Pollination success of Fraxinus excelsior L. in the context of ash dieback

Abstract

Key message: Paternity analyses show that effective pollination of ash (Fraxinus excelsior L.) in a seed orchard and a floodplain forest affected by ash dieback is more likely to be facilitated by healthier males. Thereby, natural selection can have a positive effect on the health of future generations.
Context: Ongoing ash dieback and increasing fragmentation of ash populations may result in reduced pollen flow, which can reduce pollination success of future generations of ash trees. Therefore, it is essential to further improve our understanding of gene flow patterns, especially with respect to ash dieback.
Aims: In this study, paternity analyses were conducted in a seed orchard and a floodplain forest in Germany in 2018 to explain the relationship between pollination success and the health status of ash trees and distances of effective pollen transport.
Methods: Cambium samples (i.e., from twigs and stumps) were collected from 251 ash trees (putative father and mother trees) for genotyping, and the health status of each tree was documented using a scoring system to evaluate vitality. Additionally, seeds were harvested from 12 mother trees per site. Genetic analyses using nuclear microsatellites were performed to determine paternal trees. Paternities were assigned based on the likelihood model implemented in the Cervus 3.0.7 software.
Results: Our results showed that the average pollination distance was 76 m in the seed orchard and 166 m in the floodplain forest. In general, pollination success decreased substantially with increasing distance to the mother tree. Despite the dense tree cover in the floodplain forest, pollen were transported over long distances (greater than 550 m), suggesting that non‑local sources also play a role in pollination. This is supported by the foreign pollen input identified in the seed orchard (66.5%). Self‑pollination was detected only to a very small extent, and thus had no major influence on reproduction. In addition, both healthy and slightly diseased father trees showed similar mating success. However, this was not the case for the severely diseased ash trees (more than 50% of crown damage)
because only a few offspring could be assigned to them. Nevertheless, in contrast to the floodplain forest, there was no significant correlation between damage classes and pollination success in the seed orchard.
Conclusion: Long‑distance pollen transport contributes to the connectivity of ash trees in the landscape. Additionally, both healthy and slightly diseased fathers have a greater contribution to pollination, thus potentially improving the health of the next generation of ash trees. Moreover, gene flow between stepping stone populations is necessary to ensure the positive impact on the genetic diversity of ash populations in the future.

Full text

Eisenetal. Annals of Forest Science (2023) 80:22
https://doi.org/10.1186/s13595-023-01189-5
RESEARCH PAPER
Pollination success ofFraxinus excelsior L.
inthecontext ofash dieback
Anna‑Katharina Eisen1* , Devrim Semizer‑Cuming2,3 , Susanne Jochner‑Oette1 and Barbara Fussi3
Abstract
Key message Paternity analyses show that effective pollination of ash (Fraxinus excelsior L.) in a seed orchard and a
floodplain forest affected by ash dieback is more likely to be facilitated by healthier males. Thereby, natural selection
can have a positive effect on the health of future generations.
Context Ongoing ash dieback and increasing fragmentation of ash populations may result in reduced pollen flow,
which can reduce pollination success of future generations of ash trees. Therefore, it is essential to further improve our
understanding of gene flow patterns, especially with respect to ash dieback.
Aims In this study, paternity analyses were conducted in a seed orchard and a floodplain forest in Germany in 2018
to explain the relationship between pollination success and the health status of ash trees and distances of effective
pollen transport.
Methods Cambium samples (i.e., from twigs and stumps) were collected from 251 ash trees (putative father and
mother trees) for genotyping, and the health status of each tree was documented using a scoring system to evaluate
vitality. Additionally, seeds were harvested from 12 mother trees per site. Genetic analyses using nuclear microsatel‑
lites were performed to determine paternal trees. Paternities were assigned based on the likelihood model imple‑
mented in the Cervus 3.0.7 software.
Results Our results showed that the average pollination distance was 76 m in the seed orchard and 166 m in the
floodplain forest. In general, pollination success decreased substantially with increasing distance to the mother tree.
Despite the dense tree cover in the floodplain forest, pollen were transported over long distances (greater than
550 m), suggesting that non‑local sources also play a role in pollination. This is supported by the foreign pollen input
identified in the seed orchard (66.5%). Self‑pollination was detected only to a very small extent, and thus had no
major influence on reproduction. In addition, both healthy and slightly diseased father trees showed similar mat‑
ing success. However, this was not the case for the severely diseased ash trees (more than 50% of crown damage)
because only a few offspring could be assigned to them. Nevertheless, in contrast to the floodplain forest, there was
no significant correlation between damage classes and pollination success in the seed orchard.
Conclusion Long‑distance pollen transport contributes to the connectivity of ash trees in the landscape. Addition‑
ally, both healthy and slightly diseased fathers have a greater contribution to pollination, thus potentially improving
the health of the next generation of ash trees. Moreover, gene flow between stepping stone populations is necessary
to ensure the positive impact on the genetic diversity of ash populations in the future.
Open Access
© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
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Annals of
Forest Science
Handling editor: Marjana Westergren.
*Correspondence:
Anna‑Katharina Eisen
Anna‑Katharina.Eisen@ku.de
Full list of author information is available at the end of the article

