PLOS ONE
RESEARCH ARTICLE
Genetic diversity and structure of Musa
balbisiana populations in Vietnam and its
implications for the conservation of banana
crop wild relatives
Arne Mertens 1,2ID *, Yves Bawin 2,3ID , Samuel Vanden Abeele2, Simon Kallow 1,4ID ,
Dang Toan Vu5, Loan Thi Le6, Tuong Dang Vu 5, Rony Swennen 1,7ID ID , Filip Vandelook
2,
Bart Panis 8ID , Steven B. Janssens
2,9
a1111111111
1 Laboratory of Tropical Crop Improvement, Department of Biosystems, KU Leuven, Leuven, Belgium,
a1111111111
2 Meise Botanic Garden, Meise, Belgium, 3 Ecology, Evolution and Biodiversity Conservation, Department
a1111111111
of Biology, KU Leuven, Leuven, Belgium, 4 Royal Botanic Gardens Kew, Millennium Seed Bank, West
a1111111111 Sussex, United Kingdom, 5 Research Planning and International Department, Plant Resources Center,
a1111111111 VAAS, Hanoi, Vietnam, 6 Department of Genebank Management, Plant Resources Center, VAAS, Hanoi,
Vietnam, 7 International Institute of Tropical Agriculture, Arusha, Tanzania, 8 Bioversity International,
Leuven, Belgium, 9 Molecular Biotechnology of Plants and Micro-organisms, Department of Biology, KU
Leuven, Leuven, Belgium
* arne.mertens70@gmail.com
OPEN ACCESS
Citation: Mertens A, Bawin Y, Vanden Abeele S,
Kallow S, Toan Vu D, Thi Le L, et al. (2021) Genetic Abstract
diversity and structure of Musa balbisiana
populations in Vietnam and its implications for the
Crop wild relatives (CWR) are an indispensable source of alleles to improve desired traits in
conservation of banana crop wild relatives. PLoS
ONE 16(6): e0253255. https://doi.org/10.1371/ related crops. While knowledge on the genetic diversity of CWR can facilitate breeding and
journal.pone.0253255 conservation strategies, it has poorly been assessed. Cultivated bananas are a major part
Editor: Kenneth M. Olsen, Washington University, of the diet and income of hundreds of millions of people and can be considered as one of the
UNITED STATES most important fruits worldwide. Here, we assessed the genetic diversity and structure of
Received: March 15, 2021 Musa balbisiana, an important CWR of plantains, dessert and cooking bananas. Musa bal-
bisiana has its origin in subtropical and tropical broadleaf forests of northern Indo-Burma.
Accepted: June 1, 2021
This includes a large part of northern Vietnam where until now, no populations have been
Published: June 23, 2021
sampled. We screened the genetic variation and structure present within and between 17
Copyright: © 2021 Mertens et al. This is an open Vietnamese populations and six from China using 18 polymorphic SSR markers. Relatively
access article distributed under the terms of the
high variation was found in populations from China and central Vietnam. Populations from
Creative Commons Attribution License, which
permits unrestricted use, distribution, and northern Vietnam showed varying levels of genetic variation, with low variation in popula-
reproduction in any medium, provided the original tions near the Red River. Low genetic variation was found in populations of southern Viet-
author and source are credited. nam. Analyses of population structure revealed that populations of northern Vietnam formed
Data Availability Statement: All relevant data are a distinct genetic cluster from populations sampled in China. Together with populations of
within the manuscript and its Supporting central Vietnam, populations from northern Vietnam could be subdivided into five clusters,
Information files.
likely caused by mountain ranges and connected river systems. We propose that popula-
Funding: This work was mainly funded through the tions sampled in central Vietnam and on the western side of the Hoang Lien Son mountain
Research Foundation Flanders (FWO) through the
range in northern Vietnam belong to the native distribution area and should be prioritised for
Flanders Bilateral Research Cooperation with
Vietnam project with reference “G0D9318N”, the conservation. Southern range edge populations in central Vietnam had especially high
Vietnamese National Foundation for Science and genetic diversity, with a high number of unique alleles and might be connected with core
Technology Development (NAFOSTED) under grant
number FWO.106-NN.2017.02, and the Bill and
PLOS ONE | https://doi.org/10.1371/journal.pone.0253255 June 23, 2021 1 / 22
PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
Melinda Gates foundation through the BBTV populations in northern Laos and southwest China. Southern Vietnamese populations are
mitigation project. This work was additionally considered imported and not native.
supported by the CGIAR Fund (https://www.cgiar.
org/funders/) and in particular the CGIAR Research
Program for Roots, Tubers and Bananas (CRP-
RTB). The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. Introduction
Competing interests: The authors have declared Crop wild relatives (CWR) are wild species closely related to cultivated plants, including all
that no competing interests exist. direct progenitors of crops. Because of their relatedness, CWR provide useful trait characteris-
tics for crop improvement such as stress tolerance and yield increase [1]. CWR are believed to
become even more important for breeding in the near future due to climate change and
increasing food demand [2,3]. A changing global climate may exacerbate stress levels on locally
grown crops, whereas a rapidly expanding world population and changing dietary habits put a
high pressure on food supplies [4,5]. Conservation of CWR in gene banks improves the acces-
sibility of CWR to breeders. Nowadays, highly advanced genomic methods allow faster access
to these genetic resources, and hence gene banks become even more valuable [6,7].
With an estimated production of 158.4 million tonnes in 2019, bananas (74%) and plantains
(26%) can be considered as one of the most important fruits and staple foods of the world [8].
