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\begin{document}

\title{Using semi-natural and simulated habitats for seed germination ecology}



\author[1]{Simon Kallow}%
\author[1]{Katrijn  Quaghebeur}%
\author[2]{Bart Panis}%
\author[3]{Steven Janssens}%
\author[4]{John  B. Dickie}%
\author[5]{Lavernee Gueco}%
\author[1]{Rony Swennen}%
\author[3]{Filip Vandelook}%
\affil[1]{KU Leuven Faculty of Bioscience Engineering}%
\affil[2]{Catholic University College Leuven}%
\affil[3]{Botanic Garden Meise}%
\affil[4]{Royal Botanic Gardens Kew}%
\affil[5]{University of the Philippines Los Banos}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
1. Ecologically meaningful seed germination experiments are constrained
by access to seeds and relevant environments for testing at the same
time. This is particularly the case when research is carried out far
from the native area of the studied species. 2. Here, we demonstrate an
alternative - the use of glass houses in botanic gardens as
simulated-natural habitats to extend the ecological interpretation of
germination studies. Our focal taxa were banana crop wild relatives
(Musa acuminata subsp. burmannica, M. acuminata subsp. siamea and M.
balbisiana), native to tropical and subtropical Southeast Asia. Tests
were carried out in Belgium, where we performed germination tests in
relation to exposure to sun and foliage-shading, seed burial-depth in
different heated glass house compartments, as well as seed survival and
dormancy release in the soil. We anchored the interpretation of these
studies by also conducting an experiment in a semi-natural habitat in
the species native range (M. balbisiana - Los Ba\selectlanguage{ngerman}ños, the Philippines),
where we tested germination responses to exposure to the sun and shade.
Using temperature data loggers, we determined temperature dynamics
suitable for germination in both these settings. 3. In semi-natural and
simulated-natural habitats, seeds germinated in response to exposure to
direct solar radiation. Seed burial-depth had a significant but marginal
effect by comparison, even when seeds were buried to 7cm in the soil.
Temperatures at sun-exposed compared to shaded environments differed by
only a few degrees Celsius. Maximum temperature of the period prior to
germination was the most significant contributor to germination
responses and germination increased linearly above a threshold of 23°C
to the maximum temperature in the soil (in simulated natural habitats)
of 35°C. 4. Glass houses can provide useful environments to aid
interpretation of seed germination responses to environmental niches.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\section*{Using semi-natural and simulated habitats for seed germination
ecology}

{\label{using-semi-natural-and-simulated-habitats-for-seed-germination-ecology}}

Simon Kallow\textsuperscript{1,2,3*}, Katrijn
Quaghebeur\textsuperscript{2,3}, Bart Panis\textsuperscript{2,4}, Steven
B. Janssens\textsuperscript{3,5}, John Dickie\textsuperscript{1},
Lavernee Gueco\textsuperscript{6}, Rony Swennen\textsuperscript{2,7},
Filip Vandelook\textsuperscript{3}

\textsuperscript{1} Royal Botanic Gardens Kew, Millennium Seed Bank,
Wakehurst, Ardingly, Sussex, RH17 6TN, UK

\textsuperscript{2} Katholieke Universiteit Leuven, Department of
Biosystems, Willem de Croylaan 42, 3001, Leuven, Belgium

\textsuperscript{3} Meise Botanic Garden, Nieuwelaan 38, 1860 Meise,
Belgium

\textsuperscript{4} Bioversity International, Willem de Croylaan 42,
3001, Leuven, Belgium

\textsuperscript{5} Katholieke Universiteit Leuven, Biology Department,
Kasteelpark Arenberg 31, 3001 Leuven, Belgium

\textsuperscript{6} National Plant Genetic Resources Laboratory,
Institute of Plant Breeding, College of Agriculture and Food Science,
University of the Philippines, Los Ba\selectlanguage{ngerman}ños, 4031 Laguna, Philippines

\textsuperscript{7} International Institute of Tropical Agriculture, c/o
Nelson Mandela African Institution of Science and Technology, Arusha,
Tanzania

Corresponding author: Simon Kallow (S.Kallow@kew.org)

Running title: Seed ecology in non-natural habitats

Word count: 6821

\subsection*{Keywords}

{\label{keywords}}

Botanic gardens, crop wild relatives, gap detection, seed germination

\subsection*{Abstract}

{\label{abstract}}

\begin{enumerate}
\tightlist
\item
  Ecologically meaningful seed germination experiments are constrained
  by access to seeds and relevant environments for testing at the same
  time. This is particularly the case when research is carried out far
  from the native area of the studied species.
\item
  Here, we demonstrate an alternative - the use of glass houses in
  botanic gardens as simulated-natural habitats to extend the ecological
  interpretation of germination studies. Our focal taxa were banana crop
  wild relatives (\emph{Musa acuminata} subsp. \emph{burmannica, M.
  acuminata} subsp. \emph{siamea} and \emph{M. balbisiana} ), native to
  tropical and subtropical Southeast Asia. Tests were carried out in
  Belgium, where we performed germination tests in relation to exposure
  to sun and foliage-shading, seed burial-depth in different heated
  glass house compartments, as well as seed survival and dormancy
  release in the soil. We anchored the interpretation of these studies
  by also conducting an experiment in a semi-natural habitat in the
  species native range (\emph{M. balbisiana -} Los Baños, the
  Philippines), where we tested germination responses to exposure to the
  sun and shade. Using temperature data loggers, we determined
  temperature dynamics suitable for germination in both these settings.
\item
  In semi-natural and simulated-natural habitats, seeds germinated in
  response to exposure to direct solar radiation. Seed burial-depth had
  a significant but marginal effect by comparison, even when seeds were
  buried to 7cm in the soil. Temperatures at sun-exposed compared to
  shaded environments differed by only a few degrees Celsius. Maximum
  temperature of the period prior to germination was the most
  significant contributor to germination responses and germination
  increased linearly above a threshold of 23°C to the maximum
  temperature in the soil (in simulated natural habitats) of 35°C.
\item
  Glass houses can provide useful environments to aid interpretation of
  seed germination responses to environmental niches.
\end{enumerate}

\subsection*{Introduction}

{\label{introduction}}

Ideally, seed germination ecology studies are carried out in both
natural habitats (NHs) and laboratory conditions (LCs) (Baskin \& Baskin
2014). This allows variables affecting germination to be clearly
identified and ecologically interpreted. Interpretation is usually made
in relation to spatial and temporal niches in NHs, or perhaps
semi-natural habitats (Semi-NHs) (Table 1).