Page 2 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
1 Introduction
In recent decades, ash dieback, caused by the fungus
Hymenoscyphus fraxineus (T. Kowalski) Baral, Que-
loz, and Hosoya (Baral etal. 2014) (Syno.: Hymenoscy-
phus pseudoalbidus (Queloz etal. 2011) and its asexual
stage Chalara fraxinea (Kowalski 2006), has acutely
threatened ash populations in Europe (Metzler etal.
2012; McKinney etal. 2014; Kosawang etal. 2018) and
its silvicultural future (Enderle 2019). Common ash
(Fraxinus excelsior L.) is considered a promising tree
species under climate change conditions (Kölling 2007;
Enderle etal. 2017; LWF 2019; Müller-Kroehling and
Schmidt 2019) and presents itself as a versatile and
valuable species with high ecological and economi-
cal importance (Enderle et al. 2017; Hultberga et al.
2020). However, high mortality rates have already led
to a sharp decline in many local ash populations (Lygis
etal. 2014; Marçais etal. 2017; Pliūra etal. 2017; Sem-
izer-Cuming et al. 2021). Simulations indicated that
up to 75% of ash trees in mixed stands in Europe are
expected to die over the next 30 years (Coker et al.
2019). Other studies even suggest that only approx.
1–5% of ash trees are not susceptible and show little or
no symptoms related to ash dieback (McKinney etal.
2014; Rigling etal. 2016; Enderle 2019). In this regard,
individuals with very low levels of disease symptoms
and less than 30% leaf loss are considered partially
resistant (i.e., developing less symptoms) and might
be suitable for genetic conservation or breeding pro-
grams (Lenz etal. 2012; McKinney etal. 2014). Disease
development at stand level will therefore depend on
the ability of less susceptible genotypes to spread their
genes via pollen and seeds (Lobo etal. 2015; Semizer-
Cuming et al. 2019, 2021). Partial resistance to ash
dieback was not found to be population or provenance
based but rather individual based (McKinney et al.
2011, 2014; Enderle etal. 2017; Semizer-Cuming etal.
2019). Therefore, it is essential that genetic connectiv-
ity is maintained, even in fragmented landscapes, and
that genetic diversity is sufficiently high for the repro-
duction of trees for future healthy ash populations. To
assess tree population responses and the consequences
of ecological and biological threats, it is essential to
understand pollen dispersal patterns.
Effective gene flow between partially resistant adult
trees is necessary to establish healthier next genera-
tions of ash trees (Semizer-Cuming etal. 2017, 2019;
Fussi 2020). However, ongoing ash mortality and the
increasing fragmentation of ash populations lead to a
decrease in pollen flow (McKinney etal. 2014; Semizer-
Cuming etal. 2017), which can further limit the genetic
diversity of future generations (Fussi etal. 2014). If par-
tially resistant ash trees are highly underrepresented,
less healthy genotypes will also succeed and produce
offspring. is can lead to decreased natural selection
under the prevailing environmental conditions (Eisen
etal. 2022b). Reduced genetic diversity can negatively
affect the adaptive potential of future generations of
ash trees, whereby genetic variation is particularly
important for adaptation to new pathogens due to the
long generation time of forest trees (Fussi etal. 2014;
McKinney etal. 2014). In addition, ash trees are trioe-
cious, i.e., monoecious and dioecious individuals can
occur simultaneously (Roloff 1997), which can increase
the risk of self-pollination (McKinney etal. 2014; Sem-
izer-Cuming etal. 2021).
Genetic analyses investigating the mating success of
ash individuals in populations have been performed
only in a few studies to date (Heuertz etal. 2003; Bacles
and Ennos 2008; Thomasset etal. 2014; Semizer-Cum-
ing et al. 2017). The relationship between reproduc-
tive success and the health status of ash trees in a seed
orchard has only been assessed once (Semizer-Cuming
et al. 2019): It was demonstrated that health status
and reproductive success of ash trees in a seed plan-
tation in Denmark were negatively correlated. Female
ash trees severely affected by ash dieback produced a
much lower seed quantity compared to healthy ones,
whereas damaged males could still sire some offspring.
In another study, Semizer-Cuming etal. (2017) exam-
ined the genetic connectivity of ash trees in an isolated
forest area. Thereby, it was found that about 55–64%
of the seeds and 75–98% of the seedlings descended
from local ash trees, but 26–45% of the pollen were
transported from outside the forest area. Moreover,
a positive correlation was found between seed dis-
persal distance and wind speed, while there was none
between pollen dispersal distance and wind speed.
Research on distances of effective pollen transport
varied widely among studies: Heuertz etal. (2003) con-
ducted a study in a mixed deciduous forest in Roma-
nia and determined a distance of 70–140m, whereas
Bacles and Ennos (2008) estimated the distance with
up to 2.9km in a deforested Scottish landscape. Sem-
izer-Cuming etal. (2021) found that 50% of pollen dis-
persal occurred within 140 m in a Danish forest, 5%
of pollen dispersal occurred over a distance of 1.3km,
and 1% was transported farther than 3 km. Similar
Keywords Ash dieback, Gene flow, Paternity analysis, Effective pollen transport, Pollination success

Page 3 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
results were also obtained from aerobiological investi-
gations on pollen transport and deposition using gravi-
metric pollen traps, which were conducted in two ash
seed orchards in Germany (Eisen etal. 2022a). It was
shown that 50% of the ash pollen in the air occurred
within a distance of 200m from the source, whereas
only 10% of the total pollen catch reached a distance
of 500m.
In general, pollen dispersal is influenced not only by
abiotic and biotic factors such as meteorological con-
ditions, topography, and vegetation but also by tree
height and crown size (Scheifinger etal. 2013; Puc 2012;
Adams-Groom et al. 2017; Eisen et al. 2022a). Sork
and Smouse (2006) also noted that less fragmented
landscapes have more pollen input from outside and
therefore adjacent pollen sources. e magnitude
of influential factors suggests that it is important to
improve our understanding of gene flow patterns in
fragmented and non-fragmented ash populations fur-
ther to predict accurate estimates of pollen dispersal
(Semizer-Cuming etal. 2017).
Given this background, the objectives of this study are
thus to explain the relationship between pollination suc-
cess and the health status of ash trees and to test whether
healthy fathers contribute more to the next generation, as
well as to estimate pollen dispersal at sites affected by ash
dieback. Based on the results, we discuss the implications
of the observed relationship between the extent of dam-
age caused by ash dieback and pollination success in rela-
tion to conservation and management of the species. is
could be of particular interest for the establishment of
future seed orchards where pollen emission from outside
should not influence the production of healthy offspring
in the orchards.
2 Material andmethods
2.1 Study area
The seed orchard Schorndorf (48°46′ N, 9°25′ E, 420m
a.s.l.) is located in the valley Remstal near Schorn-
dorf, Baden-Württemberg, Germany, and has an area
of approx. 2.3ha (Fig. 1a). The average annual tem-
perature is 10.3°C (DWD station Stuttgart Schnarren-
berg, 1981–2010) and the average annual precipitation
855mm (DWD station "Winterbach, Rems-Murr-Kr.",
1981–2010). The orchard is located on a NW exposed
slope with an inclination of about 6 to 10° (LGRB
2021). It consists of grafts of selected so-called “plus
trees,” which were selected for growth and stem qual-
ity prior to the outbreak of the disease and is charac-
terized by a well-spaced plot design (7m × 7 m). The
plus trees originated from the South German hill and
mountain area and the Alps and Alpine foothills in
Baden-Wuerttemberg (Enderle etal. 2014). Initially, 68
clones (416 ash trees) were planted in November 1992
in 25 rows: 36 clones with 8 female or hermaphrodite
ramets and 32 clones with 4 male ramets. Due to the
negative effects of ash dieback, the majority of the
trees (ca. 70%) died (Eisen et al. 2022a). In 2018, the
seed orchard consisted of 123 mature ash trees with
a maximum height of 17m (Fig.1b/Appendix Fig.7).
In the seed orchard, common ash is growing almost
exclusively with few other tree species such as cherry
and apple trees, wild service trees, or lime trees (espe-
cially along the sides). It is surrounded by meadows,
which are mostly bordered by a mixed forest consist-
ing mainly of beech, spruce, and pine. There are no
other ash trees in the immediate surroundings (within
a radius of about 1km). Due to ash dieback, there is
currently no commercial demand for ash seeds.
e floodplain forest is located at the Danube
between the cities Neuburg and Ingolstadt, Bavaria,
Germany (Fig.1a). e selected area for our investiga-
tions is situated near the Bergheim barrage (48°44′ N,
11°16′ E, 375m a.s.l.) and has an area of about 10ha.
e average annual temperature is 7.8 °C, and the
average annual precipitation is 715 mm (1961–1990)
(Schwab et al. 2018). e calcareous and nutrient-
rich substrate of the site enables favorable growing
conditions especially for common ash (Doben et al.
1996; Margraf 2004). Common ash is represented in
the dense floodplain forest with a share of about 15%
(Jochner-Oette etal. 2021). In the selected area, 50 ash
trees and 78 tree stumps were sampled for our analyses
(Fig.1c/Appendix Fig.8).
2.2 Sampling andvitality assessment
In October 2018, a lift truck was used to collect seeds
for genotyping in the seed orchard and in the floodplain
forest (12 mother trees selected per stand, Table1) to
quantify the proportion of pollination success of dis-
eased and healthy father trees. All investigated ash trees
(seed orchard: 123 ash trees and floodplain forest: 50 ash
trees) were classified according to the scoring system of
Lenz etal. (2012) for assessing the vitality of adult ash
trees. Trees in categories 0 and 1 (up to max. 30% leaf
loss) were classified as healthy, all other trees as diseased
(categories 2 to 4) or dead (category 5). e dead trees
were not included in the surveys. In Schorndorf, seeds
were collected from nine different clones, representing
six healthy and six diseased ash trees. From three clones,
two ramets were selected for seed harvest (Table1). A
total of five healthy and seven diseased ash trees were
selected in the floodplain forest. e number of analyzed
and genotyped seeds per mother tree varied between 48

