The invasive species Ulex europaeus modifies its seeds morphology and

germination pattern as it moves up along an elevation gradient

Juliana Portill𝐚𝟏 , Carol Garzó𝐧𝟏 , Eloisa Lass𝒐𝟏,𝟐
1. Grupo de Ecología y Fisiología Vegetal, Departamento de Ciencias Biológicas, Universidad de Los Andes
2. Smithsonian Tropical Research Institute.

Abstract Rapidly change of life history traits influence the success of biological invasions and extend
the range of habitats including suboptimal environments. Ulex europaeus is an invasive plant introduced
across a broad latitudinal gradient and found in a wide range of habitats and climates. The aim of this
study was to evaluate if changes in the germination pattern of U. europaeus are linked with their
capacity to colonize new sites at higher elevations. For this purpose, we evaluated 1) morphological
traits, as seed size, pod size and number of seed per pod and 2) germination traits, as rate and velocity
of germination. Plants from higher elevations (3300m.a.s.l) produced smaller and more numerous seeds,
but with slower germination and reduced germination rate that plants form lower elevations
(2700m.a.s.l). These changes could be linked to different invasion strategies, in populations already
stablished at lower elevations and populations trough a colonization process towards higher elevations.

Introduction

Invasive species once established in a new geographical area can cause environmental
degradation and increase the vulnerability of the native community. This negative impact has
a significant effect on biodiversity and functioning of the ecosystems, because of the rapid
dominance of invasive species and consequently the decrease of native species, being one of
the most severe threats for biodiversity, preceded only by habitat loss and fragmentation
(Foxcroft et al. 2013).

Gorse (Ulex europaeus) is one of the worst invasive species in the world according to the IUCN
invasive Species Specialist Group (ISSG) and listed in the “100 of the World’s Worst Invasive
Alien Species’ (Lowe et al. 2000). U. europaeus is a woody legume of the family Fabaceae,
native to the western coastal areas of Europe and introduced in many parts of the world at
different latitudes (Schmid et al. 1997). At present is considered invasive in New Zealand,
Australia, South and North America and it is found in a wide range of habitats and climates, at
altitudes that vary from 0 to 4000 m (Hornoy et al. 2011). The invasive nature of gorse has
been attributed to several physiological features including a long-term perennial growth that
creates a dense thicket that excludes native vegetation, higher tolerance to soil acidity than
common Fabaceae and a large persistent in the soil seed bank (Leary et al. 2006).

In Colombia, gorse is frequently found in the Andean Region, where severe infestations have
been reported in the provinces of Cundinamarca and Boyacá, especially in pastures, perturbed
areas and along borders and highways (León & Vargas, 2011). Additionally, there are reports
of gorse growing in mountain Andean forests and Paramos in Sumapaz, Chingaza and Pisba.
They represent an additional threat because the high amount of tannins contained in gorse are
flammable and increases the frequencies of fires. After a fire, only gorse is capable of
regeneration owing to his seed bank and regrowth capacity from roots, promoting the loss of
forest and Paramo native species.

Recent studies have demonstrated the adaptive potential of the germination pattern of U.
europaeus linked with their capacity to colonize new sites. In regions where gorse has been
recently introduced, substantial differences have been observed in the velocity of germination,
a reduction in the physical dormancy of the seed and less sensitivity to mould, as compared
with the native range (Udo et al. 2017). These changes in seed traits are supposed to have
developed as a strategy which favor the rapid occupation of new sites.

In the Oriental range of the Northern Andes gorse has been reported in forest and roadways
close to Bogotá, between 2700 and 3300 meters above sea level. As gorse moves up the
mountain, they may have to modify its germination potential as a strategy to expand its invasion
range to higher elevations. The aim of this study was to evaluate if gorse is changing their seeds
and germination pattern in relation to elevation, and to examine possible new life traits changes
of U. europaeus in highland forests ecosystems after its introduction. We compared several
seeds and germination traits from the extreme points of its altitudinal distribution in the Eastern
mountains of Bogotá. Traits evaluated are germination velocity, germination rate with or
without prior scarification, seed size, number of seeds per pod, and pod size.

Materials and Methods

Study species

Ulex europaeus is a 1-4 m densely branched spiny shrub that can live up to 30 years, is
characterized by their reduced leaves that looks like stiff spines (Lee et al. 1986). Flowering
begins in the second or third year and spans over several months per year. Flowers are
hermaphrodite and are pollinated by large insects such as honey bees or bumblebees (Herrera,
1999). The pods star to form immediately after the fertilization of flowers, young pods are
green and soft and become brown and hard in the maturation (Richardson et al. 1998). Each
gorse plant can produce ten thousand seeds per year, which are primarily dispersed by ejection
of an explosive dehiscence of pods. The seeds have a hard-impermeable integument which
induces physical dormancy and remain viable up to 30 years. Hard-seededness is greatest when
the seeds are first ejected and declines as the seed lines on the soil surface, where soil acidity,
friction against rock and sand and fires contribute to the abrasion of the seed coat (Ivens, 1983).