\textbf{Table 1.} Descriptions of environments for seed germination
studies.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
Name\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
Definition\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
Germination example\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
Control level\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
Interpretation level\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Natural habitat (NH)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Areas composed of viable assemblages of plant and/or animal species of
largely native origin and/or where human activity had not essentially
modified an area's primary ecological functions and species composition.
\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
(Dinsdale, Dale \& Kent 2000)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Semi-natural habitat (Semi-NH)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Ecological assemblages that have been substantially modified in their
composition, balance or function by human activities.
\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
(Stephens, Castro-Morales \& Quintana-Ascencio 2012)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Simulated natural habitat (Simulated-NHs)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
A wholly constructed environment, made to resemble a NH.\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Laboratory conditions (LC)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
A un-natural environment, variables are clearly defined and
controlled.\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
(Mattana \emph{et al.} 2016)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}= (European Investment Bank 2018)

In LC germination experiments, such as those using incubators, most
variables are kept constant (e.g. light intensity, sowing medium,
water-availability, timing of diurnal temperature cycle), and one or two
are manipulated with a few combinations (e.g. three or four temperatures
of diurnal cycles). Researchers select the conditions of variables based
on knowledge from NH microclimates, but in reality, these are
notoriously difficult to exactly define (e.g. Dinsdale, Dale \& Kent
2000). Indeed, important variables may inadvertently be omitted from
experimental designs. Interpretation of LC experiments in an
ecologically meaningful way is difficult because it will be based on
many assumptions, both in selecting variables to test and extrapolating
interpretation to NHs - especially if NHs have not been adequately
studied.

By contrast, in NH experiments, many, often unknown, variables
interplay, one or two of which may be controlled. If researchers were to
control all combinations of NH variables in LCs they would soon run out
of seeds, time and space. Interpretation of NH experiments depends on
dynamically recorded variables that cannot be well controlled, some even
being irrelevant to germination ecology. It goes without saying that NH
experiments can only truly be performed in regions where the plant is
native, whereas LC experiments can be carried out anywhere with suitable
equipment.

Alternatives to NH and LC are semi-natural habitats (Semi-NHs) or
simulated natural environments (Simulated-NHs). Examples of Semi-NHs
include fields or farm edges, in or close to a species' native region.
The term simulated-natural environment (or habitat as used here) was
used by Kaeberlein \emph{et al.} (2002) to define an environment of
natural seawater and sediment the authors placed in aquariums to culture
previously `uncultivable' marine microorganisms. Such Simulated-NHs are
less controlled than LCs, but allow better interpretation of findings
and may include important factors that are not well understood or known
and so may inadvertently be omitted from LCs. Glass houses, such as
those in botanic gardens, are examples of Simulated-NHs, as they mimic
NHs and variables are not well under control.

In botanic gardens, living collections are often arranged according to
geographic plant communities, each compartment or grouping representing
a pseudo or Simulated-NH. These living collections are a valuable
resource in studying plant ecology, particularly when NHs are
challenging to access (Perez \emph{et al.} 2019). Many botanic gardens
also hold seed banks (469 gardens), and carry out seed or spore research
(155 gardens) (BGCI 2021). As botanic gardens are biased towards
temperate regions in the Northern hemisphere (Mounce, Smith \&
Brockington 2017), there is opportunity to enhance interpretation of
seed germination studies using botanic gardens as Simulated-NHs when it
is not possible to do so in native regions (Faraji \& Karimi 2020).

Wild banana species (\emph{Musa} L.) are native to tropical and
subtropical Asia to the western Pacific (Govaerts \& H\selectlanguage{ngerman}äkkinen 2006).
Their fruit contains many hard darks seeds, 3-7 mm in diameter (Chin
1996). The conditions for germination are not well understood and
germination is notoriously inconsistent and often very low
(Kallow\emph{et al.} 2020; Panis, Kallow \& Janssens 2020; Singh
\emph{et al.}2021). LC experiments show a requirement for alternating
temperatures (Stotzky \& Cox 1962; Kallow \emph{et al.} 2021), but no NH
experiments have been executed to interpret this. For instance, is this
requirement a gap or depth detection mechanism affected by
microclimates? And do species respond differently?

Understanding seed germination ecology of wild bananas is not only of
ecological interest, it is also important for global food security. Seed
banking crop wild relatives efficiently protects genetic material and
makes it available for phenotyping and breeding (Dempewolf \emph{et
al.}2017), it is included in UN Sustainable Development Target 2.5 (UN
General Assembly 2015). Optimized germination is a vital component of
seed bank management and breeding - without it, viability is difficult
to monitor and access to plants for research and breeding is constrained
(FAO 2014; Batte \emph{et al.} 2019; Amah \emph{et al.} 2020).