Although bananas are a main export product of many countries [9], 85% of the total production
is consumed locally or sold regionally by producers [10]. Over one thousand existing varieties sig-
nificantly contribute as a staple food to the daily diet and monthly income of hundreds of millions
of people in developing regions in the subtropics and the tropics [11,12]. Next to the high caloric
intake of the fruits, other parts of the plant can be used either as food, animal fodder, fibre, or
medicine [13–15]. Despite the striking number of cultivars with different ploidy levels that have
been described [16–19], almost all are derived from hybridization events between two species:
Musa acuminata Colla (the so-called “A” genome) and Musa balbisiana Colla (“B” genome).
Only a small number of cultivars contains genetic information of other Musa species such as
Musa schizocarpa Simmonds and members of the former Australimusa section [20–23].
Musa balbisiana is a diploid (2n = 22) wild perennial herb of the “Musa” (former Eumusa)
section of the genus Musa [24]. Its native distribution area ranges from northeast India to
south China and northern Vietnam [25]. The presence of M. balbisiana on the Ryukyu islands,
the Philippines, and Papua New Guinea is assumed to be feral after the introduction of this
species by humans for cultivation purposes [26]. Asexual reproduction typically takes place
close to the parent plant through suckers emerging from a lateral rhizome bud [27]. Inflores-
cences are supported by a pendulous stalk (also called peduncle) emerging from the pseudos-
tem. Basal flowers are typically female with staminodes of varying levels of fertility, while male
flowers are found at the tip of the inflorescence [28]. The flowers are pollinated by fruit bats,
sunbirds, and likely a set of insects [29,30]. Seeds are dispersed by small foraging rodents [29].
M. balbisiana (BB) is one of the ancestors of diploid (AB), triploid (AAB, ABB), and tetra-
ploid (ABBB, AABB, AAAB) plantains, cooking, and dessert bananas [17,31,32]. Note that the
proportion of the A and B genome often deviates from the expected 1:1 ratio in the members
of cultivar groups AB and AABB or from and expected 2:1 and 1:2 ratio in members of cultivar
groups AAB and ABB respectively. This bias was recently attributed to interspecific recombi-
nation and large structural variations between the A and B genomes, highlighting a complex
origin of cultivated bananas with one or multiple backcrosses [33–36]. In contrast to cultivars
only consisting of genetic information of Musa acuminata (AA, AAA, AAAA), the presence of
the B genome has been associated with increased drought and cold tolerance [37–40], resis-
tance to Xanthomonas wilt [41–43], and tolerance against banana weevils [44]. The availability
of a high quality reference genome [45] and recent identification of large structural variations
PLOS ONE | https://doi.org/10.1371/journal.pone.0253255 June 23, 2021 2 / 22
PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
between A and B genomes [36] will aid in the development of more optimal breeding of
banana cultivars and considerable research has been done on assessing the geographic distri-
bution and genetic diversity of wild M. balbisiana populations [29,46–49]. Still, wild popula-
tions of the species in their native distribution range are still underexplored, under-conserved,
and potentially underused in breeding programs [7,50–53].
To date, only a few studies have investigated the intraspecific genetic diversity within M.
balbisiana. Using Amplified Fragment Length Polymorphism (AFLP) markers, Ude et al. [54]
assessed genetic diversity in eight M. balbisiana accessions obtained from gene banks and field
collections. Their study was of the first to detect a high level of genetic diversity among M. bal-
bisiana accessions. Ge et al. [29] and Wang et al. [49] found high levels of genetic diversity in
fifteen Chinese M. balbisiana populations using AFLP and Simple Sequence Repeat (SSR)
markers. The highest heterozygosity levels were observed in the Yunnan province, close to the
Vietnamese border. Recently, Bawin et al. [46] used 18 SSR markers and demonstrated that
seed batches sampled from additional populations of the Yunnan province in China harboured
higher levels of genetic variation compared to more feral populations in Hainan (China),
Amami (Japan), and Lae (Papua New Guinea), and to ex situ seed collections in Arusha (Tan-
zania) and Kampala (Uganda).
Species are often assumed to have larger population sizes, high genetic diversity, high gene
flow and low differentiation in the centre of their distribution range [55]. In contrast, popula-
tions at the edge of their distribution range are generally believed to be more fragmented,
smaller, and exhibit lower genetic diversity and higher genetic differentiation, though several
studies report the lack of this latitudinal trend (e.g. [56] and [57]). Edge populations are often
subjected to biotic or abiotic environmental conditions not present in these core populations
[55,58]. They may adapt in response to these conditions, being potentially interesting for crop
improvement because of unique or a larger frequency of adaptive alleles. Therefore, range-
edge populations should not be ignored when collecting germplasm for conservation. Musa
balbisiana populations at the southern edge of their native distribution range (e.g. populations
in eastern Myanmar, northern Laos or northern-central Vietnam) might be exceptionally
interesting. Populations may hold specific alleles adapted to better cope with drought stress,
interesting for cultivated material containing the B genome, especially in a changing climate.
In this study, we for the first time quantify the genetic variation in Vietnamese populations
of the banana crop wild relative Musa balbisiana. More specifically, we assessed the genetic
structure and diversity in 17 Vietnamese M. balbisiana populations using 18 SSR markers. As
large parts of northern Vietnam belong to the putative region of origin of the species, popula-
tions are expected to hold levels of genetic variation similar to those found in populations sam-
pled in China. In addition, genetic variation in 6 wild populations of neighbouring Chinese
provinces are compared with the Vietnamese populations to allow us to better understand the
native character of the Vietnamese populations. Four questions are addressed: (1) How does
genetic variation in Vietnamese M. balbisiana populations relate to that in Chinese popula-
tions? (2) How does genetic diversity vary among Vietnamese M. balbisiana populations? (3)
Are M. balbisiana populations in Vietnam genetically structured along a geographic gradient?