Page 4 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
and 49 seeds in Schorndorf and between 70 and 76 seeds
in the floodplain forest.
In spring 2019, twigs from all 123 mature ash trees (12
mother trees and 111 adult ash trees) were sampled in
the seed orchard. In the floodplain forest, twigs or wood
from 128 trees were sampled: 50 samples were derived
from standing ash trees and 78 from ash stumps. e 78
ash trees had to be removed in January 2019 in order to
ensure road safety. Since damage caused by ash dieback
had already been observed before, they were classified
as diseased. e logged trees were ash trees, which may
have served as pollen donors since they were cut after
the pollen season and may have contributed to pollina-
tion and seed development. e standing ash trees were
sampled at a 25-m radius around the mother trees, while
ash stumps were interspersed (Fig.1c/Appendix Fig.8).
After sampling, the tissue was preserved at − 20 °C until
further analyses.
Due to the identical genotypes of the clones in the seed
orchard, in most cases, a single tree could not be identi-
fied as the father (except for the cases where there was
only one potential father per clone). erefore, all ramets
of a clone that are possible pollen donors for a mother
tree were defined as potential fathers. To exclude the
trees that were not candidate fathers (i.e., females and/
Fig. 1 Study areas. a Location of the seed orchard near Schorndorf (48°46′ N, 9°25′ E, 420 m a.s.l.) and of floodplain forest (48°44′ N, 11°16′ E, 375 m
a.sl.) in Germany. b Seed orchard including investigated ash trees, labeled by tree ID: yellow stars — mother trees, blue dots — ash trees identified
as potential father trees by their clone number, gray dots — other investigated ash trees excluded as pollen donors. c Study area of floodplain forest
including investigated ash trees, labeled by tree ID: yellow stars — mother trees, blue dots — ash trees identified as father trees, orange squares —
logged ash trees identified as father trees. Source of the maps: ESRI Data & Maps

Page 5 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
or trees with no flowering), the gender of the ash trees
was determined on site (in the period 2018–2021), and
the presence of flowers or flower buds and their pheno-
logical stage was recorded thoroughly with binoculars
on 19 April 2018 (109th day of the year). Hermaphrodite
mother trees were also considered as potential father
trees. For the floodplain forest, no phenological data were
available in 2018. e gender was determined for the ash
trees alive; however, this was not possible for the ash
stumps. e evaluated attributes for all ash trees can be
found in the Appendix (Tables2 and 3). e geographic
position (UTM coordinates; 32 N) of each sampled ash
tree was recorded using DGPS (Stonex S9 III, Stonex,
Paderno Dugnano (MI), Italy) for both sites (Appendix
Fig.7/Fig.8).
2.3 DNA extraction andgenotyping ofmicrosatellite
markers
For the adult trees, cambium of the twigs and stumps
was used for DNA extraction. For the seeds, DNA was
extracted from their embryos. DNA extraction was
performed using the cetyltrimethylammonium bromide
[CTAB] method (Doyle and Doyle 1990). e DNA
content of the samples was determined using a spectro-
photometer (GeneQuant pro, Amersham Biosciences,
CA, USA). After DNA extraction, polymerase chain
reaction (PCR) was performed to examine 15 micros-
atellite loci in three different multiplexes (Appendix
Table4) (Brachet etal. 1999; Lefort etal. 1999; Gerard
etal. 2006; Aggarwal etal. 2011; Bai etal. 2011; Noakes
etal. 2014). However, reproducible results could only
be obtained for eleven markers (M2-30; Fp18437; Fem-
satl 11; Femsatl 12bis; Femsatl 4; Fp14665; Fp21064;
FRESTSSR308; FRESTSSR528; Femsatl 19; ASH2429).
Femsatl12bis had too many null alleles in floodplain
samples, while the same was the case for marker
Fp14665 in the seed orchard. ese markers were
therefore only used for the seed orchard and floodplain,
respectively. Subsequently, PCR products were sepa-
rated by high-resolution capillary electrophoresis using
Table 1 Characteristics of mother trees and number of analyzed offspring, as well as the potential longest and shortest pollen
distance from mother tree to the detected father trees
Study site Mother
tree (clone
number _tree
ID)
Vitality
classication
of mother tree
in 2018
Gender of
mother tree Number of
analyzed
ospring
Number of
ospring with
known father
(clone)
Number of
(potential)
father trees
Shortest
distance
of pollen
transport [m]
Longest
distance
of pollen
transport [m]
Seed orchard 1619_31 1 Female 48 21 34 6.3 117.8
1619_81 1 Female 48 31 17 7.7 111.9
1627_01 2 Female 48 10 29 14.7 191.1
1627_21 2 Female 48 12 26 27.7 126.9
1663_24 2 Female 48 27 24 27.9 124.3
1663_94 0 Hermaphrodite 48 9 19 19.9 137.6
1664_71 0 Hermaphrodite 49 6 13 14.8 110.8
1866_30 2 Female 49 16 26 14.4 171.7
0000_62 1 Female 49 29 38 8.2 149.9
1878_13 2 Female 48 12 17 39.7 139.1
1879_37 1 Female 49 13 25 28.3 135.1
1898_14 2 Female 48 5 16 48.1 130.4
Floodplain
forest M_01 1 Female 70 13 13 14.8 499.0
M_02 2 Hermaphrodite 76 10 10 152.0 512.5
M_03 1 Hermaphrodite 70 18 13 19.2 308.8
M_04 2 Female 73 25 14 3.9 347.2
M_05 2 Female 71 24 18 5.8 296.0
M_06 1 Female 72 24 16 32.1 462.7
M_07 0 Female 70 24 11 2.3 319.9
M_08 3 Female 71 8 7 40.6 551.6
M_09 2 Hermaphrodite 74 24 8 24.7 221.1
M_10 2 Hermaphrodite 76 16 13 16.1 253.5
M_11 1 Female 70 19 16 5.5 328.2
M_12 3 Hermaphrodite 74 19 17 0.0 479.8