In the present study we examined germination responses in a Semi-NH and
Simulated-NHs of the two primary crop wild relatives of
banana:\emph{Musa acuminata} (subsp\emph{. siamea} N.W. Simmonds, and
subsp.\emph{burmanicca} N.W. Simmonds), and \emph{M. balbisiana} Colla
(De Langhe \emph{et al.} 2009). Specifically, we aim to answer the
following questions that cannot be answered in LCs: (1) What
environments stimulate or inhibit \emph{Musa} germination? (2) Are
\emph{Musa} seeds dormant, and if so how is this broken in the
environment? (3) Can\emph{Musa} seeds remain viable in the soil?

\subsection*{Materials and methods}

{\label{materials-and-methods}}

\begin{enumerate}
\tightlist
\item
  \emph{Semi-natural habitat (nursery, Philippines)}
\item
  \emph{Plant material}
\end{enumerate}

We collected a bunch (an infructescence) of \emph{Musa
balbisiana}(accession GB61996) containing seeds from the field genebank
at the National Plant Genetic Resources Laboratory (NPGRL), Institute of
Plant Breeding, University of the Philippines, Los Baños. Seeds were
extracted by opening fruit and washing seeds in flowing water to remove
all pulp. Seeds were then left on a tray in the laboratory to surface
dry for seven days prior to sowing.

\textbf{Table 2.} Accessions used for germination experiments in
simulated natural environments, V= viability percentage from embryo
rescue tests in 2019 and 2020.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
Accession & Taxa & Source & Native distribution \textsuperscript{b} &
Year collected & V\textsubscript{2019} & V\textsubscript{2020} &
Experiment\tabularnewline
\midrule
\endhead
GB61996 & \emph{M. balbisiana} & Philippines & Trop. \& Subtrop. Asia &
2019 & NA & NA & Semi-NH\tabularnewline
bur60 & \emph{M. acuminata} subsp. \emph{burmannica} & Guadeloupe & SW.
India, China (S. Yunnan) to Indo-China & 2014 & 90 & 17 &
Simulated-NH\tabularnewline
sia61-63 & \emph{M. acuminata} subsp. \emph{siamea} & Guadeloupe &
Indo-China to N. Pen. Malaysia & 2014 & 33 & 10 &
Simulated-NH\tabularnewline
bal106 & \emph{M. balbisiana} & China (Hainan) & Trop. \& Subtrop. Asia
& 2017 & 87 & 58 & Simulated-NH\tabularnewline
bal115 & \emph{M. balbisiana} & Nigeria & Trop. \& Subtrop. Asia & 2019
& 100 & 88 & Simulated-NH\tabularnewline
\bottomrule
\end{longtable}

b = Govaerts and H\selectlanguage{ngerman}äkkinen, 2006

\emph{Solar radiation and substrate}

We used the nursery of the NPGRL (latitude 14.153, longitude 121,262),
as a Semi-NH for germination testing. We used locations either exposed
to solar radiation (`sun') - only lightly shaded in the fine screen
house, or without direct exposure to solar radiation (`shade') - in an
open-sided cabinet covered at the top also in the screen house. We sowed
seeds in plastic trays (100x40x10 cm), using two types of substrate
(clay loam soil and fine sand), and covered seeds with 5 mm of
substrate. Two replicates of 200 seeds were used for each treatment
combination. We recorded the temperature and relative humidity (RH),
every 20 minutes for 55 days in sowing locations using data loggers
(Tinytag View 2, Gemini Data Loggers, Chichester, UK). Trays were
watered daily, emergent seedlings were recorded and removed weekly. The
test was concluded after 55 days.

\emph{Simulated-natural environment (glass house compartments, Belgium)}

\emph{Plant material}

We studied germination responses in Simulated-NHs in relation to
foliage-shading and seed burial-depth using two \emph{Musa} species
(total three taxa, Table 2). Seeds were selected from the collection of
Bioversity International/KU Leuven (Leuven, Belgium) and were supplied
for scientific use with a phytosanitary and origin certificate from
China (Hainan), Guadeloupe, and Nigeria. Seeds were from open-pollinated
accessions in living collections and were air shipped from source to
Leuven as complete bunches, where they were extracted as described
above, apart from bal106 which were provided as extracted and cleaned
seeds transported to Leuven, Belgium. After ambient drying, seeds were
placed in the refrigerator in paper bags, until 2019 when they were
sealed in aluminum bags and stored in the refrigerator at approximately
6\% moisture content, fresh weight basis. Viability was assessed prior
to sowing (both in 2019 and 2020) using ER (method described by
Kallow\emph{et al.} 2020).

\emph{Foliage-shading and seed burial-depth}

We selected a total of six Simulated-NHs for germination tests. Five
were in three compartments of Meise Botanic Gardens glass house, Belgium
(latitude 50.925, longitude 4.330), and one in the full-ground glass
house of KU Leuven, Belgium (latitude 50.860, longitude 4.680).
Simulated NHs were selected to represent various heating regimes in
different compartments and levels of foliage-shading/exposure to solar
radiation (Table 3). All compartments included living banana specimens.

Seeds were sown in square plastic pots (9x9x10 cm), at two burial-depths
(1 cm and 7 cm from the surface), using potting compost (Peltracom,
composition: 70 \% white peat and 30 \% black peat; pH: 5.5- 6.5;
particle size: 0-10 mm). Two replicates of 30 seeds of each accession
were sown in separate pots which were then also buried to be level with
soil surface. Data loggers (Tinytag Transit 2 TG4080, Gemini Data
Loggers, Chichester, UK) were buried at each location and burial-depth.
Loggers were set to record temperatures every 20 minutes. Additionally
in 2020, loggers were placed at the soil surface to record light
intensity and surface temperature (HOBO Pendant MX2202, Onset, Cape Cod,
Massachusetts, US). Germination was monitored weekly and emergent
seedlings were recorded and removed. Seeds were sown in early March
2019, and again in early March 2020s, this is the end of the
winter/start of the spring season in Belgium (mean outdoor temperature
6.4°C, climate-data.org). The experiment was concluded in March 2021.

