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Int. J. Environ. Res. Public Health 2022, 19, 8160. https://doi.org/10.3390/ijerph19138160 www.mdpi.com/journal/ijerph
Article
Is Pollen Production of Birch Controlled by Genetics and
Local Conditions?
Surendra Ranpal
1,
*, Miriam Sieverts
1
, Verena Wörl
1
, Georgia Kahlenberg
1
, Stefanie Gilles
2
, Maria Landgraf
3
,
Kira Köpke
3
, Franziska Kolek
2
, Daria Luschkova
2
, Tobias Heckmann
4
, Claudia Traidl-Hoffmann
2
,
Carmen Büttner
3
, Athanasios Damialis
2,5
and Susanne Jochner-Oette
1
1
Physical Geography/Landscape Ecology and Sustainable Ecosystem Development, Catholic University of
Eichstätt-Ingolstadt, 85072 Eichstätt, Germany; miriam-sieverts@web.de (M.S.);
verena.woerl@gmx.de (V.W.); gkahlenberg@ku.de (G.K.); susanne.jochner@ku.de (S.J.-O.)
2
Environmental Medicine, Faculty of Medicine, University of Augsburg, 86156 Augsburg, Germany;
stefanie.gilles@tum.de (S.G.); franziska.kolek@tum.de (F.K.); daria.luschkova@tum.de (D.L.);
claudia.traidl-hoffmann@tum.de (C.T.-H.); dthanos@bio.auth.gr (A.D.)
3
Division Phytomedicine, Albrecht Daniel Thaer-Institute for Crop and Animal Sciences,
Humboldt-Universität zu Berlin, 10099 Berlin, Germany; maria.landgraf@agrar.hu-berlin.de (M.L.);
kira.koepke@agrar.hu-berlin.de (K.K.); carmen.buettner@agrar.hu-berlin.de (C.B.)
4
Department of Physical Geography, Catholic University of Eichstätt-Ingolstadt, 85072 Eichstätt, Germany;
tobias.heckmann@ku.de
5
Department of Ecology, School of Biology, Faculty of Sciences, Aristotle University of Thessaloniki,
GR-54125 Thessaloniki, Greece
* Correspondence: surendra.ranpal@ku.de; Tel.: +49-8421-93-23074
Abstract: Intraspecific genetic variation might limit the relevance of environmental factors on plant
traits. For example, the interaction between genetics and (a-)biotic factors regulating pollen produc-
tion are still poorly understood. In this study, we investigated pollen production of 28 birch (Betula
pendula Roth) individuals in the years 2019–2021. We sampled catkins of eleven groups of genet-
ically identical trees, which were partially topped, but of the same age and located in a seed planta-
tion in southern Germany characterized by similar microclimatic conditions. Furthermore, we mon-
itored environmental factors such as air temperature, characterized air quality (NO
2
, NO
x
and O
3
),
and assessed potential solar radiation. We especially checked for differences between years as well
as between and within clones and assessed the synchronicity of years with high/low pollen produc-
tion. We present a robust mean for the pollen production of Betula pendula (1.66 million pollen grains
per catkin). Our findings show temporal (H(2) = 46.29, p < 0.001) and clonal variations (H(4) = 21.44,
p < 0.001) in pollen production. We conclude that synchronized high or low pollen production is
not utterly site-specific and, in addition, not strictly dependent on genotypes. We suggest that ap-
propriate clone selection based on application (seed plantation, urban planting) might be advanta-
geous and encourage a long-term monitoring.
Keywords: Betula pendula; genotypes; reproduction; seed orchard
1. Introduction
Genetic variation among plant species is believed to limit the explanatory power of
abiotic or biotic influential factors on certain plant traits. Several studies have revealed
that plant traits, e.g., related to phenology, morphology, physiology, reproduction, and
distribution are associated with genetic controls. Neophytou et al. [1] found a significant
variation in the timing of bud burst among different Douglas fir progenies. Likewise, pre-
vious studies on poplar hybrids have reported that the patterns of tree biomass distribu-
tion above- and below-ground were genetically controlled [2,3]. Furthermore, naturally
regenerated birch and aspen populations showed a variation between genotypes in the
Citation: Ranpal, S.; Sieverts, M.;
Wörl, V.; Kahlenberg, G.; Gilles, S.;
Landgraf, M.; Köpke, K.; Kolek, F.;
Luschkova, D.; Heckmann, T.; et al.
Is Pollen Production of Birch
Controlled by Genetics and Local
Conditions? Int. J. Environ. Res.
Public Health 2022, 19, 8160. https://
doi.org/10.3390/ijerph19138160
Academic Editor: Paul B.
Tchounwou
Received: 15 June 2022
Accepted: 1 July 2022
Published: 3 July 2022
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and institu-
tional affiliations.
Copyright: © 2022 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://cre-
ativecommons.org/licenses/by/4.0/).
Int. J. Environ. Res. Public Health 2022, 19, 8160 2 of 19
acclimatization to soil moisture conditions by altering biomass, root and leaf morphology,
water potential, and gas exchange [4]. Rousi et al. [5] documented significant variations
in intraspecific reproduction efficiency (anther residuals and seed production) among in-
dividuals of B. pubescens in two neighboring stands in Northern Finland. In addition, in-
formation on intraspecific genetic variations plays a crucial role to improve species distri-
bution models [6]. Under varying environmental conditions, an exposed genotype has the
ability to express phenotypic plasticity [7]. Studies on birch revealed phenotypic plasticity
in leaf morphology of transplanted trees related to edaphic conditions [8] and larger phe-
notypic plasticity of juvenile above-ground growth traits in response to soil nutrient con-
ditions [9]. Such findings indicate that traits of plant individuals of the same species grow-
ing under similar or different environmental conditions must be understood with the con-
sideration of intraspecific variations.
