A peer-reviewed open-access journal

NeoBiota 60: 79-95 (2020)

= e
doi: 10.3897/neobiota.60.561 17 %) NeoBiota

http:/ / neob iota. pen soft. net Advancing research on alien species and biological invasions

Germination of the invasive legume Lupinus polyphyllus
depends on cutting date and seed morphology

Yves P. Klinger', Rolf Lutz Eckstein?, David Horlemann',
Annette Otte', Kristin Ludewig!

| Division of Landscape Ecology and Landscape Planning, Research Centre for Biosystems, Land Use and
Nutrition (iFZ), Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany
2 Department of Environmental and Life Sciences, Biology, Karlstad University, SE- 651 88 Karlstad, Sweden

Corresponding author: Yves Klinger (yves.p.klinger@umwelt.uni-giessen.de)

Academic editor: Bruce Osborne | Received 3 July 2020 | Accepted 30 July 2020 | Published 25 August 2020

Citation: Klinger YP, Eckstein RL, Horlemann D, Otte A, Ludewig K (2020) Germination of the invasive legume
Lupinus polyphyllus depends on cutting date and seed morphology. NeoBiota 60: 79-95. https://doi.org/10.3897/
neobiota.60.56117

Abstract

In semi-natural grasslands, mowing leads to the dispersal of species that have viable seeds at the right time.
For invasive plant species in grasslands, dispersal by mowing should be avoided, and information on the
effect of cutting date on the germination of invasive species is needed. We investigated the germination of
seeds of the invasive legume Lupinus polyphyllus Lindl. depending on the cutting date. We measured seed
traits associated with successful germination that can be assessed by managers for an improved timing of
control measures. To this end, we sampled seeds of L. polyphyllus on six cutting dates and analyzed the germi-
nation of these seeds in climate chambers and under ambient weather conditions. We collected information
on seed morphology (color/size/hardseededness) for each cutting date to identify seed traits associated with
successful germination. Observed germination patterns were highly asynchronous and differed between
seeds cut at different dates. Seeds cut early, being green and soft, tended to germinate in autumn. Seeds cut
late, being dark and hard, were more prone to germinate the following spring, after winter stratification.
This allows the species to utilize germination niches throughout the year, thus indicating a bet-hedging strat-
egy. Seed color and the percentage of hard seeds were good predictors of germination percentage, but not
of mean germination time and synchrony. Managers should prevent the species producing black and hard
seeds, while cutting plants carrying green and soft seeds is less problematic. Furthermore, germination pat-
terns differed between climate chambers and the common garden, mainly because germination of dormant
seeds was lower in climate chambers. More germination experiments under ambient weather conditions

should be carried out, as they can give information on the germination dynamics of invasive species.

Keywords

dormancy, grassland management, lupine, phenology, seed traits

Copyright Yves P. Klinger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

80 Yves P Klinger et al. / NeoBiota 60: 79-95 (2020)

Introduction

The timing of germination determines which environmental conditions the seedling
will experience and thereby influences a variety of plant characteristics (Casas et al.
2012). Consequently, the germination ecology of a species largely decides in which
habitats and under which climates it may establish. The introduction of species to new
ranges often leads to new germination conditions (Kudoh et al. 2007), and the ability
to germinate successfully under a variety of environmental conditions is a characteris-
tic of many successful and widespread invasive species (Baker 1974; Wainwright and
Cleland 2013). Whether seeds are viable depends largely on their development stage,
which is influenced by the timing of seed set and seed ripening.

In semi-natural grasslands, the mowing date is the environmental factor that most
strongly determines the timing of seed release. Furthermore, mowing is a way of seed
dispersal for species that have viable seeds at the right time. In most cases, the dispersal
of mature seeds after mowing is a desirable process, as it is responsible for sustaining
a high plant diversity in semi-natural grasslands (Auffret 2011; Humbert et al. 2012).
In other cases, such as weeds or non-native invasive species, dispersal of ripe seeds by
mowing is not wanted (Wilson et al. 2009) and shifts in grassland management and
the time of cutting may create opportunities for invasives to establish in these eco-
systems. Consequently, understanding the germination ecology of invasive plants is
essential for their management and control and for limiting their spread to new sites.