\textbf{Table 3} . Summary heating regimes of glass house compartments
used, temperatures are the thermostat temperature below which artificial
heating is instigated.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Compartment name & Institution & Exposure & Day (\selectlanguage{ngerman}°C) & Night
(\selectlanguage{ngerman}°C)\tabularnewline
\midrule
\endhead
Leuven & KU Leuven & Exposed & 15 & 15\tabularnewline
Mabundu & Meise Botanic Gardens & Exposed & 25 & 25\tabularnewline
Tropical & Meise Botanic Gardens & Shaded & 20 & 18\tabularnewline
Tropical & Meise Botanic Gardens & Exposed & 20 & 18\tabularnewline
Spring & Meise Botanic Gardens & Shaded & 10 & 8\tabularnewline
Spring & Meise Botanic Gardens & Exposed & 10 & 8 o\tabularnewline
\bottomrule
\end{longtable}

\emph{Dormancy and stratification in the soil}

To assess dormancy and dormancy loss in the soil (stratification), seeds
from the two \emph{M. balbisiana} accessions (bal106 and bal115) were
incubated at alternating 35\selectlanguage{ngerman}°C in the light for 6 hours and 20°C in the
dark for 18 hours (based on Stotzky, Cox \& Goos 1961; Kallow \emph{et
al.} 2021). Additionally, seeds were buried at two of the cooler
Simulated-NHs (Spring/Exposed and Leuven/Exposed) in March 2019 and
exhumed each month for a total of three months and placed in the
incubator conditions described. Seeds were enclosed in small nylon
meshed bags and were buried at 7 cm depth in March 2019. Two replicates
of 30 seeds were used for each treatment and accession. For incubation,
seeds were sown on moist sand (50 g fine sand, 14 ml deionized water) in
Petri dishes (9 cm diameter), sealed in plastic bags, at Meise Botanic
Garden. Germinated seedlings of these were recorded and removed weekly
for two months. Germination was counted as radicle emergence to 2 mm.

\emph{Survival in the soil}

At the end of the experiment, seeds planted at shaded Simulated-NHs
(locations that showed no emergent seedlings in results) were extracted
from pots and tested for viability using a tetrazolium chloride staining
test or by incubation. Seeds sown in both 2019 and 2020 were used. Pots
containing seeds were removed, seeds were separated from compost by
sieving under running water. Extracted seeds from the first replicate of
each treatment were tested for viability with tetrazolium chloride (TTC)
with a maximum of 20 seeds. The TTC tests were carried out on embryos
carefully extracted from seeds, using 0.5 \% TTC solution buffered to pH
7 (method described by Kallow \emph{et al.} 2020), incubated at 24°C for
24 hours in the dark. Extracted seeds from the second replicate were
sowed in Petri dishes on top of moist sterilized potting compost
(Peltracom, composition: 70 \% white peat and 30 \% black peat; pH: 5.5-
6.5; particle size: 0-10 mm) and placed in an incubator at a 24-hour
cycled temperature pattern (based on temperature readings from Los
Baños, the Philippines - sun exposed site, Fig. 1a). Seeds in the
incubator were monitored every two weeks, for a maximum of five weeks.
If very few seeds were extracted from the soil for a replicate, TTC
tests were prioritized above incubator germination tests.

\emph{Data analysis}

We calculated summary indices for germination tests using
the\emph{GerminaR} package in R (Lozano-Isla, Benites-Alfaro \& Pompelli
2019). These included final germination percentage (GRP, \%) (Labouriau
\& Valadares 1983), mean germination time (MGT, days) (Czabator 1962)
and synchronization index (SYN, simultaneous germination within a
replicate = 1, no overlap between seeds of a replicate = 0) (Primack
1980; Ranal \& Santana 2006).

We summarized data logger readings to extract the mean, maximum,
minimum, and range (maximum-minimum) during experimental `cue periods'.
In semi-NH this was the whole experimental period (55 days). In
simulated-NHs for each year we summarized data filtered to include
readings during `cue periods' - these were a period prior to the MGT of
each replicate, set differently for shallow and deep planting based on
the mean MGT for shallow and deep planting (shallow=32 days, deep=39
days). For locations with no germination, we used the first 39 days
after sowing. We summarized logger readings for cue periods by
calculating mean, maximum, minimum, range temperatures and light
intensity readings (in 2020). We removed light intensity readings in the
dark (\textless{}40 lux), to account for changes in day/night length
during seasons and did not calculate minimum light intensity as it would
be the same in any site.

We then carried out redundancy analysis (RDA) of summarized logger data
in cue periods, scaled to unit variance, against a corresponding matrix
of factorial variables (species, compartment, exposure, depth) using
the\emph{vegan} R package (Oksanen \emph{et al.} 2019). The minimum
adequate RDA was found by comparison of Akaike Information Criterion
(AIC), by adding variables to the minimum model.