Page 6 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
the GeXP automated sequencer (Beckman Coulter,
Inc., Fullerton, CA, USA) and analyzed by software-
assisted allele scoring.
2.4 Data analysis
For clone identification, the population genetic soft-
ware GenAlEx 6.0 (Peakall and Smouse 2012) was used.
e multilocus genotype of all adult trees was com-
pared and identical genotypes assigned to one clone in
the seed orchard. Trees with nonidentical multilocus
genotypes with any of the known clones were treated as
an individual tree and labeled “0000.” is was the case
when the rootstock had grown up to a new tree (two
variants: variant 1: e rootstock has grown up after
planting, so the ash trees are sexually mature and could
be potential fathers; variant 2: Due to ash dieback,
the trees have died, and the rootstocks have sprouted.
ese ash trees were still too young for flowering).
Paternity analysis implemented in the Cervus 3.0.7
software package (Kalinowski etal. 2007) was applied
to determine the father of each seed. In this context,
the allocation is based on Delta (Δ) (Labuschagne
et al. 2015). ereby, the difference of the likelihood-
odds ratio (LOD) score between the two most likely
parents is examined to find the actual parents. e
critical values of Δ were calculated at strict (95%) and
relaxed (80%) confidence levels during the simulations
(Labuschagne etal. 2015; Semizer-Cuming etal. 2019).
For Schorndorf, 100,000 offspring and 70% of sampled
potential fathers were simulated. For the alluvial forest,
100,000 offspring and 30% of sampled potential fathers
were simulated. e minimum number of loci was set
to eight. e error rate was kept at 0.01 (Semizer-Cum-
ing etal. 2019). In addition, self-pollination was explic-
itly taken into account in the model.
Pollen distances were calculated from the UTM coor-
dinates of the mother and father trees. In the case of the
seed orchard, the distances to the mother trees were
calculated individually for all potential fathers (ramets)
of the same clone, assuming that each of them could be
the potential father, since it was not possible to deter-
mine the distinctone. en, we averaged the sum of the
distances for each father tree (i.e., each clone). us, it
was only possible to estimate the average pollination
distances to the mother trees. In the floodplain forest,
we were able to calculate the actual pollination dis-
tances between fathers and mothers, as all had unique
genotypes. e distances were classified at intervals of
10m. In addition, the relationship between the num-
ber of offspring per father tree and the degree of dam-
age to the father trees were calculated. Since ramets of
the same clone were showing varying vitality scores,
the average vitality score over all ramets per clone was
calculated for the seed orchard Schorndorf. In order to
estimate the correlation between the damage class and
the pollination success, a chi-squared test and exact
Fisher test were applied. All analyses and visualiza-
tions were performed in RStudio (version 1.2.1335.0) or
Microsoft Excel 2016.
3 Results
3.1 Seed orchard
3.1.1 Paternity analysis: relationship betweenreproductive
success andhealth status
In total, 123 ash trees could be classified into 37 different
clones with distinct genotypes. e number of ramets per
clone varied from one to eight, with an average of four
ramets per clone in the orchard. Twelve of the trees were
selected as mother trees. Of the remaining 111 ash trees,
56 were male, 36 were female, and 15 were hermaphro-
dites. e gender of four ash trees could not be determined
accurately. Seventy-six trees had flowers or flowering buds.
us, 49 trees (36 females and 13 males/hermaphrodites
without flowers) were excluded as pollen donors (Appen-
dix Table2).
Out of the 580 examined offspring, 194 (33.5%) of them
could be assigned to their potential fathers (Fig.2a). Five
single trees and 18 clones (54 ash trees) were detected
as potential fathers, although for four clones only one
tree could be the father in each case with certainty, due
to the exclusion procedure (ash trees that were female
and/or not flowering were excluded as fathers). Due to
its hermaphroditism and the fact that paternity could be
found for this clone, one mother tree was also included as
a potential father tree. Nine ash trees had no pollination
success; three of them have not reached their reproduc-
tive age. Regarding the other six ash trees, four were her-
maphrodite, and two were male. It was striking that the
two male ash trees were very strongly affected by ash die-
back (vitality scores 3 and 4).
Fig. 2 Results of the seed orchard near Schorndorf. a Identified father clones with number of ramets (n) resp. identified fathers as well as average
vitality score per clone with the average number of offspring per father tree, b number of offspring summed allocated to the average vitality score
of the father clones, with n representing the number of genotypes, and c average number of offspring summed per potential father tree allocated
to the vitality score of the potential father trees, with n representing the number of ramts (vitality score: 0 — dark green; 1 — light green; 2 —
yellow; 3 — orange; 4 — red). d Number of father trees per distance class based on paternity analysis. All potential father trees per mother tree have
been counted
(See figure on next page.)

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Eisenetal. Annals of Forest Science (2023) 80:22
Fig. 2 (See legend on previous page.)