Established invasive species are often more challenging to manage than newly
arrived species (Simberloff 2003). Ideally, control measures would take place before seed
formation, but time windows for adequate management can be short in areas where
different conservation goals have to be matched. In the case of species invading mountain
grasslands, e.g., mowing of areas critical for the protection of ground-nesting birds has to be
postponed until nesting is finished, which means that invasive species may have produced
viable seeds by the time of mowing. Consequently, managers are looking for information
on the relationships between cutting dates, seed morphology, and seed germinability.
Lupinus polyphyllus Lindl. is a widespread perennial legume originating from North-
America. It is widely found as an ornamental plant (Fremstad 2010) and commonly used
for soil stabilization and soil melioration (Rehfuess et al. 1991). Due to its many uses,
the species is naturalized in different regions all over the world, e.g. in Europe (Fremstad
2010; Hejda 2013), New Zealand (Holdaway and Sparrow 2006) and Chile (Meier et
al. 2013). Invaded habitats include road verges (Valtonen et al. 2006), riparian terraces
(Meier et al. 2013), and mountain grasslands (Klinger et al. 2019). Due to its ability to fix
nitrogen, it is considered an ecosystem engineer and may cause unwanted ecosystem effects
(Hiltbrunner et al. 2014). In invaded habitats, L. polyphyllus is capable of overgrowing and
shading the underlying vegetation and may cause a considerable decline in the richness of
small species (Thiele et al. 2010; Hiltbrunner et al. 2014), while promoting the spread of
tall-growing, nitrogen-demanding vegetation (Otte and Maul 2005). Meadows invaded
by this species provide hay of low fodder quality, because of its high water-content and the
presence of alkaloids in L. polyphyllus (Hensgen and Wachendorf 2016).

Germination depends on cutting date 81

Despite the importance of seed ecology for the spread and establishment of species,
there is often insufficient knowledge concerning germination and ripening character-
istics of invasive species (Gallinat et al. 2018). The capability of seeds to after-ripe and
germinate, which depends on the interaction between phenology and cutting date,
may have important implications for the management of invasive species in grasslands.
Therefore, we investigated the germination of the invasive legume L. polyphyllus in
relation to the cutting date. Over the course of the vegetation period, i.e., weekly from
the beginning to the end of fruiting, we sampled seeds from five locations invaded by
L. polyphyllus. We combined two experiments to investigate the germination of L. poly-
phyllus: A common garden experiment to analyze the germination patterns under am-
bient weather conditions and a climate chamber experiment under standardized con-
ditions. We aim to provide management recommendations based on seed traits such
as seed color and hardseededness that may help to decide when fruiting lupine stands
should be cut and when plant material has to be removed from the sites after mowing.

Specifically, our research hypotheses were:

1) ‘The germination ability of L. polyphyllus seeds increases with later cutting date.
Consequently, we expect a higher germination percentage, a shorter mean germi-
nation time, and a higher synchrony of germination with later cutting date.

2) Seed traits such as seed size, seed color, and the percentage of hard seeds provide
reliable information about the germination ability of seeds sampled at different
dates. We expect larger seeds, seeds with darker color and harder seeds to show
higher germination percentage, shorter mean germination time and higher syn-
chrony compared to small, green, and soft seeds.

Methods

Seed sampling, seed handling, and experimental design

Seeds were collected in the Rhén UNESCO Biosphere Reserve, in central Germany.
The study area (from 50°26'N to 50°32'N and from 09°54'E to 10°05'E), a part of the
Biosphere Reserve, is situated between 600 m and 950 m a.s.l. It is characterized by
large and coherent semi-natural grasslands of high conservational value that are non-
intensively used as meadows and pastures (e.g., Habitats Directive 92/43/EEC, habitat
types 6520: mountain hay meadows, and 6230: species-rich Nardus grasslands). ‘These
grasslands have a centuries-long land-use history of mowing and pasturing with low
nitrogen-inputs. In the 1990s, the traditional mowing date in early July was postponed
to August and September, in order to safeguard the populations of protected ground-
nesting birds and because the meadows decreased in importance for local farmers. ‘This
allowed L. polyphyllus, already present along roadsides in the area, to produce seeds
before mowing and to spread extensively into the meadows. During the past 20 years,
parts of the region were heavily invaded, with the area covered by L. polyphyllus dou-