Experimental Design

Seeds were collected in the border of a road that connects Bogotá with Choachí over an
elevation gradient. The lowest sampling point was at 2700 m.a.s.l and the highest sampling
point at 3300 m.a.s.l. At each elevation five individuals were sampled. Seeds were collected
on January, February and April of 2019 and stored in the dark at room temperature from 3 to 7
days until germination experiment begin.

Germination experiment

Batches of 400 seed were prepared by pooling 80 seeds from each of the five individuals
collected at each elevation. Seeds were immersed in a 10% sodium hypochlorite solution for
10 minutes to sterilize the surface and washed with tap water. Half of the seed (200) were
scarificated using a sterile scalpel to remove a small portion of the seed coat and the other 200
seeds were left untouched. Then all the seeds were soaked in tap water for 24 h for imbibition
and then sown in petri dishes on filter paper soaked with 5 ml of tap water. For each elevation,
there were five replicates (five petri dishes) with 40 scarificated seeds and five replicates with
40 non_scarificated seeds. All dishes were placed in a growth chamber (PGC-14L , Percival)
were temperature was programed to varied diurnally to resemble natural conditions at the
paramo as follow: 12 C from 7:00 to 10:00, 20 C from 10:01 to 12:00, 25 C from 12:01 to
13:00, 20 C from 13:01 to 16:00, 12 C from 16:01 to 19:00 and 5º C overnight from 19:01
pm to 7:00. am. The chamber has 13 hours of light and 11 hours of darkness.

Seed germination

Seeds were monitored four times per week for five weeks. At each monitoring the germinated
seeds were counted. Seeds were registered as germinated when the radicule was visible.
Germination was assessed using two indices; the germination rate or the percentage of seeds
that germinated during the observation period (G%) and the germination velocity or median
germination time (𝑡() ), the time it takes for 50% of final maximum germination (Ranal &
Santana, 2006) as follow:

Germination Percentage:
𝑔
𝐺% = - 0 ∗ 100
𝑛

Median germination time

(𝑁 + 1)
− 𝑛5
𝑡() = 𝑡5 + 7 2 > ?𝑡= − 𝑡5 @
𝑛= − 𝑛5

Where 𝑛5 and 𝑛= are total number of seed germinated by adjacent counts at time 𝑡5 and 𝑡= , where
BCD
𝑛5 < E
< 𝑛= , and 𝑁 is the number of total seeds germinated.

Statistical Analysis

We used RStudio(RStudio Team, 2016), the lme4(Bates et al, 2015) and aov (Chambers et al.
1992) packages. We run a linear mixed effect analysis to evaluate the relationship between pod
and seed traits (pod size, seed size and seeds per pod) and elevation. We entered elevation as
fixed effect into the model, as random effect we had intercept for individuals; p-values were
obtained by likelihood ratio tests of the full model with the effect of the character against the
model without the effect in question. Germination data were analyzed in an ANOVA model,
with elevation as fixed factor.

Results

Fig. 1 Pod size (A), seed size(B) and number of seeds per pod (C) of Ulex europaeus at a highland
(3300 m.a.s.l) and lowland (2700 m.a.s.l) elevations.
Seed traits

All seed traits varied with elevation (Fig. 1). At higher elevations, pods were bigger (1.78
± 0.14 vs. 1.32 ± 0.14 cm; 𝜒 E (1) = 17.8; p = 0.0005), seeds were smaller (0.19 ± 0.02 vs.
0.22 ± 0.02 cm; 𝜒 E (1) = 9.5; p = 0.023), which results in a major number of seeds per pod
(5.30 ± 1.74 vs. 3.03 ± 1.28 cm; 𝜒 E (1) = 10.8; p = 0.013).

Fig. 2 Germination rate of Ulex europaeus for scarified and unscarified seeds of different elevations.

Fig. 3 Number of days required to reach 50% of germination (𝑡() ) for scarified seeds of Ulex
europaeus from different elevations.
Germination Percentage

As scarified and unscarified seed produced evident contrasting results, scarification was not
used as factor in ANOVA model. Instead seeds where analyzed separately with elevation as
single fixed factor. The elevation effect was significant for both scarified seeds (F(1) = 8.05;
p = 0.008 ) and unscarified seeds (F(1) = 6.06; p = 0.024), but with an inverted pattern (Fig. 2).
Scarified seeds germinated in higher numbers at the low elevation site, while non scarified
seeds germinated in higher number at the high elevation site (Fig 2).