Page 8 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
e highest pollination success was observed for the
clone 1651 (three potential fathers; average vitality
score of 2) with 35 offspring, whereas the lowest pol-
lination success was one offspring associated with 13
trees. Dividing the number of offspring per clone by the
number of ramets, the maximum number of offspring
is 11.7, and the minimum is 0.3. In addition, the aver-
age vitality score over all ramets of a father clone was
analyzed; out of the 18 father clones and five single
trees, three single trees could be assigned to an average
vitality score of 0, six clones and two single trees to an
average vitality score of 1, and ten clones to an average
vitality score of 2. Only one clone each that sired off-
spring could be assigned to the vitality classes of 3 and 4
(one offspring each). Overall, father trees in vitality class
2 produced the most offspring (127 offspring) (Fig.2b).
In vitality score classes 0 and 1, in which the ash trees
are still considered healthy, the number of identified
fathers who sired offspring was substantially below the
value of class 2 (vitality score 0: 5 offspring and vitality
score 1: 60 offspring). However, no significant correla-
tion could be detected between the damage classes and
pollination success (chi-square test: p = 0.999, Fisher
test: p = 0.997).
Considering the 54 potential father trees as individ-
ual trees (regardless of the clone affiliation), 6, 24, 18,
4, and 2 ash trees could be assigned to vitality scores 0,
1, 2, 3, and 4, respectively. With an average of 85.6 off-
spring, most of the father trees were detected in vitality
score class 2 in this case as well. However, the number
of fathers that sired offspring in scoring classes 0 and
1 was in total higher (vitality score 0: 11.1 offspring
and vitality score 1: 82.0 offspring) than the number
of offspring in scoring class 2, with an average of 93.1
offspring. Vitality scores 3 and 4 could be assigned an
average of 10.6 and 4.7 offspring from identified fathers,
respectively (Fig.2c). Since vitality classes 1 and 2 had
more ramets per clone, it can be assumed that weakly
damaged clones have a higher overall potential to pro-
duce offspring. However, no significant correlation
could be found (chi-square test: p = 0.982, Fisher test:
p = 0.904).
For the mothers, the average number of offspring that
could be assigned to a father tree was 7.5 offspring for
vitality score 0 (two mother trees), 23.5 offspring for
vitality score 1 (four mother trees), and 13.7 offspring for
vitality score 2 (six mother trees).
The analyses identified possible self-pollination for
three offspring of the mother trees 1619_31 (n = 2)
and 1898_ 14 (n = 1). In the clone 1619 (n = 8), only the
ramet 1619_86 was observed to produce male flow-
ers in addition to female flowers. Thus, due to the
number of ramets, it can be assumed that there is a
low probability (11.1%) of self-pollination of the par-
ent tree 1619_31. Clone 1898 consists of two female
and six male ash trees; thus, the probability of self-
pollination is again very low, with a value of 12.5%.
No self-pollination could be detected for the two her-
maphrodite mothers.
3.1.2 Distances ofeective pollen transport
e average distance of effective pollen transport of all
identified potential father trees to the mother trees was
76.2 m in the seed orchard Schorndorf (Fig. 2d). e
largest distance was estimated at 191.1m and could be
detected between the potential father tree 1630_90 and
the mother tree 1627_1. e shortest distance was calcu-
lated between the potential father tree 1644_32 and the
mother tree 1619_31 at 6.3m.
In general, it was found that only 5.3% of the identi-
fied paternal trees with potential pollination success
were located less than 20 m from the mother tree. A
total of 23.9% of the potential father trees were located
between 21 and 50m from the mother tree, and 45.1% of
the potential father trees were located within a distance
of 51 to 100m. For the distance between 101 and 150m,
potential pollination success was attributed to 22.2% of
the father trees and for the distance between 151 and
191.1m only to 3.5%.
For a better spatial understanding of effective pol-
len transport, pollination success was investigated with
respect to the geographical location of the mother trees.
e two mother trees, 1619_81 and 1898_14, located in
different areas of the seed orchard were selected as a rep-
resentative to describe the general observed pollen dis-
persal patterns.
For mother tree 1619_81 (vitality score 1), most of
the offspring (31 out of 48 seeds) could be assigned to
their potential fathers (17 different trees) (Table1). e
mother tree is located in the southwestern area of the
orchard, and thus, many potential father trees are within
reach (Fig.3a). e results showed that clone 1629 (four
ash trees, average vitality score 1) had the highest pol-
lination success (14 offspring) with this mother tree,
but with a distance between 54.6 and 111.9m, it is not
located in the immediate vicinity of the mother tree. At
the same time, it was found that the father clones with
only one offspring are partly very close to the mother
tree (clone 1651 — shortest distance of 34.9m and clone
1879_75 — distance of 10.5m). Both clones were linked
to an average vitality score of 2.
e mother tree 1898_14 (vitality score 2) had the
fewest offspring assigned to it (5 out of 48 seeds). How-
ever, for the five offspring, 16 possible father trees must
be considered with only a maximum of one to two

Page 9 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
Fig. 3 Mother tree. a 1619_81 and b 1898_14 with their potential identified father trees and their number of offspring in the seed orchard
Schorndorf. Orthophoto taken with drone XR6 (Airborne Robotics, London, UK) equipped with a four‑channel multispectral camera (Tetracam
Micro‑MCA 4, Chatsworth, CA, USA) on 12.07.2018; coordinate system UTM 32N

Page 10 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
offspring descended from one father clone. e rea-
son for this could be the location of the mother tree at
the northern border of the orchard (Fig.3b), since 43
offspring could not be assigned to a father clone. e
potential father trees identified are not located directly
around the mother tree but are mostly distributed in the
southern part of the orchard. e closest potential father
tree (1630_51; vitality score 3) was located at a distance
of 48.1m from the mother tree, 51.6% of the potential
father trees were located between 51 and 100 m from
the mother tree, and 44.8% of the potential father trees
were located between 101 and 130m from the mother
tree.
3.2 Floodplain forest
3.2.1 Paternity analysis: relationship betweenreproductive
success andhealth status
In the floodplain forest, all sampled trees had unique
genotypes. In total, 224 (26.14%) offspring out of 857
examined could be assigned to 73 fathers (Fig.4 a, b).
Forty-three tree stumps and 30 ash trees were detected
as father trees. From the 30 living ash trees, 2, 10, 9,
8, and 1 individuals had a vitality score of 0, 1, 2, 3,
and 4, respectively (Appendix Table3). Five of the liv-
ing ash trees were hermaphroditic; the other 25 were
male. Accordingly, no pollination success was detected
for 20 living ash trees (out of 50 initially investigated
trees), with only six of them being male and one being
hermaphrodite; the other 13 were female. Please note
that the cambium samples were collected prior to gen-
der determination. Of these seven ash trees (male and
hermaphroditic) without pollination success, two were
healthy, and five were diseased.
The highest pollination success was observed related
to the father tree tree stump 19 with 24 offspring, fol-
lowed by the father tree A_22 with 20 offspring and the
father tree A_01 with 13 offspring. Twenty-five trees
only sired one offspring each. The highest total num-
ber of offspring was found in diseased ash trees (vital-
ity scores 2–4). Thus, a significant correlation between
the damage classes and pollination success could be
found in this case (chi-square test: p = 0.001). The tree
stumps, which were regarded as diseased individuals
(n = 43), had 113 offspring (50.5%), and the diseased
ash trees (n = 18) had 47 offspring (21.0%). In vital-
ity scoring classes 0 and 1, in which the ash trees are
considered as healthy (n = 12), the number of offspring
was 64 (Fig. 4c). In addition, the average number of
offspring per number of ash trees in a vitality scoring
class was analyzed; it can be recognized that with an
average of 12 offspring, father trees of vitality score
0 could be assigned to the most offspring. The father
trees of vitality scoring classes 1, 2, 3, and 4 (n = 1) had
an average of four, three, and two offspring, respec-
tively, and the current tree stumps had an average of
three offspring (Fig.4d).
For the mothers, the average number of offspring that
could be assigned to a father tree was 24, 18.5, 19.8,
and 13.5 offspring for vitality score 0 (n = 1), 1 (n = 4),
2 (n = 5), and 3 (n = 2), respectively. Additionally, the
analyses detected self-pollination for one offspring of
the mother tree M_12 (hermaphrodite; vitality score
class 3).
3.2.2 Distances ofeective pollen transport
In the floodplain forest, the average distance of effec-
tive pollen transport was 165.6m (Fig.4e). e longest
distance was 551.6m and could be observed between
the tree stump 79 and the mother tree M_08. e short-
est distance (excluding inbreeding) with 2.3m was cal-
culated between the father tree A_02 and the mother
tree M_07.
Overall, 9.6% of the father trees with pollination suc-
cess were located less than 20m from the parent tree,
and 27.6% were located between 21 and 100 m away.
us, it can be assumed that the nearest father trees
did not pollinate most of the seeds. However, 49.3% of
the father trees with pollination success were located
within 130m of the mother tree. A total of 33.3% of
pollination success was detected for the trees at more
than 200-m distance. For the distance between 301 and
400m and 401 to 500m, pollination success could only
be detected for 6.4% of the father trees each. For the
distance of 501 to 600m, the value decreased to only
1.3%.
Detailed spatial information was acquired for two
selected mother trees, i.e., mother tree M_04 and M_08.
For mother tree M_04 (vitality score 2), 25 seeds out of
73 were attributed to their fathers. is was the mother
tree with the highest number of seeds where the respec-
tive fathers could be identified. Fourteen father trees
Fig. 4 Results of the floodplain forest. a and b Identified a living adult trees and b father tree stumps (ts) with their vitality score with the number
of offspring per father tree. c Number of offspring summed allocated to the vitality score of the father trees (n) and (d) average number of offspring
summed allocated to the vitality score of the number of father trees (n) (vitality score: 0 — dark green; 1 — light green; 2 — yellow; 3 — orange;
4 — red; tree stumps — brown). e Distribution of distance classes derived from paternity analyses for pollen dispersal. The distance classes are in
relation to the position of the pollen donor (number of father trees grouped)
(See figure on next page.)