82 Yves P Klinger et al. / NeoBiota 60: 79-95 (2020)

bling in some localities (Klinger et al. 2019). This invasion is considered a major threat
to the biodiversity of the mountain grasslands in the study region. Depending on site
conditions, L. polyphyllus can reach a height of 60 to 150 cm. In June and July inflores-
cences are formed, each consisting of 50 to 80 single flowers (Fremstad 2010; Bunde-
samt ftir Naturschutz 2017). L. polyphyllus develops seed pods with four to twelve seeds,
which burst at seed maturity and spread the seeds ballistically up to several meters (Otte
et al. 2002; Volz 2003). Per plant, up to 2500 seeds can be produced (Aniszewski 2001).
Seeds of L. polyphyllus were manually collected from five meadows (sampling locations)
over six weeks (July-August 2015; cutting dates). The distance between sampling locations
ranged between 1500 and 5000 meters. For each cutting date and location, we sampled
one inflorescence each from ten plants for the germination experiments. From each inflo-
rescence, we randomly took one pod and determined seed size, seed color, and the propor-
tion of hard seeds. For seed color, we distinguished between four colors: green, dark green,
brown and black. Seeds with different pigmentations and puncturing (see Aniszewski
2001) were integrated to the different classes according to the predominant color, seeds
were assigned the color “black” when they were considerably darker than brown seeds.
Usually, seeds of several colors were found on the same location or even within the same
seed pod. To determine the average color for each replicate, we gave ranks from one (green)
to four (black) to each color and calculated the median. For seed hardness, we classified the
seeds into five classes, from undeveloped and very soft to very hard. Based on these data,
we calculated mean seed size, average seed color and the proportion of hard seeds for each
replicate. For the germination experiments, we pooled the seeds within each sampling loca-
tion. Seeds were manually cleaned, air-dried and stored in darkness at room temperature
(app. 20 °C) until the start of the germination experiments on September 28", 2015.
Laboratory experiments are a standardized tool to investigate germination in a
controlled environment and can provide information on germination cues, dormancy,
and other factors (Baskin and Baskin 2014). Nonetheless, germination in the laborato-
ry often differs from germination under (semi-)natural conditions (Grime et al. 1981;
Holzel and Otte 2004) and thus gives only a limited representation of germination
patterns that can be observed in the field. We combined a climate chamber experiment
and a common garden experiment to study the germination of L. polyphyllus both un-
der standardized and ambient weather conditions. A factorial experimental design was
used to analyze the effects of cutting date (6 dates), sampling location (5 locations), and
temperature (day/night: 20/10 °C and 15/5 °C; only in the climate chamber experi-
ment) on seedling emergence. Germination was defined as protrusion of the radicle.
In the climate chamber experiment (from September 28", 2015 to July 28",
2016), seeds were placed into petri dishes with distilled water (25 seeds per replicate)
in climate chambers (Rumed type 3401, Rubarth Apparate GmbH). Each treatment
combination (cutting date x sampling location x temperature) was replicated five times,
resulting in 300 petri dishes. For incubation in climate chambers, we exposed the seeds
to 12 h light and 12 h darkness and two diurnally fluctuating temperatures (15/5 °C
and 20/10 °C) that represent spring and early summer temperature conditions. Similar
fluctuating temperature conditions have been applied by Elliott et al. (2011). Moisture

Germination depends on cutting date 83

content of the Petri dish was controlled during the experiment. For seeds in the climate
chambers, germination was checked once a week and seedlings were removed.