Germination Velocity

Germination velocity was calculated using 𝑇() , the time required to reach 50% of germination.
This index was calculated only for scarificated seeds, because none of the unscarified seed
batch germinated more than 50% after the 30 days of monitoring. The effect of elevation was
also significant for 𝑇() (F (1)= 16.54, p < 0.001), seeds from the low elevation site germinated
faster (16.02 ± 1.08 days) in comparison to the seeds collected at the high elevation site (18.09
± 1.65 days)(Fig. 3).

Discussion

We show that seed traits and germination of Ulex europaeus varies with elevation. Populations
at the high elevation site (3300m.a.s.l) produced smaller and more numerous seeds, but with
reduced germination rate G(%) and speed 𝑡() , than population at the lower elevation site
(2700m.a.s.l). These differences could be linked to the contrasting colonization history at
distinct elevations. For example, in sites with longer time of colonization, as in the low
elevation site at 2700 m.a.s.l, plants have had the time to adjust their seeds traits to match the
environmental conditions of the site, they produce larger seeds that can accumulate more
resources improving germinations success and better tolerance to abiotic stresses (Westoby et
al. 2002), resulting in the higher germination rate and velocity observed in this population wich
guarantees the establishment success. However, the populations at 3300 m.a.s.l , which is
probably in the front of invasion towards higher elevations, a population still going through
an acclimatization process of new environmental conditions, cannot achieve optimal
germination. For this population in the front of invasion, producing higher number of seeds
increase their dispersal efficiency ( Moles et al., 2005). Migration to the highland in this
species may occurs through patches of suitable habitat in a less suitable-matrix, as stepping
stones towards higher elevation facilitating migration and thus range expansion (Lembrechts
et al. 2018).

We also show that scarification improves germination drastically at all elevations. At al

elevation the scarified seeds were germinated in larger numbers (above 80%) whereas only
30% or less of the unscarified seeds germinated. Interestingly, we observed that unscarified
seeds germinated in larger numbers at higher elevation in comparison to lower elevations.
Dormancy and hard seed coats is considered a strategy that enables persistent seed banks and
large persistent in the soil seed bank has been reported for this species (Leary et al, 2006).
Under natural conditions hard-seedness is greatest when seed is first shed and progressively
declines as the seed lies on surfaces and becomes incorporated into the soil, due to soil acidity,
friction with rocks or abrasion by fire, until the seed is sufficiently permeable and germinate
(Guo et al. 2000, Ivens, 1983). In the case of scarified seeds, the treatment allows total
permeability of the seeds, but for seeds without scarification treatment, germination depends
on hard-seedness and hence seed permeability. Diminishing hard-seednes could represent
another strategy for rapid colonization of new environments. Changes of physical dormancy in
accordance with local climate conditions has in fact been observed in other tropical Fabacea
(Lacerda et al. 2004), furthermore other studies have revealed a reduction in physical dormancy
of gorse in areas where introduction has taken place (Udo et al. 2018).

Invasive plants are able to grow and reproduce in a wide range of environmental conditions;
success in biological invasion is facilitated by its multipurpose genotypes and the ability to
change dispersal traits quickly (Pichancourt & Van klinken 2012). The use of seed and fruit
heteromorphic to overcome different microhabitats and environmental conditions, allow the
colonization of highly unpredictable habitats (Martín-Forés et al. 2017). It has been observed
that for traits such as seed size, seed hardness, physical protection and chemical protection,
phenotypes can respond in a plastic manner when they encounter novel environments,
phenotypic diversity has been considered important for achieving dominance in a range of
diverse habitats in many invasive species (Levin, 2000).

Environmental effects on seed traits are also notable when comparing germination patterns of
gorse between native ranges and invasive ranges, but also between different sites where gorse
has been introduced. In the Andean Region of Colombia, gorse seeds are slower at germinating
than seeds at their native habitats. The median germination time 𝑡() we observed for gorse
seeds of the Andean region of Colombia was 16 days at 2700 m.a.s.l and 18 days at 3300
m.a.s.l, both values are considerably higher than the germination time observed at their native
habitats; like in France with 𝑡() of 11 days (Udo et al. 2018 ) and Spain with 𝑡() of 8 days
(Baeza and Vallejo, 2006), and other invasive sites like Reunion with 𝑡() of 9 days(Udo et al.
2018).

The great ability of gorse in changing morphology and germination pattern reinforces its
colonization success and permits rapid establishment in differing environments. Invasion at
higher elevations is already taken places, even in places beyond its climatic niche. Greater
efforts are necessary to prevention the spreading of this highly invasive species, especially in
ecosystems of high importance like paramos and high Andean forest.
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