(4) Can southern populations of Vietnam be considered as wild?
Materials and methods
Taxon sampling
Field research was carried out as a collaboration between Meise Botanic Garden (Belgium)
and the Plant Resources Center (Hanoi, Vietnam). The Plant Resources Center had permission
for working with Meise Botanic Garden under the Bilateral cooperation project: the
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
Table 1. Sampling location information.
Country population code Geographical name N Origin (province, locality) Latitude Longitude Material type
Vietnam VTN-N1 northern Vietnam 1 20 Lao Cai, Muong Cau 22.31417 104.0397 Leaf
Vietnam VTN-N2 northern Vietnam 2 16 Lai Chau, Can Cau 22.43667 103.4497 Leaf
Vietnam VTN-N3 northern Vietnam 3 11 Lai Chau, Cu Ti 22.32689 103.4861 Leaf
Vietnam VTN-N4 northern Vietnam 4 20 Lai Chau, Na Bo 22.34528 103.5175 Leaf
Vietnam VTN-N5 northern Vietnam 5 13 Lai Chau, Nam Ma Dao 22.4075 103.3156 Leaf
Vietnam VTN-N6 northern Vietnam 6 14 Lai Chau, Seo Leng 22.44528 103.2614 Leaf
Vietnam VTN-N7 northern Vietnam 7 13 Bao Thang, Suoi Thau 22.298 104.0424 Leaf
Vietnam VTN-N8 northern Vietnam 8 12 Van Ban, Khanh Yen Thuong 22.11497 104.2439 Leaf
Vietnam VTN-N9 northern Vietnam 9 16 Dien Bien, Xa Tu 21.44953 103.4403 Leaf
Vietnam VTN-N10 northern Vietnam 10 21 Dien Bien, Na Sang 21.6965 103.1113 Leaf
Vietnam VTN-N11 northern Vietnam 11 20 Lai Chau, Nam Hang 22.14219 102.9731 Leaf
Vietnam VTN-N12 northern Vietnam 12 19 Lai Chau, Bum To 22.38722 102.7806 Leaf
Vietnam VTN-N13 northern Vietnam 13 4 Lai Chau, Vang Bo 22.54954 103.297 Leaf
Vietnam VTN-C1 central Vietnam 1 20 Nghe An, Ban Phung 19.29876 104.3213 Leaf
Vietnam VTN-C2 central Vietnam 2 20 Nghe An, Khe Ngau 19.3028 104.3854 Leaf
Vietnam VTN-S1 southern Vietnam 1 18 Kontum, Mo Rai 14.35633 107.6624 Leaf
Vietnam VTN-S2 southern Vietnam 2 17 Kontum, Dak Nhoong 15.08056 107.6817 Leaf
China CHN-W1 western China 1 19 Yunnan 24.60678 97.58322 Plants grown from seeds
China CHN-W2 western China 2 20 Yunnan 24.86883 97.82441 Plants grown from seeds
China CHN-W3 western China 3 14 Yunnan 24.87246 97.85518 seed
China CHN-W4 western China 4 7 Yunnan 24.69901 97.87019 seed
China CHN-S southern China 11 Guangdong 21.1622 110.2756 seed
China HI Hainan 20 Hainan 19.51667 109.4833 Plants grown from seeds
N, number of included samples per population of wild Musa balbisiana.
https://doi.org/10.1371/journal.pone.0253255.t001
Vietnamese National Foundation for Science and Technology Development (NAFOSTED).
Field permits for Yen Bai (564/UBND-NV), Ha Giang (255/SNN-CCLN), and Lao Cai (1060/
UBND-NC) were obtained. During multiple collection missions carried out between 2017 and
2020, 274 individuals belonging to 17 populations of wild Musa balbisiana were sampled in
Vietnam (Table 1, Fig 1). Populations were selected along a north-south geographic gradient
ranging between a latitude of 22.39 and 14.37 a longitude of 102.78 and 107.68. Whenever pos-
sible, leaves from a minimum of 15 individuals per population were sampled. In order to avoid
sampling suckers from the same individual, plants located directly next to each other were not
sampled. In addition to leaf material from Vietnam, six seed collections of wild M. balbisiana
from China were also included in this study. Three seed collections (two from Yunnan and
one from Guangdong) were newly acquired and used in the present study. Finally, data from
Bawin et al. [46] for three seed collections (two from Yunnan and one from Hainan) was also
included in the analysis. Each seed collection was retrieved from one banana bunch per sam-
pling location, a common practise in collecting banana seeds.
SSR genotyping
Banana leaf material was dried for preservation in the field using silica gel [62]. Seed embryos
were excised from seed collections acquired in this study using a surgical scalpel and added to
10 μl of cetyltrimethylammonium bromide (CTAB). DNA from leaves and embryos was sub-
sequently isolated using a modified CTAB extraction protocol of Doyle & Doyle [63]. In order
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
Fig 1. Sampling locations of wild Musa balbisiana populations. Shaded area represents the northern Indochina subtropical forests, the native distribution
area of M. balbisiana. Geospatial datasets used for the creation of these maps were reprinted from [59–61] under a CC-BY 4.0 license.
https://doi.org/10.1371/journal.pone.0253255.g001
to compare our data to the genetic variation observed in Bawin et al. [46], we used the same set
of 18 microsatellite markers arranged in four multiplexes based on their polymorphic charac-
ter from multiple studies assessing both wild and cultivated material (S1 Table). To infer the
genomic coordinates from nuclear SSR markers, GenBank sequences retrieved from Wang
et al. [64] and Rotchanapreeda et al. [65] of each marker were blasted against the reference
genome sequence of Musa balbisiana “Pisang Klutuk Wulung” in the Banana genome hub
[66]. An expect cutoff value of 1e-10 was used and the single best search result was retained.