Pollen are developed in anthers (angiosperms) or in microsporangia (gymnosperms)
and their quantity per inflorescence is regarded as pollen production [10]. Pollen produc-
tion may be controlled by the genes of taxa, species or varieties. It was suggested that the
amount of pollen grains produced per anther and the number of anthers per flowers are
genetically fixed and does not vary substantially [11–13]. In addition, any further varia-
tions could be related to changes in environmental conditions [12] such as meteorology,
primarily air temperature [14–16], and edaphic factors [15,17], which alter the number of
flowers and/or pollen production per flower. However, the role of these and other varia-
bles influencing pollen production are poorly known.
Most studies on pollen production of woody plants are limited to genera or species.
Yet, a small number of studies have focused on the intraspecific level, for example, related
to Cupressus sempervirens varieties [18] or Theobroma cacao clones [19]. Although Adams
and Kunze [20] studied clonal variations in seed production in spruce, there has been little
discussion on pollen production of genetically identical trees.
In general, genetically identical trees are preferentially used for various applications
in science because it is assumed that they show the same behavior, e.g., related to phenol-
ogy [21,22]. Long-term phenological observation networks such as the International Phe-
nological Gardens in Europe (IPG) standardized phenological studies by establishing gar-
dens with cloned plant individuals to exclude genetic effects [21,23]. Such phenological
investigations based on cloned tree species assure that observed variances are due to en-
vironmental causes rather than genotypic differences between plants [24]. There have
been attempts to explore the influences or exclusion of genetics on other pollen properties
such as allergenicity. Ahlholm et al. [25] investigated the allergenicity of mountain birch
pollen collected from trees of ten half-sib families growing in northern Finland and found
that the concentration of the major birch pollen allergen (Bet v 1) is genetically controlled.
In addition, concentrations of the allergen Cry j 1 produced by pollen of Japanese cedar
were reported to be significantly different between trees of eight clones [26]. Similarly,
Fernández-Caldas et al. [27] demonstrated considerable variations in pollen allergenicity
(Ole e 1) of different varieties of Olea europaea.
However, studies related to pollen production compared for different clones in birch
are lacking and are in general very sparse related to other species of the plant kingdom.
Veilleux and Lauer [28] studied potato (Solarium phurejas) clones and suggested that
plants of the same genotype respond similarly to the environment and produce the same
amount of unreduced pollen grains. Panda et al. [29] observed a wide variation in pollen
production per anther, pollen size and pollen viability among selected banana (Musa spp.)
genotypes. Information on the variability of pollen production of genetically identical
wind-pollinated plants is, however, largely lacking.
Detailed knowledge on the pollen production of a species is crucial for improving
pollen forecasting [30]. Such forecasts have agronomical importance as seed production
and, therefore, harvest outcomes often rely on pollen production [31]. Pollen production
also plays a vital role in allergology. In the past few years, phenological, biometeorologi-
cal, and aerobiological studies on allergenic plants have become more important due to
Int. J. Environ. Res. Public Health 2022, 19, 8160 3 of 19
the high prevalence of allergies around the world. According to the World Allergy Or-
ganization (WAO) up to 40% of the global population suffers from allergic sensitization
[32], which could further increase by a parallel increase in pollen production [33–36].
Birch has a wide range of distribution in the Northern Hemisphere [37] and its pollen
are highly allergenic [38,39] presenting a major source of allergic rhinitis in Europe [40].
Due to its aesthetic value, silver birch is a frequently used tree species in urban green space
planning in Europe [41,42]. The abundance of birches, however, is problematic for many
people who are allergic to pollen [43]. Studies on genotypic variations of pollen produc-
tion of such allergenic tree species could identify clones, which are characterized by a
lower pollen production. The breeding of such clones, e.g., for planting in urban green
spaces, might also imply a reduction of atmospheric pollen concentration. On the other
hand, seed plantations, in which a high pollen production of trees is desirable for a high
quantity of seeds, may profit from those clones that are associated with a higher produc-
tion of pollen. Most important, knowledge on the genetic variability of pollen production
will allow for better evaluating the influence of environmental factors/climate change.
In this study, we assessed the pollen production of eleven groups of cloned weeping
birch (Betula pendula Roth) individuals (n = 28) in three consecutive years (2019–2021).
Since natural birch populations show a high grade of hybridization [44], we sampled in-
florescences of genetically identical trees of the same age from a seed plantation (Baden-
Württemberg, Germany), assessed the ambient microclimatic conditions and monitored
any silvicultural treatments. We especially checked for differences between years as well
as between and within clones and considered their synchronicity of pollen production
levels. Based on the results, we discussed the implications of selecting clones producing a
high/low level of pollen for seed plantations/urban planting.
2. Materials and Methods
For this study, we selected a birch seed plantation located near Wildberg (48°36′44″
N, 8°42′37″ E, 500 m a.s.l.) in Baden Württemberg, Germany (Figure 1). The average an-
nual temperature is 8.6 °C and the precipitation sum is 892 mm (German Meteorological
Service (DWD) station “Neubulach-Oberhaugstett”, 1991–2020 [45]). The plantation is lo-
cated on a west-exposed slope with an inclination of approximately 2°−6° and the soil type
is Cambisol [46]. This 1-hectare sized plantation was established in 2005 and additional
birch trees were planted in 2012, resulting in a 7 m × 7 m seedling cluster, which is man-
aged by Forst Baden-Württemberg (Forst BW; territory number 3, Nagoldtal). Initially,
215 trees belonging to 44 different clones were planted in a total of 13 rows and 17 col-
umns. The clones and trees were randomized spatially throughout the site. Until now,
almost half of the birch trees were removed as a thinning measure: 113 birch trees from 44
clones (with one to six individuals) are still present in the plantation.
Int. J. Environ. Res. Public Health 2022, 19, 8160 4 of 19
Figure 1. Study areas: (a) seed plantation near Wildberg (48°36′44″ N, 8°42′37″ E, 500 m a.s.l.) in-
cluding measurement sites: blue triangles—topped sampled trees; yellow circles—non-topped sam-
pled trees; red squares with black border—air temperature loggers; red squares with white border—
passive samplers, (b) Baden-Württemberg in Germany (red fill) and (c) location in Baden-Württem-
berg (black circle).