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Eisenetal. Annals of Forest Science (2023) 80:22
Fig. 4 (See legend on previous page.)

Page 12 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
sired 25 seeds (Table1). e mother tree was centrally
located at the study site in a clearing northeast of the
lake and was only obscured by other trees to the east
and south due to its location (Fig.5a). erefore, many
of the father trees investigated were within reach to the
mother tree. e findings indicated that this mother
tree had the highest pollination success (six offspring)
with the tree stumps 64 and 68. Both father trees were
located on the southwest side of the lake and were
not in close proximity, with the distances of 179.9 and
196.8m, respectively. In contrast, the father ash trees,
which were not more than 100m away from the mother
tree, could only be associated with one offspring. In
other words, the nearest fathers were not the most suc-
cessful fathers.
Compared to M_04, the mother tree M_08 (vitality score
3) had less offspring assigned to their fathers. Seven father
trees could be detected for the eight offspring. e mother
tree is located at the northern edge of the study area; there-
fore, all identified fathers were positioned in the south-
west of the mother tree (Fig.5b). e closest father (A_06)
with the pollination success of two offspring was located
40.6 m away from the mother tree. All other more dis-
tant father trees had only one offspring. e parent trees,
tree stump 61 and tree stump 79, were located the farthest
from the mother tree, at a distance of 374.3 and 551.6m,
respectively.
3.3 Comparative summary ofthetwo study sites
The comparison of the results obtained from the seed
orchard and the floodplain forest showed that the pro-
portion of all investigated healthy ash trees (vitality
scores 0 and 1) differed, with the orchard having 49%
healthy trees (Fig.6a) and the floodplain forest having
only 15% (Fig.6c) or 38% if excluding the tree stumps
(Fig.6e). The results were similar in terms of the pro-
portion of ash trees that could be identified as (poten-
tial) fathers (Fig.6b, d). In this case, the proportion
of healthy ash trees was 50% in the seed orchard and
17% in the floodplain forest. However, considering the
number of offspring, the analyses showed that at both
sites, healthy fathers (vitality scores 0 and 1) as well
as those not severely affected by ash dieback (vitality
score 2) had almost the same mating success. In con-
trast, highly diseased fathers (vitality scores 3 and 4),
whose proportion in the seed orchard was 14% and in
the floodplain forest 30% (excluding the tree stumps;
Fig.6f), could only be assigned to a few offspring. Nev-
ertheless, effective gene flow also takes place between
highly susceptible trees.
Although the seed orchard offers the potential to
capture most potential father trees, the proportion
of offspring whose father could be identified was not
noticeably lower in the floodplain forest either (orchard:
33.5%; floodplain forest: 26.1%). Our results on genetic
connectivity showed that the average distance between
identified father and mother trees was 76m in the seed
plantation and 166m in the floodplain forest. Pollina-
tion success generally decreased substantially with
increasing distance to the mother tree. Despite the
dense tree coverage in the floodplain forest, pollen were
transported over long distances (greater than 550 m).
us, it indicates that local sources were not the only
ones playing a role in the pollination. is is supported
by the 66.5% of foreign pollen input found in the seed
orchard. Nevertheless, we were able to identify the
differences in pollen dispersal patterns, especially in
the seed orchard. For instance, for mother trees at the
border of the plantation (1627_01, 1878_13, 1898_14,
1627_21, 1866_30, 1663_94), only an average of 22%
of the offspring could be assigned to their potential
fathers, whereas the proportion for mother trees close
to the center (1663_24, 1619_31, 1879_37, 0000_62,
1664_71, 1619_81) was 44%. A similar pattern was also
found in the floodplain forest, though it was not as pro-
nounced (near the center: 30%; at the edge: 21%).
4 Discussion
4.1 Relationship betweenreproductive success andhealth
status
Overall, our results showed that the ash trees of vital-
ity score 2 produced the most offspring on average,
both in the seed orchard and in the floodplain forest
(excluding tree stumps). But, taking into account the
number of individuals, healthy trees with no or little
damage (vitality scores 0 and 1) were not associated
with substantially lower pollination success. However,
it should be noted that in the case of the Schorndorf
seed orchard, only the average vitality score class of
the clones could be considered for our assessments
in the paternity analyses. Due to the identical geno-
type of the ramets per clone, the software cannot
detect a distinct father tree. As the ramets per clone
can have varying vitality scores, the average vital-
ity score has been used. Therefore, it is possible that
healthy ash trees will produce more offspring than
diseased trees in a clone or vice versa. In the flood-
plain forest, some of the father trees were only pre-
sent as tree stumps due to the tree removal before
sampling. Therefore, we cannot accurately estimate
their vitality score but assume that they were dam-
aged by the fungus to some extent (vitality score > 2).
Thus, based on the results, both healthy and relatively

Page 13 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
Fig. 5 Mother tree a M_004 and mother tree b M_008 with their identified father trees and their number of offspring in the floodplain forest.
Source of the maps: ESRI Data & Maps