The genomic coordinates from this search were then plotted inside chromosome tracks in R
[67].
PCR of microsatellite fragments was carried out with Qiagens Type-it Microsatellite PCR
Kit (Qiagen, Venlo, the Netherlands). Subsequently, 1 μl of diluted PCR sample was added to
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
12 μl HiDi Formamide mixed with 0.4 μl of the MapMarker 500 labelled with DY-632 (Euro-
gentec, Seraing, Belgium), after which 1.5 μl of this product was genotyped on an ABI 3730 sys-
tem (Applied Biosystems, Foster City, California) at the Université Libre de Bruxelles (ULB),
Belgium. For more recent samples, 20 μl of PCR product was sent to Macrogen (Macrogen
Europe, Amsterdam, the Netherlands) for genotyping on an ABI 3730 system. A minimum of
ten samples per genotyping run were ran in duplicate to deal with potential differences in frag-
ment sizing. Raw data were scored and checked for errors in Geneious Prime 2021.0.3 (Bio-
matters, New Zealand) with a third order least squares method. To ensure a uniform scoring
of alleles, raw data obtained from the two populations of Yunnan and one from Hainan from
the study of Bawin et al. [46] were re-scored using the same sizing method. Samples with
ambiguous patterns were genotyped twice to resolve erroneous scoring, and samples with
more than 10% missing data were excluded from the analyses. To screen our data for outliers,
the GENETIX software [68] was used and one population was removed from the dataset (Lao
Cai, Nam Ma commune) based on an AFC-3D visualisation of the genotypic data (S1 Fig).
Genetic diversity indices
The average number of alleles per locus with a frequency larger than 5% (Na), the average
number of private alleles unique to a single population (Np), Shannon’s Diversity Index (H),
proportion of polymorphic loci (P), Unbiased expected Heterozygosity (uHe), and Observed
Heterozygosity (Ho) were calculated with the GenAlEx 6.51 Excel Package [69]. Inbreeding
coefficients were additionally calculated with the “fs.dosage” function in R package “hierfstat”
[70].
Population genetic structure
To explore our genetic data and to visualize the genetic structure of populations, we assessed
the pairwise genetic differentiation between populations through codominant genotypic dis-
tances and used a Principal Coordinate Analysis (PCoA) as visualisation. With GenAlEx, an
analysis of molecular variance (AMOVA) was run using 999 permutation steps to assess the
genetic variation present within and between sampled populations while taking the sampled
region into account. Here, genetic differentiation was measured as FST. Within and between
population variation including all samples was additionally visualised by a PCoA constructed
using a Euclidean distance matrix with the “dudi.pco” function in R package “ade4” [71].
Population genetic structure was assessed using Bayesian clustering in STRUCTURE 2.3.4
[72]. All runs using STRUCTURE were done on the online Galaxy platform [73] using an
admixture model to allow samples to be assigned to one or multiple genetic sources (clusters).
Because unequal population sampling may lead to poor estimations of the optimal number of
genetic clusters (K), we followed the methodology of Wang [74] and used the uncorrelated
allele frequency model with a separate ancestry prior alpha (α) for each cluster while setting
the initial α to 1/K.
First, the optimal number of genetic clusters to which individuals could be assigned to was
determined by testing 10 independent runs for a K value ranging from one to 20 using a
model with correlated allele frequencies and a default initial α of 1.00 for all clusters. For each
independent run, 100,000 Markov Chain Monte Carlo (MCMC) iterations were sampled after
a burn-in of 100,000 iterations. The optimal number of genetic clusters was then determined
based on ΔK and the log posterior probability of the replicates over each K [75]. This value of
K was additionally compared to the optimal number of clusters based on the MedMeaK
(median of means), MaxMeaK (maximum of means), MedMedK (median of medians), and
MaxMedK (maximum of medians) as described in Puechmaille [76]. These descriptors were
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
calculated on the online web server of StructureSelector [77]. The second set of analyses was
then run using the same parameter configurations, but this time allowing separate α values for
each cluster and an initial α of 1/K, with a K inferred from the first run, with independent allele
frequencies. Samples with a cluster assignment probability < 0.8 were considered to be
admixed and were not assigned to one specific inferred cluster.
Two datasets were used to run the clustering analyses in STRUCTURE. The first dataset
contained all samples from all populations after removing any outlying samples, allowing us to
directly compare populations of Chinese with those of Vietnamese origin. The second dataset
consisted of only Vietnamese populations in their native distribution range (northern Indo-
china). This allowed us to evaluate the population genetic (sub)structure of these populations.
Structure plots with the optimal K were made with the CLUMPAK software as implemented
in StructureSelector [78].