The study was conducted in three successive years (2019–2021). We focussed on 28
trees, all planted in 2005, representing eleven clones from six different geographic origins
(Table 1). These clone origins are, however, located nearby, within approximately 45 km
to 130 km from the study site. The trees were selected based on the reachability of twigs
and, therefore, inflorescences. The number of studied trees per clone, therefore, varied
between one to four.
Table 1. Studied clones and their geographic origins.
Clone
Number
Number of
Individuals
Per Clone
Origin Latitude (N) Longitude (E)
55–04 1
Lahr 48°21′ 7°52′
55–07 4
55–10 4
55–46 2
55–21 4 Nürtingen 48°37′ 9°20′
55–24 2 Karlsruhe 49°00′ 8°24′
55–30 2 Kehl 48°35′ 7°51′
55–33 1
55–38 3 Kandern 47°43′ 7°39′
55–42 3
55–47 2 Villingen-Schwenningen 48°04′ 8°24′
Male catkins were harvested in March after the beginning of catkin elongation and
prior to anthesis. Samples were collected from different branches at 1.5 to 2 m above
ground from all cardinal directions. In addition, we measured growth traits: (a) the pe-
rimeter at breast height, (b) the height of the tree and crown by use of Suunto PM-
5/1520PC Height Meter, and (c) the crown diameter, which was calculated by averaging
two perpendicular diameters of the crown at its widest portion.
Int. J. Environ. Res. Public Health 2022, 19, 8160 5 of 19
We counted the number of catkins within a sampling cuboid (50 cm × 50 cm × 50 cm)
in the crown, which was considered to characterize the average distribution of catkins in
the tree [18]. We selected an ovoid shape of the crown to estimate pollen production per
tree.
In July 2018, tree topping (cutting of the apical parts of the main trunk), which is an
intended measure to increase seed production [47], was carried out in the seed plantation.
Therefore, the sampled trees were categorized as topped (n = 12) and non-topped (n = 16).
Six more sampled trees were topped in July 2020; however, male catkins were already
formed in those trees and, therefore, no large effect on pollen production was assumed.
Air temperature and precipitation data were obtained from a 5 km distant DWD cli-
mate station “Neubulach-Oberhaugstett” [45]. In addition, we installed five temperature
loggers (HOBO Pro v2 U23-001, Onset, Bourne, MA, USA) from spring 2019 (8 April) until
summer 2021 (20 June). One logger was installed in the center and four at the northern,
eastern, southern, and western borders of the plantation (red squares with black border
in Figure 1) to determine temperature differences within the site. Each logger was placed
in a radiation shield and mounted at a height of 2 m at the northern side of a birch tree.
The loggers’ data were retrieved and processed using HOBOware (Version 3.7.23) from
Onset, Bourne, MA, USA.
The air quality of the study site was characterized by the measured values of nitrogen
dioxide (NO2), nitrogen oxides (NOx) and ozone (O3) concentrations monitored directly at
the stem of the birch trees (n = 2, red squares with white border in Figure 1). Passive sam-
pling of these pollutants lasted one week in summer 2020 (25 June to 2 July). The passive
samplers were supplied and evaluated by Passam AG (Männedorf, Switzerland).
For estimating the potential solar radiation around each tree, the unmanned aerial
vehicle Phantom 4 Pro, DJI, Nanshan, Shenzhen, China was used, which features an
onboard RGB camera with a sensor resolution of 12 megapixels and a focal length of 24
mm. The flight altitude was 35 m above ground level. During the flight, which took place
on 10 August 2019 and lasted approximately 17 min, 712 photos were taken with an over-
lap of 80%. A digital elevation model was generated using Metashape Professional (Ver-
sion 1.8.1) from Agisoft LLC, St. Petersburg, Russia. In ArcGIS Pro (version 2.7.0) software
from ESRI, Redlands, CA, USA, the spatial analyst tool “Solar radiation (area)” was used
to calculate the potential solar radiation (W/m2) on the surface depending on the time of
day and position of the sun as well as the latitude for each pixel of the digital elevation
model. We calculated solar radiation for each pixel as a sum for the period 1 May until 31
August as this period is critical for the start and development of the following year’s cat-
kin [48]. We selected a buffer of two meters around each tree and calculated the mean
solar radiation. We assume only minor differences in the canopy of the surrounding forest
and, therefore, use the data gained in 2019 for a general site characteristic for the whole
study period.
To extract pollen grains, we adapted the method proposed by Damialis et al. [18]. For
each year, one average-sized inflorescence from each cardinal direction and per tree was
selected, its length and width were measured (at the widest point), and the number of
flowers was counted. Then, each catkin was soaked in a 10% KOH solution [31,49] and
boiled at 120 °C the following day. Afterwards, the plant material was crushed with a
glass rod to break up plant tissues and to allow pollen release. To prevent pollen clumping
[50], we added glycerol (70%), a bipolar solvent, to a volume of 20 mL; safranin was added
as a stain. Two aliquot samples (10 µL each) per suspension were obtained using a
VITLAB® micropipette while stirring it vigorously to ensure homogeneity. Subsequently,
the extraction was put on microscope slides and covered with slips. Pollen grains on these
slides were subsequently counted at 100× magnification (Zeiss AXIO Lab.A1, Germany).
In case of a large difference between the pollen counts obtained from these two slides
(>30%), the procedure was repeated in order to increase the homogeneity of the suspen-
sion.
Int. J. Environ. Res. Public Health 2022, 19, 8160 6 of 19
We estimated pollen production at various scales [18]: The number of pollen grains
per catkin (Pca) was calculated using Equation (1):
𝑃
=𝑉
𝑉
𝑝 (1)
where 𝑉
and 𝑉
are the volumes of the suspension (in mL) and the sample taken (in µL),
respectively, and p is the number of pollen grains counted per 10 µL solution.