In the common garden experiment, germination was observed under ambient
weather conditions from September 17", 2015 to July 14", 2016. The seeds were placed
ona 1:1 mixture of sand and commercial potting soil (Fruhstorfer Erde, Type P, Indus-
trie-Erdenwerke Archut GmbH, Lauterbach/Germany) in trays (18 x 28 cm) in a com-
mon garden at the research station Linden-Leihgestern of the Justus-Liebig University
(50°32'N, 8°41'E). Per tray, 25 seeds were used (n = 5 for each cutting date x sampling lo-
cation combination, resulting in 150 trays). Seeds were protected from predation using
wire cages. For seeds in the common garden, germination was checked once every seven
to fourteen days. After three months of incubation, germination decreased in both ex-
periments and thus was checked every other week. After ten months of incubation, the
experiments ended since no further germination was observed. By the end of the experi-
ments, the remaining seeds were covered by mold and collapsed when pinched by hand.
Thus, the remaining seeds were considered dead (following Baskin and Baskin 2014).

Germination variables and statistical analyses

As response variables, we calculated the germination percentage (%), mean germination
time (days) and synchrony of germination (unitless) per replicate (according to Ranal
and Santana 2006; Ranal et al. 2009). The germination percentage is the proportion of
germinated seeds of the total number of seeds. Mean germination time and synchrony of
germination were calculated based on seedling counts over time (Ranal et al. 2009). Mean
germination time is a measurement of the weighted average time required for germina-
tion (Ranal and Santana 2006). The synchrony index is a measure for the overlapping of
germination that ranges from 0 (when no two seeds germinated at the same time) to 1
(when all germinating seeds germinated at the same time; for details see Ranal et al. 2009).

Seeds from the climate chamber experiment and from the common garden experi-
ment were analyzed separately. The effects of the experimental variables cutting date,
sampling location and temperature on the response variables germination percentage
and germination time were analyzed using linear mixed-effect models (LMM) and
synchrony of germination using generalized linear mixed-effect models (GLMM) for
binomial distributions. The factors cutting date and temperature were included as fixed
factors in the first models. As there was no effect of the temperature, the final models
only included cutting date or seed color fixed factors. We added an error term for re-
peated measures to the models to account for variation within each sampling location.
Furthermore, we added a general linear hypothesis and multiple comparisons (glht) to
determine significant differences between groups.

To identify seed traits associated with germination success, we checked for cor-
relation of seed traits with the factor cutting date using Pearson's R?. This was the case
for seed size, seed color, and proportion of hard seeds. We then fitted models with these
traits as fixed factors (both in combinations and as single-factor models) and sampling

84 Yves P Klinger et al. / NeoBiota 60: 79-95 (2020)

location as random factor. To choose the best seed traits or trait combination to explain
germination success of L. polyphyllus, we compared these models via AIC and pairwise
model ANOVA. To assess model quality, we calculated Nagakawa and Schielzeth’s R?
for linear mixed-effect models (Nakagawa and Schielzeth 2013). We visually checked
for normality of residuals and homogeneity of variances using diagnostic plots (Zuur
et al. 2010). Mixed-effect models were carried out using the ‘Ime4’ (Bates et al. 2015)
and ‘ImerTest’ (Kuznetsova et al. 2017) packages, post-hoc-tests were calculated using
the ‘multcomp’ package (Hothorn et al. 2008), graphs were created using the ‘ggplot2’
package (Wickham 2016) in R (R Core Team 2016).

Results

During the sampling period, seed color became darker (changing from green via dark
green and brown, to black) and the proportion of hard seeds increased gradually. Mean
seed size ranged from 3.9 mm (date six, August 11") to 6.4 mm (date three, July 21").
It increased during the first three weeks of cutting and then decreased thereafter as
seeds became drier. Seed color and the proportion of hard seeds were correlated, as
hard seeds usually were darker than soft seeds. There were no differences in the total
germination percentages between different sampling locations, although the germina-
tion peaks shifted by up to two weeks between different locations.