Isolation by distance
To estimate whether genetic distance also increases when populations are geographically more
distant (Isolation by distance), a geographic distance matrix was calculated based on the geo-
graphic coordinates of each population. This was done for the two different datasets, one
including all sampled populations and one only including populations sampled in northern
and central Vietnam. For both datasets, a paired Mantel test analysis was performed between
the geographic and genetic distance matrices (Edward’s distance) with R package “ade4” to
assess whether genetic isolation by distance was significant [71]. All R packages were used in R
version 4.0.2 [67].
Results
Genomic coordinates
Blasting SSR marker sequences against the reference genome showed that they are spread
across 8 out of 11 chromosomes (S2 Table). No markers covered chromosomes 1, 5, and 7 and
some of the remaining chromosomes were covered by multiple markers. Plotting the geo-
graphic coordinates revealed that some markers were located in close proximity to each other
and that large parts of chromosomes are not covered (S2 Fig).
Genetic diversity
The average number of alleles per locus with a frequency larger than 5% was low in all sampled
populations, ranging from 1.11 in populations VTN-N8 and VTN-S1 to 1.94 in VTN-C1
(Table 2). Fourteen populations did not contain private alleles, and the highest average num-
ber of private alleles was found in VTN-C1 and CHN-S. The Shannon diversity index was low-
est in VTN-S1 and highest in VTN-N10. The proportion of polymorphic loci ranged from
11% to 67% and was low (<20%) in the southern populations (VTN-S1, VTN-S2) and some
northern populations (VTN-N3, VTN-N8). Expected and observed levels of heterozygosity
ranged between 0.05 to 0.28, with an average of 0.19 and 0.16 respectively, though strongly var-
ied between populations. Low levels of inbreeding were found across all populations, but six
M. balbisiana populations had an excess in heterozygotes (negative values) (VTN-N3,
VTN-N8, VTN-N9, VTN-N13, VTN-S1, and CHN-W3).
Genetic structure
In the Principal Coordinate Analysis of the full dataset, the first two axes explained together
26.6% and 17.8% of the genetic variation present between the populations respectively
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
Table 2. Intra-population genetic diversity indices of Musa balbisiana populations.
Pop Na Np H P (%) He Ho FIS
VTN-N1 1.222 0.000 0.085 0.222 0.054 0.056 -0.015
VTN-N2 1.556 0.000 0.253 0.389 0.157 0.147 0.054
VTN-N3 1.167 0.000 0.115 0.167 0.087 0.152 -0.818
VTN-N4 1.500 0.056 0.263 0.556 0.169 0.172 -0.020
VTN-N5 1.722 0.000 0.323 0.500 0.208 0.218 -0.048
VTN-N6 1.611 0.000 0.331 0.444 0.218 0.218 -0.001
VTN-N7 1.222 0.111 0.101 0.222 0.067 0.051 0.247
VTN-N8 1.111 0.000 0.077 0.111 0.058 0.111 -0.968
VTN-N9 1.278 0.000 0.193 0.278 0.143 0.278 -1.000
VTN-N10 1.889 0.111 0.427 0.444 0.250 0.257 -0.026
VTN-N11 1.778 0.000 0.331 0.500 0.200 0.206 -0.027
VTN-N12 1.722 0.000 0.330 0.444 0.204 0.202 0.011
VTN-N13 1.278 0.000 0.193 0.278 0.159 0.278 -1.000
VTN-C1 1.944 0.056 0.401 0.556 0.250 0.269 -0.078
VTN-C2 1.889 0.222 0.423 0.667 0.254 0.256 -0.005
VTN-S1 1.111 0.000 0.075 0.111 0.055 0.080 -0.482
VTN-S2 1.167 0.056 0.109 0.167 0.079 0.085 -0.030
CHN-W1 1.778 0.056 0.372 0.611 0.233 0.241 -0.064
CHN-W2 1.556 0.000 0.285 0.500 0.171 0.183 -0.078
CHN-W3 1.278 0.056 0.153 0.333 0.106 0.175 -0.697
CHN-W4 1.778 0.000 0.358 0.556 0.243 0.243 0.012
CHN-S 1.556 0.167 0.319 0.444 0.223 0.210 0.067
HI 1.556 0.000 0.251 0.444 0.152 0.161 -0.061
Na, number of different alleles with frequency >5%; Np, average number of private alleles unique to a single population; H, Shannon’s Diversity Index; P (%),
proportion of polymorphic loci; He, expected heterozygosity; Ho, observed heterozygosity; FIS, inbreeding coefficient.
https://doi.org/10.1371/journal.pone.0253255.t002
(Fig 2A). The Chinese and northern Vietnamese populations were separated from each other
in the PCoA plot, whereas additional sub-clustering was observed within Vietnamese popula-
tions. VTN-S1 did not clearly group with any of the other populations. The AMOVA showed
that the among population variation was lower (32%) in comparison with the genetic variation
present among individuals within different populations (53%). Fifteen percent of the observed
genetic variation was attributed to the sampling regions (S3 Fig). This is also evident from the
PCoA with all samples included (S4 Fig). In the dataset containing only Vietnamese popula-
tions in northern Indo-Burma (VTN-N and VTN-C), the first two axes of a PCoA explained
together 35.6% and 27.9% of the genetic variation respectively (Fig 2B). Some population
structuring was found. Populations VTN-N1, VTN-N3, VTN-N7, and VTN-N8 were grouped
together as well as VTN-N2, VTN-N4, and VTN-N13. VTN-N9 was more distant to the other
sampled populations.