The number of pollen grains per flower (𝑃
) was estimated as follows (Equation (2)):
𝑃
=𝑃
𝑓
𝑙 (2)
where fl is the number of flowers per catkin.
The number of pollen grains per volume unit (m3) of crown (𝑃
) was estimated using
Equation (3):
𝑃
=𝑃
𝐶
𝑀 (3)
where 𝐶 is the number of catkins per crown sampling unit (cuboid) and 𝑀 is the volume
of the sampling unit.
The number of pollen grains per individual (𝑃) was estimated using Equation (4):
𝑃 =𝑃
𝑉 (4)
where 𝑃
is the number of pollen grains per crown volume unit (see Equation (3)) and 𝑉
is the total volume (in m3) of the crown. The volume of an ovoid tree Crown can be calcu-
lated as follows (Equation (5)):
𝑉=𝜋𝑑𝑑ℎ
6 (5)
where 𝜋 ≈ 3.14, 𝑑 and 𝑑 are two perpendicular diameters of the crown, at its widest part,
and ℎ is the crown height.
Pollen production per flower, catkin, and volume unit of crown, as well as flowers
per catkin and catkins per crown sampling unit, were descriptively analyzed. These re-
productive metrics were non-normally distributed according to Shapiro–Wilk test. We
checked for differences among sampling years and clones using the Kruskal–Wallis test
and post-hoc (Dunn) test. Correlation analyses between the reproduction metrics and be-
tween solar radiation and pollen production were conducted using Spearman’s correla-
tion test. The differences between topped and non-topped trees were analyzed using
Mann–Whitney U test. The variation within non-topped clones was assessed by compar-
ing the coefficient of variances (CVs). For indicating if one specific clone can be proposed
as “good” or “poor” regarding pollen production, we averaged the crown metrics (crown
height and crown width) of all non-topped trees and calculated a mean crown volume.
We considered that this computed crown dimension would represent an average non-
topped birch tree in the seed plantation. Similarly, we calculated the mean Pca and mean
Csu obtained from the non-topped trees during the study years. These values allowed us
to quantify the total Pin for an average tree (using Equation (5)). Further, we used average
Pca and Csu of each clone along with the crown volume of an average tree to calculate the
pollen produced by each clone under mean growth parameters to compare the pollen pro-
duced by each clone to an average birch tree. All statistical analyses and visualizations
were performed in RStudio (version 4.1.2) from RStudio, PBC, Boston, MA, USA, ArcGIS
Pro (version 2.7.0) or Microsoft Excel 2016 from Microsoft, Washington, DC, USA.
Int. J. Environ. Res. Public Health 2022, 19, 8160 7 of 19
3. Results
3.1. Descriptive Statistics and Correlation Analyses among Reproductive Metrics
The average pollen production per catkin (P
ca
) for all selected 28 trees and all study
years (2019–2021) was 1.66 ± 1.28 million pollen grains (see Table 2). P
ca
varied within a
wide range from 48,000 to 8.27 million pollen grains, especially in the year 2019. P
ca
in 2020
was 11% higher and 28% lower compared to 2019 and 2021 when regarding mean values.
P
ca
in 2021 was 54% higher compared to 2019.
Table 2. Descriptive statistics of pollen production per catkin (P
ca
) and catkins per crown sampling
unit (C
su
; 0.125 m
3
) (minimum, maximum, mean, median and standard deviation) estimated from
28 selected birch trees of the seed plantation Wildberg during 2019–2021.
Year Minimum Maximum Mean Median Standard Deviation
Pollen production per catkin (P
ca
)
2019 48,000 8,270,000 1,359,049 983,500 1,245,134
2020 108,000 4,172,000 1,511,170 1,360,000 892,862
2021 184,000 8,018,000 2,090,888 1,796,000 1,495,281
2019–2021 48,000 8,270,000 1,658,846 1,356,000 1,277,605
Catkins per crown sampling unit (C
su
)
2019 10 45 23 22 8
2020 10 120 44 35 26
2021 3 60 19 20 12
2019–2021 3 120 29 23 20
The number of catkins in a crown sampling unit (C
su
; 0.125 m
3
) ranged between 3 and
120 with an average of 29 catkins. C
su
in 2020 were 191% and 232% higher compared to
2019 and 2021 and 17% lower in 2021 compared to 2019. Statistics for all analyzed levels
(P
ca
, P
fl
, P
cr
, fl and C
su
) are presented in the Appendix A (Table A1).
We detected a statistically significant difference between P
ca
among the three study
years (H (2) = 46.29, p < 0.001). A post-hoc test revealed that there were significant differ-
ences between all pairs of years (Figure 2). The same applied for C
su
(H (2) = 200.78, p <
0.001; boxplots not shown).
Figure 2. Boxplots based on pollen production per catkin (P
ca
) (eight replications per tree) estimated
for 28 trees in the seed plantation in Wildberg for 2019, 2020 and 2021. 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, points indicate outliers, lines above box-
plots indicate pair of years, which were significantly different (Kruskal–Wallis test and Dunn’s mul-
tiple comparison’s tests).
Int. J. Environ. Res. Public Health 2022, 19, 8160 8 of 19
Correlations between different reproductive metrics from all study years are shown
in Table 3. The highest Spearman’s correlation coefficient was found for Pfl and Pca (rs =
0.980, p < 0.001). fl was associated with a negative correlation with Pfl (rs = −0.230, p < 0.001)
and a positive correlation with Csu (rs = 0.200, p < 0.001). Pfl or Pca did not show any signif-
icant correlations with Csu.
Table 3. Spearman correlations between averaged reproduction metrics for all studied years and 28
birch individuals of the Wildberg seed plantation. rs: Spearman’s correlation coefficient, p: signifi-
cance.
Reproductive Metrics Pfl Pca fl
rs p rs p rs p
Pca 0.980 0.000
fl −0.230 0.000 −0.040 ns
Csu −0.060 ns −0.020 ns 0.200 0.000
The temporal variations of pollen production (Table 2, Figure 2) probably include
some abiotic and biotic influential factors, which are described below.