In climate chambers, 16.3% of all collected lupine seeds germinated (Fig. la, b).
Germination percentage was lowest after the first cutting date (July 7°, 8.6%) and in-
creased until the third date July 21") where it peaked at 26% (Fig. 1a). Afterwards, we
observed a significant decrease from week three (July 21") to four (July 28"; to 13.4%;
Table 1). Mean germination time was 114 days and varied from 3 days to 303 days in cli-
mate chambers (Fig. 1c, d), with seeds collected on the first date having the longest mean
germination time (141 d; Fig. 2d). Mean germination time decreased until week three
(98 d), then increased again and had its overall minimum in week six (74 d). Synchrony
of germination was quite low with an average of 0.08 over all cutting dates (Fig. le, f).

In the common garden, 51.7% of seeds germinated and mean germination time
was 153.6 days (Fig. 2). Thus, seeds in the common garden germinated to a higher
degree compared to seeds in climate chambers, but slower. Germination percentages in
the common garden were lowest in seeds sampled during the first two weeks (17.0%
on July 7" and 30.6% on 14"), reached the highest level in week three (63.2% on
July 21") and stayed high afterwards (Fig. 2a, Table 1). In the common garden, mean
germination time was similar for all cutting dates and averaged 153.6 days. Synchrony
of germination in the common garden was quite low with an average of 0.12 over all
treatments and on all cutting dates (Fig. 2e, f).

There were significant differences in germination percentages between seeds of differ-
ent color (Figs 1b, 2b, Table 2). In climate chambers, dark green seeds showed the highest
germination while in the common garden, germination percentages increased steadily as
seeds darkened (Figs 1b, 2b). In climate chambers, germination percentage peaked when
60% of collected seeds were hard and decreased when the amount of hard seeds was lower

Germination depends on cutting date 85

Ger mination percentage (% )

July7 July14 July21) July28 August4 August 11 green dark green brown black

Mean ger mination time (d)

July7 July14 July21 July28 August4 August 11 green dark green brown black

Synchron y (Z)

July7 July14 July21 July28 August4 August 11 green dark green brown black
Cutting Date Seed colour

Figure |. The effect of the factors cutting date (weekly from July 7* to August 11") and seed color on germi-
nation percentage (a, b), mean germination time (¢, d), and synchrony of germination (e, f) in seeds stored
in climate chambers averaged over the two temperature regimes. Bars show mean values + standard errors.

Table |. Differences in germination percentages of L. polyphyllus seeds between six cutting dates assessed
in two germination experiments (climate chamber and common garden). Differences were assessed using
mixed effect models for each experiment separately with sampling location as random factor (formula:
Germination percentage ~ Cutting Date + (1|Sampling location).

Climate chamber n = 300 R? einai 9-20 R*. srionat= 9°25
Estimate Std. Error Df t Value p Value
Date 1 (July 7; Intercept) 8.64 1.86 22.56 4.65 < 0.001
Date 2 (July 14) 9.36 21 295 4.45 < 0.001
Date 3 (July 21) 17.36 2.11 295 8.25 < 0.001
Date 4 (July 28) 4.8 21-1 295 2.28 0.023
Date 5 (August 4) 10 2.11 295 4.75 < 0.001
Date 6 (August 11) 4.64 2.11 295 2.20 0.028
Common garden n= 150 R? scoinat 9°63 RR? sicionat™ O+7 1
Estimate Std. Error Df t Value p Value
Date 1 (July 7; Intercept) 16.96 3.88 14.21 4,37 < 0.001
Date 2 (July 14) 13.6 3.86 145 BDz < 0.001
Date 3 (July 21) 46.24 3.86 145 1.99 < 0.001
Date 4 (July 28) 47.68 3.86 145 12.37 < 0.001
Date 5 (August 4) 52 3.86 145 13.49 < 0.001

Date 6 (August 11) 48.8 3.86 145 12.66 < 0.001

ioe)
ON

Ger mination percentage (% )

July7

Mean ger mination time (d)

July 7

Synchron y (Z)

July 7

July14 July 21

July14 July 21

July14 July 21

Yves P Klinger et al. / NeoBiota 60: 79-95 (2020)

July28 August4 August 11

July28 August4 August 11

July28 August4 August 11

Cutting Date

green

green

green

dark green

dark green

dark green

brown

brown

brown

Seed colour

black

black

black

Figure 2. The effect of the factors cutting date (weekly from July 7" to August 11%) and seed color on
germination percentage (a, b), mean germination time (c, d), and synchrony of germination (e, f) in

seeds stored under ambient weather conditions. Bars show mean values + standard errors.

