using the Kruskal-Wallis test conrmed no
statistically signicant differences between the four scoring classes.
Correlations
Regarding the correlation analyses (Fig.5) conducted using the percentage of germinated and viable
pollen per tree in 2022, BY1 and BY3 showed a positive (non-signicant) correlation, whereas BY2
demonstrated a negative (non-signicant) correlation (BY1: rho = 0.323, p = 0.153; BY2: rho = -0.178, p =
0.417; BY3: rho = 0.191, p = 0.461).
A positive association indicates that trees with a high number of viable pollen are characterised by a
higher germination capacity. However, in the case of the study site BY2, less pollen germinated relative to
the percentage of viable pollen. Note that an outlier inuenced this pattern.
4. Discussion
In this study, we observed no statistically signicant differences in pollen viability between mildly and
severely affected ash trees, both using the TTC test and pollen germination assessment. Nevertheless,
there was a tendency towards higher pollen viability for more vital ashes. This phenomenon was
particularly evident in pollen germination, where increased germination rates were evident in trees
exhibiting milder symptoms of ash dieback.
In prior studies, the effect of plant diseases on pollen viability has rarely been investigated so far.
Kazimierski and Kazimierska (1965) explored the effects of viral infections on pollen viability in
Lupinus
luteus
L., revealing that healthy plants exhibited signicantly higher rates of pollen germination and
growth compared to diseased counterparts. Conversely, viral infection led to degeneration of
components within the embryo sac at various developmental stages. Eisen et al. (2024) investigated the

Page 8/20
inuence of ash dieback on the pollen viability of
Fraxinus excelsior
L. in two seed orchards and a
riparian forest in southern Germany. Similar tendencies of reduced pollen viability were observed in
severely affected ash trees. Nonetheless, a statistically signicant distinction was only detectable
between mildly and severely diseased ash trees within the riparian forest. It can be concluded that ash
trees are able to produce viable pollen with potential for successful fertilization irrespective of their
health status. Therefore, we suggest that ash dieback disease does not universally impede fertilization.
Even pollen from ash trees severely affected by ash dieback and exhibiting a potential genetic
predisposition for high susceptibility to the disease are able to successfully fertilise other ash trees.
However, as ash dieback induces the development of additional epicormic shoots during disease
progression, further investigations explicitly studying the impact of epicormic shoots on pollen viability
would give more insights. However, severely affected trees exhibited a substantial reduction in ower
production, as demonstrated by Eisen et al. (2024). This observation was conrmed in our study, as
pollen samples could only be obtained from nine out of the total 119 trees from vitality score class 4
across all study sites and years. Consequently, this reduces the likelihood of pollen from severely
diseased trees contributing to the fertilization of other ash trees. These ndings align with prior research
using genetic analyses and mating models that showed that ash dieback is linked to a diminished
individual reproductive success of ash trees (Semizer-Cuming et al., 2021; Eisen et al., 2023).
Overall, the mean viability of
Fraxinus excelsior
pollen assessed in this study exceeded that reported by
Eisen et al. (2024) in southern Germany and by Castiñeiras et al. (2019) in Spain (81.1% vs. 73% and 65%,
respectively). This variance may be attributed to diverse local environmental factors inuencing pollen
viability across the study sites in Germany and Spain (Razzaq et al., 2019; Vasilevskaya, 2022). Notably,
pollen viability can be inuenced by elevated temperatures (Djanaguiraman et al., 2013; Iovane et al.,
2022). This could explain the lower viability observed in Spain and in seed orchards, where the pollen are
probably exposed to higher values of solar radiation compared to enclosed forest environments.
Buchner et al. (2022) demonstrated the susceptibility of
Fraxinus excelsior
L. pollen to high
temperatures, resulting in a rapid decline in viable pollen, particularly when coupled with prolonged UV
radiation exposure. High UV radiation has been shown to adversely affect pollen viability (Bohrerova et
al., 2009; Hidvégi et al., 2009). Thus, prevailing meteorological conditions can signicantly impact
viability (Ge et al., 2011). According to Ge et al. (2011), the viability of
Panicum virgatum
L. pollen
diminished vefold more rapidly under sunny conditions than under overcast conditions. In our study,
twigs with male owers, from which pollen were extracted, were stored in the laboratory under uniform
light conditions without direct sunlight exposure. Thus, it is reasonable to presume that the inuence of
radiation on the viability of the examined pollen was neglectable. In other words, this experimental
setting seems to be adequate to test for the inuence of ash dieback since the inuence of solar
radiation was limited.
Air pollution, such as carbon monoxide (CO), sulphur dioxide (SO2) and nitrogen dioxide (NO2), were
linked to negative impacts on the viability of pollen (Duro et al. 2013). Investigations into pollen viability
conducted in both highly polluted regions characterized by heavy trac and in unpolluted areas have