3.2. Meteorological Differences in the Study Years
Figure 3 shows the meteorological conditions at Neubulach-Oberhaugstett, near the
plantation site, for the period 2018–2020. In addition, we calculated averages for months
that are especially important for the initiation and formation of catkin of the following
year (i.e., May until August of the preceding summer; [48]). We estimated the lowest av-
erage Pca in 2019 (Table 2, Figure 2), which was following a relatively high temperature
(17.6 °C) and moderate precipitation (63.5 mm) during those specified four months in 2018
(compared to 2019 and 2020). Mean Pca was higher in 2020 and linked to a preceding pe-
riod with a moderate temperature (16.3 °C), but a high precipitation sum (77.1 mm) was
recorded during May–August 2019. The average numbers of pollen grains per catkin es-
timated in 2021 was the highest among all study years; the preceding period in 2020 was
associated with the lowest temperature mean (16.1 °C) and precipitation sum (54.7 mm)
compared to 2018 and 2019. The selected period of the year was on average warmer but
received less precipitation in all study years compared to 1991–2020 (15.7 °C; 82.3 mm).
Figure 3. Monthly average temperature (lines) and monthly precipitation sum (bars) for the years
2018 (blue), 2019 (orange) and 2020 (grey) recorded at a nearby weather station (DWD station Neu-
bulach-Oberhaugstett). x-axis: months, left y-axis: monthly mean temperature in °C, right y-axis:
monthly precipitation sum in mm. Mean values (1991–2020) are displayed as black dashed lines
(temperature) and crosses (precipitation).
0
20
40
60
80
100
120
140
160
180
200
-5
0
5
10
15
20
25
Monthly pricipitation sum [mm]
Monthly mean temperature [°C]
Int. J. Environ. Res. Public Health 2022, 19, 8160 9 of 19
Site-specific temperature data (8 April 2019 to 20 June 2021) at five different locations
within the plantation (see Figure 3) were found to be not significantly different according
to ANOVA tests (daily mean temperature: F (4, 4020) = 0.73, p = 0.570, monthly mean tem-
perature: F (4, 125) = 0.03, p = 0.990). In addition, air pollutants sampled at two sites (see
Figure 1) were almost identical: site 1—NO
2
< 6.5 µg/m
3
, NO
x
= 2.4 µg/m
3
and O
3
= 33.2
µg/m
3
; site 2—NO
2
< 6.5 µg/m
3
, NO
x
= 2.6 µg/m
3
and O
3
= 36.2 µg/m
3
.
Incoming shortwave radiation, expressed as the sum of radiation in the months May
to August in W/m
2
, varies within the seed plantation due to the surrounding forested area
and is generally lower in the southern part (Figure 4). However, we found no statistically
significant correlation between mean P
ca
(2019–2021) and solar radiation (r
s
= −0.111, p =
0.574) when regarding all 28 selected birch trees. For single years, we detected an alter-
nating (but still not significant) signal: in 2019 and 2020, the correlations were positive (r
s
= 0.201, p = 0.304 and r
s
= 0.076, p = 0.702, respectively) and in 2021 the correlation was
negative (r
s
=−0.149, p = 0.4489).
Figure 4. Solar radiation map and synchrony of pollen production levels of non-topped trees (n =
16). Circles—group 1 (n = 6, trees with maximum P
ca
in 2020); triangles—Group 2 (n = 4, trees with
P
ca
extraordinarily high in 2021) and stars—group 3 (n = 6, almost constant P
ca
values and/or mini-
mum values in 2020).
3.3. Tree Condition
The differences in pollen production between trees that were topped in 2018 and non-
topped trees were compared for 2020 and 2021 (Table 4). Pollen production in 2019 was
considered unaffected by tree topping since this intervention was carried out after the
formation of catkins.
Table 4. Reproductive metrics (mean and median) of topped (n = 12) and non-topped trees (n = 16)
in 2020 and 2021 and comparisons (Mann–Whitney U test) between them.
Reproductive Metrics Group 2020 2021
Mean Median p Mean Median p
P
ca
Topped 1,252,938 1,116,000 0.000 2,143,096 2,098,000 0.016
Non-topped 1,704,844 1,654,000 2,048,469 1,486,000
P
fl
Topped 10,564 9,617 0.000 19,271 17,362 0.039
Non-topped 15,935 14,742 19,266 13,309
P
cr
Topped 485,992,500 399,840,000 ns 405,397,846 306,200,000 0.000
Non-topped 541,242,500 384,200,000 238,281,125 169,840,000
fl Topped 122 117 0.000 112 112 0.030
Int. J. Environ. Res. Public Health 2022, 19, 8160 10 of 19
Non-topped 112 112 107 107
Csu Topped 53 45 0.009 23 20
0.002
Non-topped 38 35 16 17
Mann–Whitney U tests revealed that there were significant differences between
topped compared to non-topped trees. The first year with potential effects of tree topping
(2020) was associated with a significantly lower pollen production and a significantly
higher flower and catkin formation compared to non-topped trees. For example, Pca was
27% lower, Pfl was 34% lower, fl were 9% higher and Csu were 40% higher for these dam-
aged trees. The effect of tree topping was most obvious in 2021 since all metrics were as-
sociated with significantly higher mean values. For example, Pca was 5% higher, Pcr was
70% higher, fl were 5% higher and Csu were 44% higher for topped compared to non-
topped trees. In 2021, the effect on Pcr was most pronounced, especially when bearing in
mind that this last study year presented a year with poor catkin formation (see Table 2).
3.4. Synchrony of Pollen Production Levels
Due to the effects of topping, the assessment of synchrony in pollen production levels
was carried out for all non-topped trees (n = 16) for which the temporal development was
evaluated and classified into three different groups (Figure 5). The classification was per-
formed visually according to the maximum in pollen production and the variation among
years.
Figure 5. Pollen production per catkin (Pca) (y-axis) in 2019–2021 assessed for the selected non-
topped trees at the seed planation Wildberg and categorized in three groups with similar temporal
behavior. The color of the lines symbolizes trees of the same clone.