Final germination, for each experiment, was assessed using counts of
germinated seeds at the end of experiments against seeds that did not
germinate, thus accounting for sample size variance. Sample sizes were
adjusted in the analysis to only include viable seeds, estimated from ER
results of that year, using the formula:

\begin{equation}
adjusted\ sample=seeds\ sown\ x\ viability\ of\ year\ sown\nonumber \\
 \end{equation}

These data were used in generalized linear modelling (GLMs) of binomial
data with logit link function. If overdispersion was present in binomial
GLMs we used quasibinomial error structure. Minimum adequate models
(MAM) were produced by removing variables from maximum models after
comparisons with ANOVA and \emph{X} \textsuperscript{2} test. Estimated
marginal means were made from models and used for post-hoc analysis
using the \emph{emmeans} R package (Lenth 2020). Additionally, we used
GLMs for factorial variables on MGT and SYN using gamma error structure.
We assessed the effect of environmental variables in cue periods on
binomial germination outcomes using GLMs as described. Following this we
tested variables for breakpoints to assess temperature thresholds or
optimums. We did this on GLMs produced for each microclimate variable
separately by using the algorithm with bootstrapping in
the\emph{segmented} R package (Muggeo 2017). We used starting points
estimated by plotting GRP against microclimate variables and trend lines
using the non-linear regression method Loess.

We compared viability of seed survival of seeds exhumed after 1-2 years
in the soil from the incubation test, TTC test, and maximum achieved
from Simulated-NHs, against the original ER viability. We again used a
binomial GLM for this, as described, and post-hoc contrasts against
original ER with Dunnet tests. All analysis was performed in R (R Core
Team 2020).

\subsection*{Results}

{\label{results}}

\begin{enumerate}
\tightlist
\item
  \emph{Effects of exposure to solar radiation and substrate on seed
  germination}
\end{enumerate}

In the semi-NH exposure to direct solar radiation (sun) rather than
shade, significantly increased germination rates (z=20.963,
p\textless{}0.001, Fig. 1a). There was no significant effect of
substrate on germination outcome according to the GLM. Final germination
percentage in the sun was 72\% (soil), 71\% (sand) whereas in the shade
it was 19\% (soil), 12\% (sand).

The MGT in the shade was 22\selectlanguage{ngerman}±2 days (mean, standard deviation, used
hereafter), whereas in the sun it was slightly faster germinating in
18±2 days, albeit only few seeds germinated in the shade.
Synchronization index in the sun was 0.25±0.10 and in the shade
0.27±0.07.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig. 1} (a) Cumulative germination of \emph{Musa
balbisiana}seeds sown in the semi-natural habitat (nursery, Los Ba\selectlanguage{ngerman}ños,
the Philippines), seeds were sown in the sun and shade, and in sand and
soil (n=200, 2 replicates, mean values shown); (b) typical diurnal
temperature and (c) relative humidity profiles logged at sowing site
during germination test (Los Baños, the Philippines,
16\textsuperscript{th} November 2019).

Mean daily temperature in sun and shaded exposure was identical, but
standard deviation was approximately 1°C greater in the sun (27.3±2.8°C
sun, 27.3±1.9°C shade, Fig. 1b, Table 4). Most notably, this relates to
warmer maximum temperatures in the day of around 4°C in the sun. Mean
diurnal range in the sun was therefore also greater (8.9°C sun, 5.1°C
shade). Humidity in the sun and the shade was broadly similar, ranging
diurnally between 75 and 100\% RH (Fig. 1c), a function of temperature.

\textbf{Table 4.} Summaries of the daily temperature and humidity
recordings in the sun and shade at the location of the seed germination
experiments. Values in brackets are standard deviations of means.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
& Sun & Sun & Sun & Sun & Shade & Shade & Shade & Shade\tabularnewline
\midrule
\endhead
& Temperature (\selectlanguage{ngerman}°C) & Temperature (\selectlanguage{ngerman}°C) & Relative humidity (\%) &
Relative humidity (\%) & Temperature (\selectlanguage{ngerman}°C) & Temperature (\selectlanguage{ngerman}°C) & Relative
humidity (\%) & Relative humidity (\%)\tabularnewline
Daily & Night & Day & Night & Day & Night & Day & Night &
Day\tabularnewline
Mean & 24.5 (\selectlanguage{ngerman}±0.8) & 33.4 (\selectlanguage{ngerman}±3.6) & 81.3(\selectlanguage{ngerman}±16.6) & 100 (\selectlanguage{ngerman}±0.1) & 25.2
(\selectlanguage{ngerman}±0.7) & 30.3 (\selectlanguage{ngerman}±2.0) & 75.0 (\selectlanguage{ngerman}±9.8) & 97.5 (\selectlanguage{ngerman}±2.3)\tabularnewline
Max & 26.2 & 42.2 & 100 & 100 & 26.7 & 38.5 & 94.3 & 100\tabularnewline
Min & 23.0 & 24.7 & 10.1 & 99.6 & 23.6 & 25.8 & 56.9 &
90.9\tabularnewline
\bottomrule
\end{longtable}

\emph{Effects of foliage-shading and seed burial-depth on seed
germination}

\emph{Microclimate in simulated natural habitats}

Cue period microclimate variables were constrained by the factors room
and exposure in the minimum adequate RDA, and notably not by seed
burial-depth or species (r\textsuperscript{2}=0.88, Fig. 2). Exposure
had the greatest influence on the microclimate RDA (df=1, F=262.15,
p\textless{}0.001), room had more variance (df=3, F=144.
P\textless{}0.001). Unsurprisingly, compartments with higher thermostat
temperature thresholds (Table 3) were clustered with higher mean and
minimum temperatures. These clusters did not include variables relating
to extremes i.e., maximum temperatures, temperature ranges and soil
surface values (for temperature and light). These were clustered
separately and correspond to exposed microclimates. Shaded microclimates
varied less than exposed, hence the different shapes of ellipses in Fig.
2.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 2} Redundancy analysis ordination of microclimates used as
simulated natural habitats in glass houses for seed germination
experiments (r\textsuperscript{2}=0.88), ellipses are exposed and shaded
sites (confidence limit= 0.95), green text is explanatory factors
(Comp=compartment, Exp=level of exposure to sun), blue text are
microclimate variables achieved from data loggers at site (s=from data
logger at soil surface, temp=temperature).