Table 2. Differences in germination percentages of L. polyphyllus seeds between four seed colors (median

seed color per sample with four levels: green, dark green, brown, and black) assessed in two germination

experiments (climate chamber and common garden). Differences were assessed using mixed effect models

for each experiment separately with sampling location as random factor (formula: Germination percent-

age ~ Seed color + (1|Sampling location).

Climate chamber

Green (Intercept)

Dark green
Brown

Black

Common garden

Green (Intercept)

Dark green
Brown

Black

a= 300 Rae 0.15 conditional — 0.22

Estimate Std. Error Df t Value p Value
9.45 1.88 16.2 5.03 < 0.001
14.34 1.94 298.48 7.4 < 0.001
7.71 DelZ 299,21 3.64 < 0.001
5.64 1.74 297.85 25 < 0.01

oo 150 Rae 0.58 conditional — 0.65

Estimate Std. Error Df t Value p Value
L229 4.11 12.24 4.21 < 0.01
27.78 3.82 146.4 Teak < 0.001
47.13 4.18 146.84 11.27 < 0.001
50.14 3.42 146.09 14.65

< 0.001

Germination depends on cutting date 87

or higher while in the common garden, germination percentage increased continuously
with the amount of hard seeds. In both experiments, seeds of different color had relatively
similar germination times with black (99 d) and dark green (109 d) seeds germinating
most rapidly in climate chambers (Fig. 1d). In the common garden, there were no sig-
nificant differences in mean germination time between seeds of different colors (Fig. 2d).

While in climate chambers, germination peaked early and decreased afterwards
(Fig. 3), two peaks (in autumn and spring) characterized germination in the common
garden (Fig. 4). There were no significant differences between colors in climate cham-
ber, while synchrony in the common garden increased slightly with the increase in the
percentage of hard seeds.

Germination percentage (in both experiments) and mean germination time (only in
climate chambers) responded significantly to cutting date, while there was no effect of the
different temperature regimes. For germination percentage and mean germination time,
the best explanatory models (see Suppl. material 1: Model Tables) each contained solely
one fixed factor, mainly due to high correlations between explaining factors. Germination
percentage in climate chambers was best explained by seed color and showed highly sig-
nificant differences between colors (R? = 0.15). Germination percentage in the common
garden was well explained by both seed color (R? = 0.58) and proportion of hard seeds.
For mean germination time, the best explaining factors were either color or proportion
of hard seeds, while both models performed poorly overall. Synchrony was not affected
significantly by any factor and there was no model of significant explanatory value.

Month Month Month
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Figure 3. Germination patterns of L. polyphyllus in climate chambers conditions (15/5 °C and 20/10 °C di-
urnally fluctuating temperatures) sampled weekly on six cutting dates (July 7 to August 11") after seed set.

88 Yves P Klinger et al. / NeoBiota 60: 79-95 (2020)

Month Month Month
Oct Nov Dec Jan Feb Mar Apr May Jun Jul Oct Nov Dec Jan Feb Mar Apr May Jun Jul Oct Nov Dec Jan Feb Mar Apr May Jun Jul
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250 300 3500 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 0 50 100 150 200
Oct Nov Dec Jan Feb Mar Apr May Jun Jul Oct Nov Dec Jan Feb Mar Apr May Jun Jul Oct Nov Dec Jan Feb Mar Apr May Jun Jul
8 SS SEE EEE EEE OO a a,
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Day of the Year Day of the Year Day of the Year

Figure 4. Germination patterns of L. polyphyllus under ambient weather conditions sampled weekly on
six cutting dates (July 7* to August 11") after seed set.