Group 1 includes the trees with maximum Pca in 2020 (n = 6). Group 2 consists of trees
whose Pca was extraordinarily high in 2021 (n = 4). Group 3 has almost constant Pca values
and/or minimum values in 2020 (n = 6). Only one clone (clone number 21) with three rep-
lications was always categorized to the same group (group 1). The trees of all other clones
were distributed in more than one group.
These three different groups are highlighted in Figure 4 by different symbols. A one-
way ANOVA did not reveal significant differences in cumulative solar radiation between
the groups (F (2, 13) = 0.637, p = 0.545).
0
1,000,000
2,000,000
3,000,000
4,000,000
5,000,000
6,000,000
2019 2020 2021 2019 2020 2021 2019 2020 2021
Group 1 Group 2 Group 3
Pca
Int. J. Environ. Res. Public Health 2022, 19, 8160 11 of 19
3.5. Differences within and among Clones
Based on the results that showed significant differences between topped and non-
topped trees (Table 4), we selected five clones (12 trees) having at least two or more non-
topped trees to further investigate the differences among clones, i.e., clone number 7 (n =
3), 21 (n = 3), 24 (n = 2), 30 (n = 2) and 42 (n = 2). The estimated pollen production of each
year from those trees was analyzed to derive mean values and coefficient of variance (CV)
(Table 5).
Table 5. Mean values for reproductive metrics and associated coefficients of variance (CV) of clones
with non-topped trees in the seed plantation Wildberg for 2019–2021.
Clone fl Pfl Pca Csu Pcr
7 96 (0.10) 15,366 (0.66) 1,478,347 (0.71) 27 (0.48) 333,661,556 (0.95)
21 106 (0.13) 13,164 (0.71) 1,354,889 (0.69) 29 (0.49) 385,906,000 (1.06)
24 98 (0.11) 20,362 (0.70) 1,929,146 (0.65) 25 (0.88) 331,433,333 (0.87)
30 120 (0.11) 9732 (0.81) 1,169,417 (0.81) 39 (0.60) 444,390,000 (1.26)
42 127 (0.13) 15,787 (0.54) 1,967,167 (0.57) 18 (0.30) 312,689,333 (0.79)
16 non-topped trees 112 (0.17) 14,994 (0.84) 1,612,250 (0.81) 26 (0.56) 332,198,375 (1.04)
According to the calculated CV across all study years, clone 42 is the most consistent
clone regarding Pca (CV = 0.57). Clone 42 was also found to produce the highest mean
value for Pca and the lowest Csu linked to the smallest CV (CV = 0.30). Similarly, clone 30
produced the lowest average pollen but was associated with a higher coefficient of vari-
ance (CV = 0.81) and a higher Csu with a moderate coefficient of variation (CV = 0.60) com-
pared to other clones.
Flowers per catkin (fl) were linked to lower CV values and was therefore most con-
sistent compared to other reproductive metrics. Clone 7 had the lowest mean (96 fl) and
CV (CV = 0.10) and clone 42 had the highest mean (127 fl). Clone 21 and clone 42 were
linked to the highest CV (CV = 0.13) at the level of fl.
Interestingly, the CV is higher (for fl) and equal or higher (for Pca) when not splitted
for each clone but when calculated for all the 16 non-topped trees (Table 5). Nevertheless,
it is moderate in the case of Csu.
There was a statistically significant difference between Pca estimated for clones (H (4)
= 21.44, p < 0.001) (see Figure 6). The post-hoc tests revealed that clone 30 was significantly
different from clone 24 (p = 0.008) and clone 42 (p = 0.001). Clone 21 and clone 42 were also
significantly different (p = 0.023).
Int. J. Environ. Res. Public Health 2022, 19, 8160 12 of 19
Figure 6. Boxplots based on pollen production per catkin (P
ca
) (eight replications per tree) estimated
for five selected clones with at least two non-topped trees in the seed plantation in Wildberg for
2019, 2020 and 2021. Clones 7 and 21 consist of three trees each. Clone 24, 30 and 42 consist of two
trees each. The interquartile range is represented by the height of the boxes, maximum and mini-
mum values by the upper and lower whiskers, the median by bold horizontal lines in the boxes,
points indicate outliers, lines above boxplots indicate pair of clones, which were significantly differ-
ent (Kruskal–Wallis test and Dunn’s multiple comparison’s tests).
For an average non-topped birch tree in the seed plantation, a mean crown volume
was calculated as 81.55 m
3
(average crown height = 6.28 m and average crown width =
4.98 m). This mean crown volume along with mean values of C
su
and P
ca
(in Table 5) were
used to calculate mean number of catkins and mean P
in
for different clones and for an
average birch tree (Table 6).
Table 6. Mean number of catkins and mean pollen production for different clones and for an average
tree. The last column shows the equivalence of the selected clones’ trees to an average tree.
Clone Mean Catkins Per Tree Mean Pollen Production Per
Tree (P
in
) Equivalent to 100 Average Trees
Average tree 16,962 27,347,187,742 100
7 17,615 26,040,366,183 105
21 18,919 25,633,543,942 107
24 16,310 31,463,863,118 87
30 25,443 29,753,697,858 92
42 11,743 23,100,462,441 118
Table 6 demonstrates that clone 42 reproduces fewer catkins per tree (11,743) com-
pared to other clones and 118 trees would be needed to produce the same amount of pol-
len produced by 100 average trees (based on mean values of all 16 trees). Clone 24 was
found to produce almost the same number of catkins per tree as an average tree; however,
it produces more pollen per tree. Therefore, 87 trees of clone 24 could produce the same
amount of pollen as 100 average trees. Clone 30 produces a higher number of catkins per
tree (25,443) and 92 trees would be needed to produce the same amount of pollen as 100
average trees.
Int. J. Environ. Res. Public Health 2022, 19, 8160 13 of 19
4. Discussion
Our study investigating pollen production of 28 birch trees in three consecutive years
is unique since we examined a large number of male birch inflorescences and assessed the
internal variability of pollen production regarding genetic differences and similarities. In
addition, this study excludes (major) environmental differences as well as age effects.