Shaded microclimates obviously received less light intensity (731\selectlanguage{ngerman}±240
lux shaded, 4491±1621 lux exposed) (Fig. S1) and had a lower maximum
temperature (25.8±1.0°C shaded, 40.7±7.1°C). In the soil, the maximum
temperature, again, was less at shaded sites compared to exposed sites
(19.5±2.2°C shaded, 27.1±4.6°C exposed). The relationship between light
intensity and temperature at the soil surface was asymptotic, above 2000
lux there was very little increase in temperature (Fig. S2).

\emph{Effects of microclimate factors on seed germination}

In simulated-NHs, no seeds germinated in any shaded microclimates. These
were then excluded from statistical analysis. Compartment and depth
remained in the MAM, species and year were excluded after testing (Fig.
3a). Seeds had significantly higher probability of germinating in the
two compartments that have a higher heating regime (compartments Mabundu
, z=4.531, p\textless{}0.001, and Tropical z=6.586, p\textless{}0.001).
Across all rooms, shallower buried seeds were more likely to germinate
(z=6.491, p\textless{}0.001).

Seedling emergence happened sooner after sowing in the compartments with
warmer heating regimes (Mabundu, t=8.557, p\textless{}0.001 and Tropical
6.805, p=\textless{}0.001, Fig. 3b). Predictably, shallow buried seeds
at 1 cm emerged quicker than those buried at 7 cm (t=2.657, p=0.010).
There was no effect of species on time to germination (seedling
emergence), so this was excluded from the model, leaving compartment and
burial-depth.

Synchronization was significantly greater in the cooler compartments
(Spring and Leuven) for \emph{M. balbisiana} (Fig. 3c), particularly in
the pairwise contrast between Leuven and Mabundu (z=3.110, p=0.010).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig. 3} Estimated marginal means of minimal adequate GLMs on
germination of \emph{Musa} seeds buried in Simulated-NHs in Belgium (a)
Binomial germination outcome; (b) mean germination time; (c)
synchronization index.

\emph{Effects of microclimate variables on seed germination}

The effect of each microclimate variable on final germination percentage
was visualized (Fig. S3). These were then used to estimate breakpoints
on GLMs produced from binomial germination outcomes (Fig. 4). Results of
this show temperatures (at seed burial level) operate a threshold
mechanism, above which germination increases linearly (on the logit
scale), within the limits of temperatures achieved in simulated-NHs.
Therefore, for germination to occur mean soil temperature needs to be
above 19\selectlanguage{ngerman}°C, maximum temperature above 23°C, minimum temperature has much
less of an effect. At the soil surface, the effect of mean temperature
is positively linear to an optimal 24°C after which germination is
reduced. The effect of maximum temperature is positive above a threshold
of 28°C. Germination increases with soil surface minimum temperature to
15°C, then the positive slope is less steep. Soil warming relates to
light intensity asymptotically (as described above); germination
increased above 1076 lux (mean). In relation to maximum light intensity,
germination increased to a breakpoint at 54000 lux, after which the
effect on germination is negative. Comparisons of AICs of these models
(Table S1) show at seed burial level the maximum temperature is the best
fit; at soil surface level mean temperature is the best fit, and mean
light intensity is a better fit than the maximum value during cue
periods.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig. 4} Segmented GLMs of binomial germination outcomes
back-transformed to the response probability scale as response to
microclimate variables in simulated-NHs, dashed lines are breakpoints
and pink shading is standard error (0.95), residuals are red circles.

\emph{Survival and dormancy loss in the soil}

Overall, 40\% of seeds that were buried in the soil for two years and
68\% for one year were successfully exhumed from the soil. The
probability of finding seeds was modelled against year, burial-depth,
and accession (Fig. 5a). Seed burial-depth was the most important
factor, deep-buried seeds were more likely to be successfully exhumed
(z=16.722, p\textless{}0.001), followed by year (z=13.379,
\textless{}0.001). Surprisingly, seeds with low viability when sown had
high probability of being exhumed (bur60 z=7.242, p\textless{}0.001,
sia61 z=7.025, p\textless{}0.001).

Viability of exhumed seeds were tested by TTC and incubation. Viability
tests from these, original ER and maximum in Simulated-NHs, was modelled
in a GLM. In contrast to the results of the above, accession was
excluded from the MAM, year remained. Contrasts were made per year
against original ER. Viability from the incubator test was not lost
during one year in the soil. After two years in the soil, viability was
reduced in both incubator and TTC tests. The TTC test consistently
underestimated viability.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig. 5} Seed survival in the soil; (a) Probability of finding
seeds in 2021 that were sown in 2019 and 2020 according to burial-depth
and accession; (b) viability of seeds found in 2021 using a germination
test in an incubator (IN) and the tetrazolium chloride test (TTC), with
reference to viability at the start of the experiment using embryo
rescue techniques (ER), and maximum germination achieved in a
Simulated-NH; p values show contrasts (Dunnett test) against original
viability (ER) for each year.

Seeds that were removed from storage and incubated under suitable
conditions (20/35\selectlanguage{ngerman}°C), displayed dormancy i.e., they did not germinate at
all. However, if they were buried in the soil at 7 cm for up to three
months, germination significantly increased to probability 0.4 (t=2.518,
p=0.021, Fig. 6).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Fig. 6} Germination probability in an incubator (at 35/20 \selectlanguage{ngerman}°C,
light/dark, 18/6 hours cycled) of seeds buried and then exhumed from the
soil for up to three months. Seeds were buried in the exposed Spring
compartment.