Discussion

The germination patterns of the invasive legume L. polyphyllus differed between dif-
ferent cutting dates, partially confirming our first hypothesis. Seeds collected early,
while being green and soft, germinated to a lower degree and more slowly compared
to seeds collected later. While seeds of early-cut L. polyphyllus plants germinated in
autumn, seeds of late-cut plants were more prone to germinate in spring. This relation-
ship may be associated with their progression through different phases of seed develop-
ment. During morphogenesis the embryo develops, then during maturation, storage
compounds are synthesized in the growing endosperm and thereafter, seeds may go
through a phase of desiccation, in which they dry and eventually enter dormancy
(Angelovici et al. 2010). Consequently, the different cutting dates of our experiment
covered the phases of maturation and desiccation. Until late July, L. polyphyllus seeds
were in the phase of maturation. Afterwards (end-July to mid-August), seeds were in
the desiccation phase. Although dormancy per se was not tested in our study, the ob-
served germination patterns and differences between climate chambers and the com-
mon garden strongly indicate that seeds from late cut L. polyphyllus plants expressed
dormancy, which is also supported by our observation that seeds decreased in size and
became harder. Physical dormancy is common in legumes (Russi et al. 1992a), but
whether an individual plant produces dormant seeds at a given point in time depends
on a variety of factors, such as temperature and moisture conditions during seed ripen-
ing (Masaka and Yamada 2009; Bolingue et al. 2010; D’hondt et al. 2010). ‘Thus, the

expression of dormancy can vary strongly in legume seeds, even within plants of the

Germination depends on cutting date 89

same population (D’hondt et al. 2010), which may consequently lead to asynchronous
germination patterns.

Despite pronounced peaks of germination in autumn and spring, germination of
L. polyphyllus seeds was highly asynchronous. In both experiments and under all cut-
ting dates, some seeds germinated over the whole duration of the experiments, over
300 days. The timing of germination determines which environmental conditions the
seedling will experience and may influence plant characteristics, such as growth and
reproduction (Donohue 2002; Casas et al. 2012). The timing of germination itself
may be influenced by plant life-history traits, e.g. the phenology of flowering, seed
maturation, and seed dispersal (Galloway 2001; Donohue 2002). Variations in germi-
nation depending on the time of seed collection have been observed by other authors
(e.g., Greipsson and El-Mayas 2003; Samarah 2005; El-Keblawy and Al-Rawai 2006;
Broback 2015), but there is little information on the long-term germination patterns
of species and seasonal effects that are associated with this factor. In invasive species,
asynchronous germination can lead to the exploitation of open germination niches
throughout the year, which might contribute to their invasion success (Wolkovich
and Cleland 2011; Gioria et al. 2016). In the case of L. polyphyllus, this effect may be
amplified by its high seed production (Volz 2003), its long-lasting flowering, by its
ability to resprout and produce seeds after early cutting (Broback 2015), and by the
observation that the ballistic seed dispersal of the species takes place over many weeks if
stands are left untouched (Klinger et al., unpublished data). The observed germination
patterns of L. polyphyllus thus suggest a bet-hedging strategy (Cohen 1966), which may
partly explain its invasion success and its capability to colonize many different habitats.

Our second hypothesis can be verified, as seed color and the percentage of hard seeds
were good predictors of germination percentage and give information on the germination
patterns that can be expected. Soft and green seeds germinated to the lowest degree and in
autumn. However, germination percentages of these seed batches were relatively high, giv-
en their early developmental phase. High germination rates in immature seeds have been
found in some legumes, e.g., in Lotus and Scorpiurus (Cristaudo et al. 2008), and Vicia
(Samarah 2005), but germination failed in others, such as in green seeds of Lupinus noot-
katensis (Greipsson and El-Mayas 2003). Black and hard seeds germinated to a high degree
and in spring. In temperate climates, seedlings germinating in autumn face harsh environ-
mental conditions during winter combined with low competition, while spring germina-
tion is associated with more favorable environmental conditions, but higher competition
(Masuda and Washitani 1992). Since soft and green seeds mostly germinated in autumn,
the winter survival of the emerging seedlings may be low, as L. polyphyllus seedlings seem to
be sensitive to freezing and showed high mortality when exposed to —10 °C (Arfin-Khan
et al. 2018). Furthermore, unripe seeds of roadside L. polyphyllus stands in Sweden were
prone to mold infection that led to very low germination rates (Broback 2015). The last
cutting date represents the state in which seeds are shed by the plant. Both ballistic seed
dispersal as well as the expression of physical dormancy go along with the drying of the
pods and the seed coat. Black and hard seeds are more prone to germinate in spring and
may thus have higher survival rates compared to green seeds. Furthermore, as L. polyphyllus
follows a c-strategy (Grime et al. 1988), it may be able to cope with higher competition