Page 14 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
healthy fathers have high pollination success. How-
ever, the effect observed by Gassner etal. (2019) that
the highest ash pollen emissions occurred 1 to 2years
after the first symptoms of ash dieback noticed in a
region was very weakly perceived in our study. Simi-
lar effects of excessive flowering with increasing tree
damage were also noted in the context of the forest
dieback in the 1980s (Gassner et al. 2019). Thus, it
Fig. 6 Vitality of the ash trees in the study sites in 2018. Vitality of (a all investigated ash trees, which could be a pollen donor versus b all assigned
potential fathers in the seed orchard and c all investigated ash trees, which could be a pollen donor versus d all assigned fathers in the floodplain
forest (vitality score: 0 — dark green; 1 — light green; 2 — yellow; 3 — orange; 4 — red; tree stumps with unknown vitality — brown)

Page 15 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
appears that Hymenoscyphus fraxineus infection could
at least temporarily increase pollen emission. This
can be explained by the fact that stress symptoms in
affected trees could lead to increased flowering (Wada
and Takeno 2010), which can be regarded as a com-
promise between reproduction and defensive reaction
of the tree (Denancé etal. 2013). Since disease resist-
ance can be partially inherited from parents to off-
spring (Kjær etal. 2012; Lobo etal. 2015), increased
flowering of damaged trees may have a negative effect
on the natural regeneration. Thus, Kjær etal. (2012)
suggested that only about 1% of the ash trees have
the potential to produce offspring with an expected
crown damage of less than 10% under the current
disease pressure. Since our study revealed low polli-
nation success for the severely damaged father trees
and we observed a poor flower development, specifi-
cally on severely damaged ash trees in previous stud-
ies (Eisen etal. 2022a), it can be assumed that severe
damage is associated with decreased pollen produc-
tion. Nevertheless, even severely damaged male ash
trees were capable of producing offspring, which is in
line with the findings of Semizer-Cuming etal. (2019;
2021). On the other hand, crown damage caused by
ash dieback reduces individual reproductive success
(Semizer-Cuming et al. 2021) and thus has a posi-
tive effect on natural selection. Therefore, next gen-
erations will probably develop a higher resistance to
ash dieback. This emphasizes the importance of not
removing healthy ash trees during thinning as well as
of enriching the gene pool of existing forests through
targeted planting of less susceptible ash trees.
Additionally, self-pollination does not seem to have
a major effect on reproduction in ash. According to our
results, only in a very small proportion of the offspring,
self-pollination could be detected with high probability
(one offspring in the floodplain forest). Moreover, the
study by Saumitou-Laprade et al. (2018) showed that
Fraxinus excelsior L. might be self-incompatible, prevent-
ing loss of genetic diversity, which is critical for the future
of ash, especially in the light of ash dieback (Fussi 2020).
Concerning the mother trees, in the seed orchard, the
majority of the offspring (52.6%) whose fathers could be
identified were assigned to mothers of vitality class 1,
and in the floodplain forest, the proportion was 56.3%.
Since the total number of seeds produced per tree was
not investigated at this point, it is not possible to make
a statement about the relationship between health status
and reproductive success of the mother trees. However, a
couple of studies previously reported that female ash trees
severely affected by ash dieback produced much lower
amounts of seeds compared to healthy ones (Semizer-
Cuming etal. 2019, 2021). Overall, our research indicates
a relationship between reproductive success and health,
with healthy and only slightly damaged male ash trees
(vitality score ≤ 2) contributing more to pollination.
4.2 Estimation ofpollen transport distances
Our results showed that ash trees in Schorndorf had the
highest pollination success rate (70%) within a radius of
100m. In the floodplain forest, over 50% of pollination
success occurred within 140m, whereas only about 8%
of father trees had pollination success over distances of
more than 400m. e longest detected distance of polli-
nation in the floodplain forest was more than 550m, cor-
responding nearly to the maximum extent of the study
area. us, our findings are very consistent with previ-
ous research; the study by Heuertz etal. (2003), which
revealed that effective pollen transport occurred at dis-
tances ranging from 70 to 140m, fits well with our results
from Schorndorf. e highest amount of successful polli-
nation occurred at a distance of 76m between father and
mother trees in the seed orchard. In contrast, Semizer-
Cuming etal. (2021) reported that 50% of effective pol-
len transport occurs within 140m. is observation was
reflected in the results of efficient pollen transport in the
floodplain forest. In both cases, a sub-area of a large con-
tiguous mixed forest was investigated, where ash occurs
with other forest tree species.
Despite the fact that 2018 was a masting year at the
Schorndorf seed orchard (Eisen et al. 2022a), a 66.5%
foreign pollen input was detected. Seed orchards are
collections of breeding populations that have been care-
fully selected to produce high-quality reproductive
material (Fussi etal. 2014). erefore, pollen input from
ash trees from outside the orchard can affect seed qual-
ity. A seed orchard should be distinguished above all by
the fact that the selected plus trees within the orchard
mutually fertilize each other, with preferably no foreign
input. Although no ash trees were found in the immedi-
ate vicinity of the orchard, it is possible that ash pollen
from the further surrounding area contributed to fertili-
zation. is has already been demonstrated in previous
studies on effective pollen transport, which detected dis-
tances between 1.3 and 2.9km (Bacles and Ennos 2008;
Semizer-Cuming et al. 2021). In the 300-ha landscape
heavily deforested described by Bacles and Ennos (2008),
pollen immigration represented also between 43 and 68%
of effective pollination (depending on the assignment
method). Furthermore, Semizer-Cuming et al. (2017)
found that 26–45% of pollen were transported from out-
side when they studied the genetic connectivity of ash
trees in an isolated forest site in a fragmented landscape
(2ha). e cause of not local or even long-distance pol-
len transport could be wind related, which, as already
demonstrated in other studies, is the primary factor