In both sets of genetic clustering analyses using STRUCTURE with correlated allele fre-
quencies, the optimal number of clusters was different for the three assessment criteria, mak-
ing it hard to determine the optimal number of clusters (S3 Table). Because the high optimal
number of clusters suggested by most criteria (K ranging from 11 to 19) were very hard to
interpret, we considered the Evanno method (ΔK/K) to assess the number of clusters. For both
datasets, this resulted in an optimal number of K = 2 clusters (S5 Fig). However, it has been
shown that the Evanno method is often biased towards K = 2 as the most probable number of
genetic clusters [79]. Therefore, population stratification was also assessed using the second-
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
Fig 2. Principal coordinate analysis of wild populations of Musa balbisiana based on a Codom genotypic genetic distance matrix. (A), full dataset; (B), Native
Vietnamese populations. Colours are based on cluster assignments of the STRUCTURE analyses below (see Fig 3).
https://doi.org/10.1371/journal.pone.0253255.g002
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
highest value for ΔK/K and by comparing the STRUCTURE results for all K values to the
PCoA. For the full dataset, no K value lower than K = 16 was found to confidently represent
the population structure thus K = 2 was considered to be most optimal, which was also sup-
ported by the PCoA (Fig 2A). For the dataset with only native Vietnamese populations, K = 5
was considered most reliable based on this method and the PCoA (Fig 2B). These K values
were used to run the final set of analyses with uncorrelated allele frequencies with a separate α
for each cluster and with an initial α = 0.5 and α = 0.2 for the full and restricted analyses,
respectively, using independent allele frequencies. In the full analysis (K = 2), only 17 individu-
als had an assignment probability of< 0.8 and were therefore not assigned to a specific cluster.
Most of them were individuals from populations sampled in central Vietnam (VTN-C1 and
VTN-C2). Almost all individuals from native populations from Vietnam (except some from
VTN-C2) were assigned to the same cluster, while individuals from Chinese populations and
from populations sampled in south Vietnam were all assigned to the other cluster (Fig 3A).
Using a K = 5 for the native Vietnamese populations resulted in 35 individuals from 9 different
populations with an assignment probability to a specific cluster of< 0.8. Most individuals of
four populations (VTN-N1, VTN-N3, VTN-N7, VTN-N8) were mainly assigned to cluster 1,
most of three other populations to cluster 2 (VTN-N6, VTN-N11, VTN-N12), and individuals
of another four populations to cluster 3 (VTN-N2, VTN-N4, VTN-N5, VTN-N13). All indi-
viduals from VTN-N9 were assigned to their own cluster 4. Plants from central Vietnam were
almost all assigned to cluster 5. Some individuals of VTN-N10 were assigned to clusters 1, 2, or
4, but most individuals of this population could not be assigned to a specific cluster (Fig 3B).
Mantel tests between geographic and genetic distances revealed a significant pattern of
genetic isolation by distance between wild M. balbisiana populations (p< 0.01) when assessing
all populations. However, the regression curve only accounted for 15% of the observed
response (R2 value of 0.153) (Fig 4A). When assessing only the Vietnamese populations, no
pattern of IBD was found (p = 0.769) (Fig 4B).
Discussion
Genetic diversity in Musa balbisiana populations
In this study, we assessed for the first time the genetic variation of Vietnamese populations of
Musa balbisiana and complemented them with populations sampled in the Yunnan and
Guangdong province of China, as well as a population of Hainan island. For this, we used a set
of 18 polymorphic microsatellite markers that were combined and in four multiplex PCRs in a
previous study [46]. This allowed us to compare genetic diversity of Vietnamese populations
with populations sampled in the regions that have been suggested to be part of the native dis-
tribution region of M. balbisiana [29,49]. Blasting the markers for which the sequences were
available against the reference genome showed that most chromosomes were covered. How-
ever, some were only covered by one marker and large parts of chromosomes are not optimally
covered using SSRs. Still, compared to the 11 out of 18 markers Bawin et al. [46] found to be
polymorphic, an additional two (13 in total) were polymorphic for our dataset, validating that
these markers can detect sufficient genetic variation. While a direct comparison is not possible
with the populations used in these studies due to differences in genetic markers (AFLP or
other SSR markers) and incomplete information on data processing, general patterns in
genetic diversity can be compared. In relation to studies that similarly assess the genetic varia-
tion of populations of other CWR species using SSRs (e.g. [80–82]), the genetic variation we
found in all M. balbisiana populations was rather low, with observed heterozygosity levels
ranging from 0.05 to 0.28 with an average of 0.19. Low genetic variation in populations can be
expected in small, fragmented populations or with low gene flow between populations and
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
Fig 3. Bar plots indicating cluster assignments obtained with STRUCTURE. Bar plots are presented for the full
dataset (A) and the reduced dataset only including native Vietnamese populations (B) with K = 2 and K = 5,
respectively. Pie charts indicate cluster assignment values of individuals within each population. White pieces indicate
the partition of individuals that could not be assigned to one specific cluster, using an assignment probability threshold
of 0.8. Geospatial datasets used for the creation of these maps were reprinted from [59–61] under a CC-BY 4.0 license.
https://doi.org/10.1371/journal.pone.0253255.g003
high levels of inbreeding [83]. Comparable to population genetic studies of M. balbisiana and
other crop species with clonal reproduction capacities [84,85], we found negative inbreeding
coefficients (heterozygotic excess) in six populations and overall high levels of within popula-
tion variation (53%) versus among population variation (32%). In bananas, excess of heterozy-
gosity could be explained mainly by three mechanisms. First, while the species is not self-
incompatible, selfing is limited in M. balbisiana due to the way the flowers develop. The flow-
ers at the base of the inflorescence are typically female, followed by hermaphrodite flowers
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
Fig 4. Isolation by distance plot. Geographic distance is displayed on the x-axis and genetic distance on the y-axis for (A) the
complete dataset, and (B) the reduced dataset including only native Vietnamese populations.
https://doi.org/10.1371/journal.pone.0253255.g004
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
with aborted female and male organs at the tip of the inflorescence. Due to different flowering
times, self-pollination is largely prohibited. Second, the species can propagate clonally. Clonal
reproduction might result in a heterozygosity excess because of a low number of genotypes
present in a population (low expected heterozygosity) while maintaining heterozygosity for a
large proportion of the assessed loci [86]. Lastly, levels of heterozygosity could be maintained
through the accumulation of somatic mutations over long periods of time, but it is very
unlikely that, if this is the case, can be picked up with 18 SSR loci [29,84,85,87].