Page 9/20
demonstrated distinct differences, with notably diminished viability observed in more polluted areas
(Gottardini et al., 2004; Ouyang et al., 2016; Talukdar et al., 2018; Ramírez-Aliaga et al., 2022; Bazhina and
Sedaeva, 2023). However, the effects are species-dependent, with certain pollen types exhibiting greater
tolerance to pollution than others (Iannotti et al., 2000; Duro et al., 2013). While air pollution was not
specically examined in this study, its potential inuence on pollen viability warrants consideration. The
study sites Monheim (BY1) and Isen (BY3) were situated distantly from major roads, thus the samples
from these locations are less likely to be affected by substantial air pollution. However, the ash trees
located in Bruckberg (BY2), adjacent to an industrial area, exhibited a higher total pollen viability across
both analysed years, notably in 2022. Consequently, it can be assumed that the inuence of air pollution
is not fundamental at this location either. However, it is likely that pollen viability depends on different
phenotypes. Gupta et al. (2017) found that in plants with dichasial inorescences (comprising three
stages of owers that sequentially bloom), functionally male/hemaphrodite owers have increased
pollen viability. A similar effect may occur in ash trees due to their trioecious nature. It has to be noted
that a higher proportion of male/hermaphrodite compared to male ash trees were sampled at the BY2
site (data not shown). Moreover, the sexual characteristics of ash trees can vary from year to year
(Douglas et al., 2013; Westergren et al., 2020), which could further explain the differences in viability, as
determined by TTC, in 2021 and to 2022.
Methodologically, the TTC test serves as an indicator of pollen germination potential. The germination
capacity, in turn, reects the actual capability of pollen to germinate and allows for an assessment of
potential productivity (Duro et al., 2013). Consequently, a direct comparison between the results of the
TTC test and pollen germination is not feasible. A lower percentage of germinated pollen compared to
the viability indicated by the TTC test is therefore anticipated and was also observed in our study
(averaged for all sites in the year 2022 10.03%
vs
. 89.41%). Impe et al. (2020) further suggested that
pollen germination may be partly under genetic control. Previous studies investigating the relationship
between pollen viability and germination have yielded mixed results: Oberele and Watson (1953)
reported weak correlations between viability and germination. Additionally, Part and Gansehan (1989)
and Sulusoglu and Cavusoglu (2014) conrmed that a positive correlation between these two
reproductive metrics is not always evident. Only two out of the three study sites in our study exhibited a
positive (although non-signicant) correlation between these two pollen quality parameters. At BY2,
previously described inuences may have exerted additional impacts on pollen germination. Thus, this
study underscores the complex interplay of factors impacting pollen viability and consequently the
reproductive ecacy of ash trees, emphasizing the necessity for further research activities.
5. Conclusions
Pollen, as the central vector of gene ow, were found to be not signicantly affected in their viability by
ash dieback. Although there was a tendency for more severely affected trees to produce slightly less
viable pollen, we suggest that male trees maintain the capability to potentially fertilize female ashes
equally. Consequently, other metrics such as ower and pollen production gain more importance in the

Page 10/20
reproduction ecology of ash. As a further decline in ash will lead to a reduced pollen availability, further
studies on the aerobiology of ash pollen are highly recommended.
Declarations
Acknowledgements
We gratefully acknowledge the Bavarian State Forestry and the forest administration of each study site
for permitting us to utilize their forests for our research endeavours. In addition, we thank Johanna
Jetschni for her technical support in the eld.
Fundinginformation
The project receives funding via the Waldklimafonds (WKF) funded by the German Federal Ministry of
Food and Agriculture (BMEL) and Federal Ministry for the Environment, Nature Conservation, Nuclear
Safety and Consumer Protection (BMUV), administrated by the Agency for Renewable Resources (FNR)
under grant agreement number 2219WK20H4.
Conicts of Interests
The authors declare that there are no conicts of interest. There are only non-nancial research interests,
related directly or indirectly to this work submitted for publication. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in
the decision to publish the results.
Statement of contribution
SJ-O was responsible for funding and the study design. Field work and data analyses was done by GK;
writing, reviewing and proof-reading by GK, LB, SJ-O and A-KE. All authors have read and agreed to the
nal version of the manuscript.
References
1. Baral HO, Queloz V, Hosoya T (2014)
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Figures
Figure 1
Study sites: (a) the location of the three study sites in Bavaria, Germany, (b) Monheim BY1, (c) Bruckberg
BY2 and (d) Isen BY3. The yellow dots indicate the investigated ash trees

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Figure 2
Boxplots of ash pollen viability for the study sites BY1 (Mohnheim), BY2 (Bruckberg) and BY3 (Isen) in
the year 2021 and 2022. The interquartile range is represented by the height of the boxes, maximum and
minimum values by the upper and lower whiskers, the median by bold horizontal lines in the boxes, and
points indicating outliers

Page 18/20
Figure 3
(a) Boxplots showing the percentage of viable pollen for the two groups related to a different health
status (scoring class 1/2: mildly affected; 3/4: severely affected) and (b) Boxplots showing the
percentage of viable pollen for all groups related to the health statuses 1 to 4 for the study sites BY1
(Mohnheim), BY2 (Bruckberg) and BY3 (Isen) in the year 2021 and 2022. The interquartile range is

Page 19/20
represented by the height of the boxes, maximum and minimum values by the upper and lower whiskers,
the median by bold horizontal lines in the boxes, and points indicating outliers
Figure 4
Boxplots showing the percentage of germinated pollen for the two groups related to a different health
status (scoring class 1/2: mildly affected; 3/4: severely affected) (a) and the percentage of the
germinated pollen for all groups related to the health status 1 to 4 (b) for the study sites BY1 (Monheim),
BY2 (Bruckberg) and BY3 (Isen) in the year 2022. The interquartile range is represented by the height of
the boxes, maximum and minimum values by the upper and lower whiskers, the median by bold
horizontal lines in the boxes, and points indicating outliers
Figure 5

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Scatterplots of the percentage of viable pollen [%] and the percentage of germinated pollen [%] at study
sites BY1, BY2 and BY3 in 2022