90 Yves P Klinger et al. / NeoBiota 60: 79-95 (2020)

in spring, especially in habitats with weak competitors, such as semi-natural grasslands.
Additionally, water impermeable/hard seeds are more prone to being carried over into the
seed bank (Russi et al. 1992b) or dispersed via endozoochory (Otte et al. 2002; D’hondt
and Hoffmann 2011). Although LZ. polyphyllus may not have invaded the seed bank of
meadows in our study region yet (Ludewig et al., unpublished data), a carry-over of seeds
should be avoided, as it makes invasive species management lengthier and more expensive.
Consequently, managers should target plants that still have green and soft seeds, which can
be considered less problematic despite germination percentages being relatively high.
Germination patterns differed between climate chambers and the common garden,
particularly after seeds darkened and became harder. Overall, germination percentages
in the climate chamber experiment (ca. 16%) were similar to the emergence rates found
by Sober and Ramula (2013) (21.5%), but relatively low compared to other studies on
L. polyphyllus (Elliott et al. 2011; Arfin-Khan et al. 2018; over 60%). We suggest that this
is at least partly due to the fact that seeds were not scarified and that dormancy was prob-
ably not broken by imbibition in the climate chamber experiment. This is also supported
by the results of the common garden experiment, in which germination percentages were
considerably higher than under laboratory conditions, mainly due to a second germina-
tion peak in spring after winter-stratification in situ. However, germination of L. polyphyl-
lus only slightly increased when seeds were pre-treated by cold in another study (Elliott
et al. 2011). Our results show that, while laboratory experiments give valuable informa-
tion on the environmental factors influencing germination, the germination patterns ob-
served under artificial conditions may diverge from germination dynamics under ambient
weather conditions (Hélzel and Otte 2004). A better understanding of invasive species
germination under natural conditions is necessary, as it can potentially reveal windows
of opportunity for invasive species management. We thus recommend to complement
germination experiments in climate chambers with common garden or field experiments.

Conclusions

Seeds of L. polyphyllus are capable of after-ripening and germinating even if plants are
cut while most seeds are still green and soft. Germination capability increased strongly
during the first weeks after seed set with a maximum when most seeds were brown to
black and not fully hardened. Therefore, L. polyphyllus stands should be cut before seed
set, if possible. If this is not feasible due to different limitations, we recommend cutting
while plants carry green and soft seeds. When stands with black and hard seeds are cut,
the plant material should be removed immediately to reduce propagule pressure on site.

Acknowledgments

We would like to thank Dr. Reinhard Stock, Dr. Volker Wachend6rfer, and Dr. Franz-
Peter Heidenreich for continuous interests and suggestions as well as constructive sup-
port of our study. Furthermore, we thank Torsten Kirchner (Wildlandstiftung Bavar-

Germination depends on cutting date i

ia), Michael Geier and Tobias Gerlach (Bavarian Administration of the Biosphere Re-
serve Rhén), as well as Torsten Raab (Hessian Administration of the Biosphere Reserve
Rh6n). We greatly thank Josef Scholz vom Hofe for assistance with propagule and
data collection both in the field and in the lab, Sabrina Rothen for data preparation,
Sarah Harvolk-Schéning for statistical support, and Melanie Schindler for valuable
comments on the final version of the manuscript. We are grateful to two anonymous
referees for their insightful comments on an earlier version of the manuscript.

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Germination depends on cutting date 95

Supplementary material |

Model Tables

Authors: Yves P. Klinger, Rolf Lutz Eckstein, David Horlemann, Annette Otte, Kristin

Ludewig

Data type: table xlsx-file

Explanation note: Model summary tables.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/neobiota.60.56117.suppl1