\subsection*{Discussion}

{\label{discussion}}

\begin{enumerate}
\tightlist
\item
  \emph{Use of semi-natural and simulated habitats for germination
  ecology experiments}
\end{enumerate}

In the present study we used a Semi-NH from the native range of \emph{M.
balbisiana} , and several Simulated-NHs in glass houses located in a
temperate region, to examine wild banana seed germination ecology. We
established that with such approximations of NHs, it is possible to link
germination responses to ecological factors such as foliar-shading and
burial-depth. Hence with this approach we overcame limitations when
access to experimental NHs was not possible, this allowed for greater
ecological interpretation than with LCs alone.

\emph{Temperature}

We found that \emph{Musa} seed germination is stimulated by exposure to
the sun. It was the maximum part of the temperature fluctuation, in our
results, that is most closely associated with germination. Above the
threshold of 23°C, germination increased to a maximum at 35°C (in
Simulated-NHs and 42°C in Semi-NHs) in exposed conditions. Our findings
are broadly consistent with previous LC results, where optimal maximal
and minimal temperature for germination were diurnal cycles of
35/18-20°C respectively, for both \emph{M. acuminata} and \emph{M.
balbisiana} (Stotzky \& Cox 1962; Kallow \emph{et al.} 2021).
Interactions between the elements of warming and cooling cycles play an
important role in simulating germination.

\emph{Light}

One might think that as germination responses were directly associated
with soil exposure to sun and, in simulated-NHs, light intensity,
germination is stimulated by light. Additionally, we also found that
seeds germinated to a greater extent from 1 cm compared to 7 cm.
However, light waves cannot usually penetrate the soil to greater than
4-5 mm depth (depending on soil moisture and particle size), and not to
any amount that can illicit germination responses to light sensitive
seeds (Woolley \& Stoller 1978; Tester \& Morris 1987), but in our
experiment, seeds germinated from a depth of 7 cm. We therefore infer
that whilst \emph{Musa} seed germination may be correlated with factors
associated with light (light intensity, shallow burial) theses are
correlations rather than causal, and it is temperature that regulates
germination.

\emph{Gap detection}

\emph{Musa} seed germination in response to sun exposure demonstrates
adaptation to detect suitable niches for seedling establishment
following disturbance in forest NHs. Conversely, inhibition of
germination in shade is also an adaptation for seedling survival (Kos \&
Poschlod 2007; Poschlod \emph{et al.} 2013). \emph{Musa} germination
responses were sensitive to sun/shade even when microclimates were very
similar. For instance, mean temperatures were the same and range
differed by only a few degrees in sun and shaded semi-NHs. Germination
is therefore finely tuned to respond to microclimate such as that which
would occur when a forest gap is formed (Pearson \emph{et al.} 2002;
Pearson \emph{et al.} 2003). The effect of forest disturbance on
temperature dynamics was studied by Harwick \emph{et al} . (2015). The
authors measured soil (10 cm depth) and air temperature (1.5m height) at
three levels of forest disturbance in Borneo\emph{.} Diurnal
temperatures in oil palm plantations (formerly forested) were around 7°C
greater at the hottest part of the cycle compared to old growth forests
and soil temperatures were around 3 °C warmer at this point and around 1
°C cooler in the night; these were similar to our results in semi-NHs.
Temperatures in logged forests were somewhat in between these two, but
more similar to old growth forest. One could imagine that even in small
forest gaps, microclimates could therefore also vary considerably
(Pearson \emph{et al.} 2002). These responses are in line with
adaptations of other disturbance-adapted species that also require
alternating temperature cycles rather than constant temperatures to
germinate (Vázquez\selectlanguage{english}-Yanes \& Orozco\selectlanguage{english}-Segovia 1982; Vazquez-Yanes \&
Orozco-Segovia 1993; Pearson \emph{et al.} 2002; Seiwa \emph{et
al.}2009).

\emph{Seed burial-depth}

When seeds were in the shade, burial-depth made no difference to
germination, they did not germinate irrespective of burial-depth. In
exposed sites, shallow buried seeds were more likely to germinate than
deeply buried seeds. This was because temperature dynamics are likely
buffered by burial depth. For some species, sensitivity to alternating
temperatures is an adaptation to detect burial depth (Thompson, Grime \&
Mason 1977; Thompson \& Grime 1983). Pearson et al. (2002) found, for
large-seeded species, diurnal temperature sensitivity was more likely to
related to forest gap size than seed burial-depth. This was also the
case for large-seeded \emph{Musa} , in that exposure was by far the most
significant factor in simulated-NHs, and burial-depth was only
secondary. For small seeds it is important to detect burial-depth as
seedlings must reach the surface with small endosperm reserves; for
larger seeds with greater nutrient reserves this is less of a limiting
factor for survival.

\emph{Survival and dormancy loss in the soil}

We found seeds can persist and remain viable buried in the soil for at
least two years. In fact, there was no loss of viability after one year,
there was however loss of actual seeds. Seed loss was more pronounced
with shallow burial, suggesting it is the result of predation or perhaps
splashing during watering, rather than decomposition. This is also
supported by the fact that accessions with less viable seeds were more
likely to be found i.e., seeds were not lost by decomposition of dead
seeds.

For seeds adapted to disturbance, seed persistence in the soil seed bank
is important. \emph{Musa} clearly invest considerably in seed coat
defenses (Graven \emph{et al.} 1996), to survive the intense pressure
present in the soil community (Dalling \emph{et al.} 2011).

Not only do seeds persist in the soil, but dormancy is reduced during
this process. In our results, when seeds were stratified for three
months, or when they were in the soil for a year, germination increased.
A stratification requirement is in keeping with results from our
previous study (Kallow \emph{et al.} 2021). Although, stratification was
not required for freshly extracted \emph{M. balbisiana} seeds in
Semi-NHs, implying drying induces secondary dormancy in
\emph{Musa}seeds, as proposed by Chin (1996).