Despite the relatively low observed levels of genetic variation, clear differences were found
between populations. Excluding the population from Hainan, Chinese populations on average
had moderate to high percentages of polymorphic loci, expected and observed levels of hetero-
zygosity as well as low levels of inbreeding. In previous studies on genetic diversity in Chinese
M. balbisiana populations, high relative levels of genetic diversity were also found in popula-
tions sampled in Yunnan, Guanxi and southern Guangdong, especially for the population of
Yunnan in the northern Indochina subtropical region [29,49]. They suggest that the high
genetic diversity in the more western Chinese M. balbisiana populations is potentially caused
by the presence of long-tongued fruit bats (Macroglossus sobrinus) that act as a long-distance
pollinator in western China.
In contrast to the relatively high genetic variation observed in Chinese populations (Ho
ranging from 0.175 to 0.243), the genetic diversity in Vietnamese Musa balbisiana showed
larger differences between populations (Ho ranging from 0.051–0.278). Populations sampled
in south Vietnam, near the Red River Basin (VTN-N1, VTN-N7, VTN-N8) as well as VTN-N3
in Lai Chau had low genetic diversity, with a low number of different alleles, a limited number
of private alleles, a low number of polymorphic loci, and low heterozygosity levels. Three pop-
ulations had high levels of these indices and low levels of inbreeding (VTN-N10, VTN-C1,
VTN-C2), while the other populations had moderate levels. Most notable is that the popula-
tions of central Vietnam (VTN-C1, VTN-C2) had the highest levels of different alleles and
number of polymorphic loci and in particular VTN-C2, which had the highest proportion of
private alleles compared to the other populations. Compared to the Chinese populations, simi-
lar levels of genetic diversity were found in northern and central Vietnam, excluding VTN-N1,
VTN-N3, VTN-N7, and VTN-N8. Noteworthy, according to the bats checklist of Vietnam
[88], Macroglossus sobrinus has only been recorded in the close vicinity of those central Viet-
namese M. balbisiana populations (VTN-C1,VTN-C2). However, other sources also report M.
sobrinus bats further North (including where VTN-N9 and VTN-N10 occurred) [89].
In the PCoA considering all sampled populations, Vietnamese populations could clearly be
distinguished from Chinese populations (Fig 2A). This was supported by STRUCTURE analy-
ses, with the exception of high admixture between the two clusters in VTN-C2 and populations
of southern Vietnam clustering with Chinese populations. We did not observe a pattern of iso-
lation by distance in the northern and central Vietnamese populations, and only a low (though
significant) R2 value of the IBD pattern for all assessed populations. This can be caused by
either limited gene flow due to geographic barriers or a lack of suitable long-distance pollina-
tors on the one hand, or excessive gene flow homogenising genetic variation among popula-
tions on the other hand. The former has been linked with a potential decline in fruit bats due
to habitat loss, bushmeat hunting, and climate change [49,90]. Because bananas can easily
propagate via suckers, a long-distance seed dispersal event might result in the establishment of
a healthy population genetically similar to the source population, but with no or limited gene
flow after colonisation. Apart from VTN-N3 from Lai Chau and the populations near the Red
River (VTN-N1, VTN-N7, VTN-N8), northern and central M. balbisiana populations can be
considered to be a part of the native distribution area of the species based on their levels of
observed heterozygosity. However, the origin of the two remote M. balbisiana populations
PLOS ONE | https://doi.org/10.1371/journal.pone.0253255 June 23, 2021 13 / 22
PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
located in southern Vietnam (Kon Tum province) remains more uncertain. Populations in
southern Vietnam might be more connected to M. balbisiana populations in Laos and Cambo-
dia than to other populations in Vietnam, which may explain the genetic differentiation of the
southern populations from other Vietnamese populations included in this study. Nevertheless,
the distribution of M. balbisiana in these neighboring countries is obscure, impeding the infer-
ence of any potential link with the populations in southern Vietnam. Alternatively, the south-
ern M. balbisiana might be feral, originating after the recent human introduction of the
species in the region as a source of food, medicine, fibre, and animal fodder. M. balbisiana was
known by locals in northern Vietnam because of its higher tolerance to cold, drought and
potential plant diseases, encouraging its cultivation in other parts of the country [38,39,42].
The southern populations of M. balbisiana, especially VTN-S1, were also found close to culti-
vated varieties. Moreover, these populations were located far outside the range of the northern
Indo-Burma subtropical forests, the native habitat of the other Vietnamese M. balbisiana pop-
ulations. Taken together and in absence of information on M. balbisiana in neighboring coun-
tries, southern Vietnamese populations of M. balbisiana are supposedly no part of the native
distribution area of the species.