Page 16 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
responsible for the dispersal of pollen (Laaidi 2001; Eisen
et al. 2022a). According to Puc (2012), larger effective
pollen transport distances are linked to stronger winds.
Semizer-Cuming etal. (2017) found a positive correlation
between prevailing wind direction and the mean direc-
tion of pollen dispersal in ash. In addition, factors such
as tree height and canopy width also influence pollen
dispersal (Adams-Groom et al. 2017). us, pollination
within the orchard may have suffered from many dead
trees due to ash dieback. us, spacing between trees
increased, possibly leading to an increasingly mixed pol-
len cloud and higher pollen income from outside to pol-
linate the flowers. is also corresponds well with our
pollen dispersal patterns: In the case of mother trees in
marginal positions, a greater pollen input from outside
was observed, especially in the orchard. erefore, seeds
should be harvested preferably from central trees hav-
ing a higher probability that both parents originate from
plus trees within the orchards. In Germany, the Forest
Reproduction Act (FOVG 2002; BGBl. I.S. 1658) recom-
mends that seed orchards maintain a distance of 400m
from phenotypically weak stands of the same species or
a species that can be crossed with it (gGA 2019). us,
one measure to increase pollination within the orchard
is to ensure that the distance between foreign ash trees
and the seed orchard is substantially greater than 400m.
In addition, protecting seed orchards primarily with
conifers such as spruce planted at the borders may aid
in reducing pollen input from the outside and avoiding
cross-pollination. Deciduous trees or shrubs might also
reduce pollen input but are more permeable because leaf
formation often begins after flowering of ash or are low
in height (in case of shrubs). Another fact is the synchro-
nization of flowering time between clones in the seed
orchard. Flowering of male and female flowers has to
occur approximately at the same time in order to ferti-
lize successfully. When selecting plus trees from different
regions, the flowering time of the plus trees combined in
the seed orchard may differ from each other. If there is
only little overlap between the flowering time of male and
female flowers, pollen from outside may be more fertile
at certain times (Mondal etal. 2019). erefore, we rec-
ommend that a large orchard isestablished with as many
trees as possible. is increases the likelihood that the
trees flower synchronously and thus ensures that most
pollination will occur within the seed orchard.
In the floodplain forest, only 26.1% of the seeds
could be assigned to their fathers, implying that pol-
len are transported over distances greater than 550m.
However, in the approaches based on paternity analy-
ses, it is often difficult or infeasible to sample all trees
in a forest that represent a potential pollen source for
reproduction (Bacles and Ennos 2008). Hence, it must
be noted that a higher number of parent trees would
possibly also shift the mean values, maxima and min-
ima. However, as already discussed, since healthy
and only slightly damaged male ash trees (vitality
score ≤ 2) contribute more to pollination, there is
a chance for gene flow between somewhat isolated
individuals or groups of healthy ash trees. Increas-
ingly fragmented ash populations that are expected
in the future, as well as already isolated ash trees
that are less susceptible to H. fraxineus, may still
be in genetic exchange with one another, increasing
the chance of a healthier next generation. Individu-
als of the natural regeneration are the result of natu-
ral selection and might be able to withstand inter- or
intraspecific competition and could therefore be dis-
ease resistant (Metzler etal. 2012). Thus, in the long
term, those genotypes that can better withstand the
negative effects of the fungus will prevail and have
the highest fitness under the prevalent environmen-
tal conditions (Fussi etal. 2014). However, Buchner
etal. (2022) found that the environmental conditions
prevailing during long-distance pollen transport play
an important role in the successful pollination of
female ash flowers. It was demonstrated that pollen
viability decreased faster with increased or prolonged
UV radiation and under warmer conditions. Instead,
viable pollen could still be observed after 28 days
at moderate temperatures. Thus, prolonged heat
extremes caused by climate change can have serious
consequences for the pollen’s viability during long-
distance pollen transport. Nevertheless, it has been
shown that gene flow can occur over longer distances,
and successful pollination is possible.
5 Conclusion
In summary, it can be concluded that effective pol-
len transport occurs over long distances, although with
decreasing pollination success. ere is still hope for the
future of ash since healthy and slightly diseased fathers
make a greater contribution to pollination, meaning that
gene flow will facilitate the healthier next generation of
ash in the future. However, in order to reduce pollen
input from outside and avoid cross-pollination on the
seed plantations, it is important to establish large seed
orchards protected by planting around the border. In
addition, female trees should be planted mainly in the
center of the orchard to enhance short-distance pollina-
tion within the orchard. We recommend harvesting seeds
only during masting years due to intensified pollination
between plus trees in the orchard.

Page 17 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
Appendix
Fig. 7 Seed orchard including investigated ash trees, labeled by tree ID: yellow stars – mother trees, blue dots—ash trees identified as potential father
trees by their clone number; gray dots –investigated ash trees excluded as pollen donors. Source of the map: ESRI Data & Maps
Fig. 8 Study area of floodplain forest including investigated ash trees, labeled by tree ID: yellow stars — mother trees, blue dots — ash trees identified
as father trees, and orange squares — logged ash trees identified as father trees. Source of the map: ESRI Data & Maps

Page 18 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
Table 2 Ash trees of the seed orchard near Schorndorf
Gender assignments (m, male; f, female; h, hermaphrodite), the presence
of owers on the 109th day of the year (+ or −), and vitality classications
according to Lenz etal. (2012) (categories 0 and 1, healthy; categories 3–4,
diseased; category 5, dead). Color codes are as follows: pink indicates the
mother trees, blue shows the potential fathers assigned to ospring, green
denotes the potential fathers unassigned to any ospring, and white shows all
other ash trees excluded as potential fathers
Table 3 Ash trees of the floodplain forest
Gender assignments (m, male; f, female; h, hermaphrodite), the presence
of owers on the 109th day of the year (+ or −), and vitality classications
according to Lenz etal. (2012) (categories 0 and 1, healthy; categories 3–4,
diseased; category 5, dead). Color codes are as follows: pink indicates the
mother trees, blue shows the potential fathers assigned to ospring, green
denotes the potential fathers unassigned to any ospring, and white shows all
other ash trees excluded as potential fathers

Page 19 of 21
Eisenetal. Annals of Forest Science (2023) 80:22
Table 4 Specifications of the fifteen nuclear microsatellite markers used in the study. Markers with no reproducible results are shown
in Italics
Acknowledgements
We gratefully acknowledge the Forst Baden‑Württemberg and Forstliche
Versuchs‑und Forschungsanstalt Baden‑Württemberg for providing the seed
orchard as study site. In addition, we thank Wittelsbacher Ausgleichsfonds for
the permission to conduct scientific research in the riparian forest. We thank
Tristan Marshall, Johanna Jetschni, Johanna Weidendorfer, and Lisa Thamm for
technical assistance.
Authors’ contributions
Conceptualization, BF and SJO; methodology, BF, DSC, SJO, and AKE; formal
analysis and investigation, AKE; data curation, AKE; visualization, AKE; writing
— original draft preparation, AKE; writing — review and editing, AKE, SJO, BF,
and DSC; funding acquisition, SJO and BF; and supervision, SJO. The authors
read and approved the final manuscript.
Funding
This research was funded by the Bavarian State Ministry of Food, Agriculture,
and Forestry through the Bavarian State Institute for Forests and Forestry
(LWF) as part of the project “P035–Quo vadis Pollen? Untersuchungen zur
(effektiven) Pollenausbreitung und Pollen‑ und Samenqualität als Beitrag zur
Generhaltung bei der Esche.” Open Access funding enabled and organized by
Projekt DEAL.
Availability of data and materials
The datasets generated during and/or analyzed during the current study are
available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
All authors gave their informed consent to this publication and its content.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Physical Geography/Landscape Ecology and Sustainable Ecosystem Develop‑
ment, Catholic University of Eichstätt‑Ingolstadt, 85072 Eichstätt, Germany.
2 Forest Research Institute of Baden‑Württemberg (FVA), 79100 Freiburg, Ger‑
many. 3 Bavarian Office for Forest Genetics (AWG), 83317 Teisendorf, Germany.
Received: 11 November 2022 Accepted: 10 May 2023
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