There was greater germination synchronization in cooler Simulated-NHs as
seeds responded to threshold temperatures when sun was stronger during
the summer. When temperatures were consistently warmer, synchronicity
was reduced - this may again be a disturbance adaptation. We found this
response more evident in \emph{M. balbisiana} than in \emph{M.
acuminata,} suggesting it may also relate to seasonality as \emph{M.
balbisiana} has a large distribution that includes subtropical seasonal
climates (Mertens \emph{et al.} 2021).

\subsection*{Conclusions}

{\label{conclusions}}

Studying germination ecology has intrinsic challenges, possibly the
biggest being access to seeds and experimental set-ups in suitable
conditions and timeframes. In the present study we demonstrate an
approach for dealing with such difficulties in studying tropical seed
germination ecology, which is a challenge when researchers are outside
of a plant's native region. Using Semi-NHs and Simulated-NHs we found:
(1) foliage-shading inhibits germination of non-dormant seeds, and
exposure to sun stimulates germination; this response is most closely
associated with maximum temperature variation found under direct
sunlight; this effect is marginally buffered by deep burial in the soil;
(2) freshly extracted seeds are non-dormant, but stored seeds lose their
dormancy during burial in the soil; (3) \emph{Musa} seeds remain viable
in the soil for at least a year without any loss in viability. Thus,
wild banana species are well adapted to exploit canopy gaps following
disturbance.

\subsection*{Acknowledgments}

{\label{acknowledgments}}

We thank Isla Kallow, Jasmin Kallow, Manuela Garcia Zuluaga and Kevin De
Pauw for assisting with the setting up the glass house experiments. We
gratefully acknowledge the help of John Mark Barios, Lyka Yanos and
Paulo Jerome Lopez at NPGRL for setting up and monitoring the
germination experiments in the Philippines and for Michelle Lyka V.
Descalsota for assistance in viability assessing seed collections at
NPGRL. We also thank the late Daniele Roques (CIRAD, Centre de
Ressources Biologiques Plantes Tropicales (CRB-PT), Guadeloupe),
Josephine Agogbua (International Institute of Tropical Agriculture
(IITA), Nigeria) and Zhiying Li (Institute of Tropical Crop Genetic
Resources, CATAS Tropical Crops, Danzhou, China) for providing seeds to
use. This work was funded as a sub-grant from the University of
Queensland from the Bill \& Melinda Gates Foundation project `BBTV
mitigation: Community management in Nigeria, and screening wild banana
progenitors for resistance' {[}OPP1130226{]}. The authors thank all
donors who supported this work also through their contributions to the
CGIAR Fund (http://www.cgiar.org/funders), and in particular to the
CGIAR Research Program Roots, Tubers and Bananas (RTB-CRP). This study
was supported by a bilateral grant between the Research Foundation -
Flanders (FWO-Vlaanderen) and the Vietnamese National Foundation for
Science and Technology Development (NAFOSTED) under grant number
G0D9318N.

\subsection*{Author contributions}

{\label{author-contributions}}

SK: conceptualization, methodology, software, formal analysis,
investigation, data curation, writing-original draft, writing-review and
editing, visualization; KQ: investigation; BP: conceptualization,
methodology, resources, writing-review and editing, supervision, funding
acquisition; SBJ: conceptualization, methodology, resources,
writing-review and editing, supervision, funding acquisition; JD:
writing-review and editing, supervision, funding acquisition; LG:
resources; RS: writing-review and editing, supervision; FV:
conceptualization, methodology, resources, writing-review and editing.

\subsection*{Conflict of interest}

{\label{conflict-of-interest}}

The authors declare that there is no conflict of interest associated
with this article and research.

\subsection*{Data availability}

{\label{data-availability}}

All data available at Kallow, Simon (2021): Using semi-natural and
simulated habitats for seed germination ecology. figshare. Dataset.
https://doi.org/10.6084/m9.figshare.14884470.v1

\subsection*{}

{\label{section}}

\subsection*{Supplementary figures and
tables}

{\label{supplementary-figures-and-tables}}

\subsection*{}

{\label{section-1}}

\subsection*{}

{\label{diagram-table-description-automatically-generated}}

\textbf{Fig. S1.} Average and standard deviations of temperature and
light intensity during cue periods of exposed and shaded environments,
temperatures are degC, light intensity is lux.

\subsection*{}

{\label{chart-scatter-chart-description-automatically-generated}}

\textbf{Fig. S2.} Light intensity and temperature measured at simulated
natural environments in glass houses during germination tests;
asymptotic non-linear regression shown (residual standard error 4.119 on
417,661 degrees of freedom).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Fig. S3.} Final germination percentages and microclimate
variables from simulated-NHs, dashed trend lines plotted with Loess
non-parameter regression, shaded area is 95\% standard error, light
variables are in lux, temperature are \selectlanguage{ngerman}°C, s=value from logger at the
soil surface, otherwise they are at seed burial depth.

\textbf{Table S1.} Model fits of germination outcome in response to
microclimate variables in simulated-NHs in segmented GLMs.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Variable & AIC & Residual deviance & df\tabularnewline
\midrule
\endhead
temp.mean & 1749 & 1741 & 3388\tabularnewline
temp.max & 1612 & 1604 & 3388\tabularnewline
temp.min & 1890 & 1882 & 3388\tabularnewline
s.temp.mean & 799 & 791 & 1220\tabularnewline
s.temp.max & 817 & 810 & 1220\tabularnewline
s.temp.min & 961 & 953 & 1220\tabularnewline
s.lightemp.mean & 803 & 796 & 1220\tabularnewline
s.lightemp.max & 887 & 880 & 1220\tabularnewline
\bottomrule
\end{longtable}

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