We estimated pollen production values at the level of catkins ranging from 48,000
pollen grains to 8.3 million pollen grains (mean 1.66 million). Some studies have already
estimated pollen production values for Betula pendula (syn. Betula alba, Betula verrucosa).
Erdtman [51] reported an estimate of 5.5 million pollen grains per inflorescence for B. ver-
rucose. Jato et al. [30] estimated values ranging between 8.2 million and 4.8 million pollen
grains per inflorescence, sampled from six trees of B. alba in northwestern Spain in 2002
and 2003, respectively. Piotrowska [43] estimated a mean value of 10 million pollen grains
per inflorescence on the basis of 30 catkins deriving from three individuals. Although
these studies have reported higher values compared to the mean Pca estimated in this
study, they were based on either a few trees or estimated only for a single or two study
years. Consequently, it is not known if sampling took place in a masting or non-masting
years. For this reason, our study can be regarded as important since we have sampled 28
trees for three years and present a robust estimate for the mean pollen production of Betula
pendula.
We found that birch catkins with fewer flowers produce more pollen and vice versa.
This could be considered as an internal compensation since the plant aims at upregulating
pollen production when the flower amount is low. Molina et al. [52] studied ten anemoph-
ilous species of aerobiological importance (Betula ssp. not included) and found a signifi-
cant decrease in pollen per flower with a higher number of flowers per inflorescence. They
suggested that there is a more or less constant amount (within a defined margin) for pollen
production in anemophilous tree species. These species tend to compensate for reproduc-
tive characteristics (e.g., pollen per anther, flowers per tree, and inflorescences per tree)
by increasing some and decreasing others. Our analysis showed that the number of flow-
ers is the most homogenous value since a low coefficient of variance was associated to this
measure, e.g., in clonal comparisons.
Our study shows an annual variation in pollen production with the lowest mean val-
ues in 2019 and the highest in 2021. Such alterations could be caused by yearly changes in
the meteorological conditions of the locality. Some studies examining the relationship be-
tween temperature and pollen production suggested that warmer conditions result in
higher pollen quantities. For example, experimental studies indicated that an increase in
temperature [16] but also an increase in atmospheric CO2 concentration [16,53,54] was as-
sociated with a higher pollen production of common ragweed (Ambrosia artemisiifolia).
However, it was also found that pollen production of birch (Betula pendula Roth) along an
urban-rural gradient was negatively correlated with temperature [55]. The authors argue
that the physiological performance of birch, which mainly grows at lower temperatures
in mid to high latitudes, might be affected by (very) high temperatures and in turn react
with a decrease in pollen production, as also suggested by Ziello et al. [56]. However, any
differences in pollen production found in natural environments might also be affected by
other factors, which attenuate or diminish the influence of temperature. In addition, the
response to temperature might also be species-specific and strongly dependent on the
methodologies used.
Although many studies have examined the temporal change in birch pollen concen-
trations based on pollen trap monitoring, there is no study presenting long-term changes
in pollen production assessed using the same birch trees. Detecting the influence of tem-
perature on pollen production based on the data presented in this study is not feasible,
since (a) we only cover a period of three years and (b) a small spatial extent (1 ha) with
similar temperature conditions, as documented using five installed temperature loggers.
Many other environmental factors such as soil type and edaphic conditions as well as air
pollutants are regarded to be similar as well. Especially the latter is also supposed to affect
Int. J. Environ. Res. Public Health 2022, 19, 8160 14 of 19
pollen production, as documented by Jochner et al. [55]. In their study, atmospheric NO2
levels were negatively associated to pollen production.
However, we found differences in solar radiation, which arise mainly from the for-
ested surrounding of the seed plantation. During the study years, the correlations between
pollen production and solar radiation did not vary much in magnitude, but they did shift
in sign. Therefore, we calculated the correlation coefficient for mean (2019–2021) pollen
production, but the association to solar radiation was no longer discernible. Thus, solar
radiation, which is known to lead to higher stem and tissue temperatures [57] might also
be inadequate to explain variations of pollen production at a small spatial scale. This was
also evident when comparing solar radiation values with the association of birch trees to
groups with similar pollen production patterns across the study years.
We did not detect a high synchrony of pollen production levels of birch trees within
the birch plantation since we found that six trees exhibited the highest pollen production
in 2020, four trees a very high pollen production in 2021 and six trees an almost constant
pollen production across the study years. The birch trees allocated to one of these three
groups did not necessarily belong to one clone. Thus, a coherence on the level of clones
was not evident, except for one clone group.
Masting behavior, the inherent year-to-year variation in pollen production by plant
populations [58,59], can be observed in several tree species [60,61] including birch [14,30].
Flowering and annual pollen sums in birch were reported to fluctuate from year to year
[62]. Using aerobiological data gathered from pollen traps that assess the pollen concen-
tration of the ambient outdoor air, a biennial [63] as well as triennial rhythm [64] of mast-
ing can be observed. Related to Betula species, Ranta et al. [59] found that male flowering
shows synchronized annual fluctuations among stands at a regional scale; however,
stand-specific catkin number during the masting year varies considerably, which in turn
might also influence the pollen produced. This is also in accordance with our findings
since the numbers of catkins varied (mean Cs (SD) = 23 (8), 44 (26) and 19 (12) in 2019, 2020,
and 2021, respectively, Table 2) within the plantation.
Asynchronous pollen production levels, which were found in our study might be
caused by the resource balance of an individual tree. If the initial resource stock and the
resource gained afterwards differ from one individual to the next in the stand, masting
synchronization might not occur [65,66], even under the same environmental conditions
[65]. In addition, plant-pathogen and plant-mycorrhizosphere interactions may reduce or
enhance the impacts of abiotic stress on resource allocation [67] which could be specific to
each tree.
Effects on pollen production and catkin formation were especially obvious two years
after topping. Topping and pruning have been considered as adequate tree crown man-
agement techniques to enhance seed production, specifically in conifer seed orchards, or
to promote the branching of the trees [47,68,69]. Viherä-Aarnio and Ryynänen [47] studied
seed production of silver birch individuals that were topped in the second year in a green-
house experiment. In the fourth year, a ten times higher amount of seeds per plant (com-
pared to the previous year) was obtained. This was followed by a year with poor flower-
ing and seed production. In our study, we cannot conclude on any effects in upcoming
years; therefore, we recommend a longer monitoring of pollen production after topping
in further studies.
Birch clones characterized by on average lower pollen production could be an oppor-
tunity to reduce the prevalence of allergies. In an experiment, transgenic birch grown in a
greenhouse showed the ability to prevent flowering in silver birch trees [70]. However,
such preventions might be associated with adverse side effects such as aberrant branching
and growth disturbance. Therefore, we suggest selecting birch clones associated with low
pollen production. We estimated Pca ranging between 1.17 million (clone 30) and 1.97 mil-
lion (clone 42) pollen. Clones producing less pollen might contribute to lower pollen con-
centrations in the atmosphere. Therefore, clone 42 could be recommended for urban plan-
Int. J. Environ. Res. Public Health 2022, 19, 8160 15 of 19
tations. Similarly, clone 24 needs 87 trees to produce the same pollen amount as 100 aver-
age trees. This clone could be suitable in seed plantations to increase seed production.
Since variations within clones were especially obvious when comparing pollen produc-
tion levels across years (Chapter 3.4), we highly recommend monitoring pollen produc-
tion for a longer term in order to create robust averages for different clones.
5. Conclusions
Our study revealed considerable differences in pollen, flower and catkin productions
by birch trees among the study years. Moreover, we found topped birches were associated
to higher reproductive outputs, especially two years after the intervention. We conclude
that synchronicity of pollen production levels is not utterly site-specific and, in addition,
not strictly dependent on genotypes. The detected variations in solar radiation within the
plantation were found to be not responsible for asynchrony. Since we revealed significant
differences in pollen production between clones, we propose that a wise selection of plants
depending on their application (seed plantation, urban planting) might be advantageous.
As these conclusions are based on three years of investigation, we recommend a longer
monitoring period to further extend our knowledge related to pollen production of ane-
mophilous tree species. In addition, further experimental studies with intended treatment
such as pruning and topping under different climatic conditions are highly desirable.
Author Contributions: Conceptualization, S.J.-O.; methodology, S.J.-O., S.R., A.D. and G.K.; soft-
ware, S.R. and G.K.; validation, S.J.-O., S.R., A.D. and T.H.; formal analysis, S.R.; investigation, S.R.,
M.S., V.W. and G.K.; resources, S.J.-O. and S.R.; data curation, S.R.; writing—original draft prepara-
tion, S.R.; writing—review and editing, S.R., S.J.-O., A.D., G.K., S.G., T.H., M.L., D.L., C.B., C.T.-H.,
M.S., V.W., K.K. and F.K.; visualization, S.R. and G.K.; supervision, S.J.-O.; project administration,
S.J.-O.; funding acquisition, S.J.-O. All authors have read and agreed to the published version of the
manuscript.
Funding: This research was funded by the German Research Foundation (Deutsche Forschungsge-
meinschaft) (DFG) as part of the project “pollenPALS: Biotic and abiotic effects on pollen production
and allergenicity of birch and related health impacts (655850)”. The open access publication of this
article was supported by the Open Access Fund of the Catholic University Eichstätt-Ingolstadt. The
study was partly implemented in the frame of the EU-COST Action ADOPT (New approaches in
detection of pathogens and aeroallergens), Grant Number CA18226 (EU Framework Program Hori-
zon 2020).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank Jens Holzmüller, Lisa Buchner, Sabine Fürst, Johanna Jetschni, An-
nika Rippert, Verena Wiethüchter, Celina Riedl, Florian Weber, and Sagun Ranpal for technical as-
sistance. In addition, we thank Forst Baden-Württemberg for providing the seed plantations as
study sites.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the
design of the study; in the collection, analyses, or interpretation of data; in the writing of the manu-
script, or in the decision to publish the results.
Int. J. Environ. Res. Public Health 2022, 19, 8160 16 of 19
Appendix A
Table A1. Descriptive statistics of pollen production per catkin (Pca), pollen production per flower
(Pfl), pollen production per volume unit of crown (Pcr), flowers per catkin (fl) and catkins per crown
sampling unit (Csu; 0.125 m3) (minimum, maximum, mean, median and standard deviation) esti-
mated from 28 selected birch trees of the seed plantation Wildberg during 2019–2021.
Reproductive Metrics Minimum Maximum Mean Median Standard Devia-
tion
All years
Pca 48,000 8,270,000 1,658,846 1,356,000 1,277,605
Pfl 407 80,291 15,018 12,093 12,008
Pcr 4,992,000 2,333,440,000 359,736,647 244,592,000 348,107,774
Fl 77 173 113 112 18
Csu 3 120 29 23 20
2019
Pca 48,000 8,270,000 1,359,049 983,500 1,245,134
Pfl 407 80,291 12,001 8691 11,360
Pcr 4,992,000 1,654,000,000 250,112,679 164,292,000 261,586,850
Fl 77 173 116 113 19
Csu 10 45 23 22 8
2020
Pca 108,000 4,172,000 1,511,170 1,360,000 892,862
Pfl 788 37,250 13,633 12,093 8729
Pcr 12,960,000 2,333,440,000 517,563,929 388,160,000 409,661,684
Fl 82 173 117 117 19
Csu 10 120 44 35 26
2021
Pca 184,000 8,018,000 2,090,888 1,796,000 1,495,281
Pfl 2000 71,589 19,268 16,102 14,030
Pcr 8,880,000 1,731,360,000 313,195,517 207,240,000 300,234,937
Fl 78 142 109 107 16
Csu 3 60 19 20 12
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