Genetic structure of native Vietnamese Musa balbisiana
By inferring genetic clusters solely among the Vietnamese populations that occur in the north-
ern Indochina subtropical forests, the northern and central Vietnamese M. balbisiana popula-
tions could be subdivided into five different groups. This result was supported by both the
PCoA and the STRUCTURE analyses. Interestingly, the genetic diversity indices followed to a
large extent this subdivision. In this section, we discuss the observed patterns of genetic struc-
ture among the northern and central Vietnamese M. balbisiana populations.
In northern Vietnam, four populations (VTN-N1, VTN-N3, VTN-N7, VTN-N8; Fig 3B)
clearly grouped together in all analyses, especially in the STRUCTURE analysis. They shared
very low levels of genetic diversity. These populations, apart from VTN-N3 sampled in Lai
Chau, were located between the east side of the Hoang Lien Son mountain range and the Red
River, and showed limited genetic admixture with the other clusters. The northwest-southeast
orientation of the Red River valley separated these populations from the northeastern montane
regions. This suggests that the Red River acted as geographic barrier that prevents gene flow
between populations in the east, a pattern that has been observed in two frog species [91] and a
species of Cycas [92]. As a southern extension of the Himalayas with multiple peaks above
2,500m and even up to 3,143m on Fansipan mountain, the Hoang Lien Son mountain range
might act as a barrier to the west [93]. How and why VTN-N3 was assigned to this cluster is
uncertain. We propose that this might be the result of a one-time long dispersal event that was
mediated by either animals or humans, with limited connectivity to neighbouring populations.
Another hypothesis is that all populations belonging to cluster 1 have a history of human influ-
ence. For example, M. balbisiana is known to be introduced from China to the Ryuku isles in
Japan already in the 16th century for fiber use, making it hard to consider all sampled popula-
tions as wild, especially when they have low genetic variation and are located close to cultivated
areas [26].
On the western side of the Hoang Lien Son mountain range, populations VTN-N2,
VTN-N4, VTN-N5, and VTN-N13 (cluster 2, Fig 3) are geographically close to each other and
follow to some extent a small river complex (Nam So, Nam Na, Nam Ma, Fig 1). The link
between populations of cluster 3 and the Black River (Song Da, Fig 1) is clear, with a direct
connection between VTN-N11 and VTN-N12 and an indirect connection with VTN-N6 via
the Nam Na river that also connects to some extent to the Nam so. This could potentially
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PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
explain the assignment of some individuals of VTN-N6 to either cluster 2 or cluster 3. The
Nam Muc river indirectly connects the highly admixed VTN-N10 to the Black River system.
The high genetic diversity found in this population might be due its proximity to VTN-N9
that was assigned to its own cluster as well to the Black River system or to populations in Laos.
Though all individuals from VTN-N9 were assigned to its own cluster, this population had a
relatively high level of observed heterozygosity and an inbreeding coefficient of -1, suggesting
that this population might be for a large part reproducing clonally [87,94]. The link between
river systems and genetic clustering could be the heterogenous topography of northwestern
Vietnam with peaks over 2,000m a.s.l. that can act as a physical barrier for both seed dispersal
and pollination by bats and insects via land [95,96].
Populations sampled in central Vietnam showed the highest number of different alleles, the
highest proportion of private alleles and the highest percentage of polymorphic loci. Both popu-
lations also had among the highest levels of heterozygosity of all sampled Vietnamese popula-
tions. Both populations clearly clustered together in the STRUCTURE analyses. Geographically,
they can be considered to be at the southern range edge of the northern Indo-Burmese region.
In theory, populations at the edge of the distribution range are expected to be more isolated and
smaller, with lower levels of genetic diversity within populations but higher genetic differentia-
tion between populations due to reduced gene flow [55]. This can result in high levels of
regional diversity at the range edge populations, hence worth preserving. Moreover, local adap-
tations and unique alleles present in these populations make them even more interesting for
conservation [97]. However, while the proportion of unique alleles and the genetic variation
between populations was high in the populations from central Vietnam, we also found a high
genetic variation within populations and a low level of inbreeding. This might be explained by
the fact that northern Indo-Burma was not entirely covered by ice-sheets during the Pleistocene
and this area might have served as a long-term climatically stable refugia [98,99]. Moreover, the
Hoang Lien Son mountain range is a north-south oriented extension of the Himalayas with a
heterogeneous topography, allowing populations to easily adapt to climate fluctuations via small
shifts in their altitudinal and latitudinal range. North to south migrations during cooling and
warming events are believed to be much more difficult in east-west oriented mountain ranges,
making the local extinction of populations and the associated loss of genetic variation much
more likely [97,100]. Another possibility is that these populations were closely connected to
other, unsampled populations, especially in floristically similar regions such as northern Laos,
which may have increased their genetic variation via gene flow [93].
It is clear that these populations at the southern edge of the native distribution area,
together with those from northern Vietnam, contain unique and new genetic variation poten-
tially interesting for crop improvement. Not only should more germplasm be collected from
these regions, other parts of the northern Indo-Burma region (e.g. northern Laos and eastern
Myanmar) should be studied as well, as they are likely suitable for Musa balbisiana.
Conclusions
For the first time, Vietnamese populations of Musa balbisiana were genetically assessed and
considerable differences between sampled populations were identified. Populations in central
Vietnam, as well as a subset of populations in northern Vietnam, exhibited relatively high lev-
els of genetic diversity comparable to native populations in China. Interestingly, Vietnamese
populations showed no pattern of isolation linked to distance, indicating either excessive gene
flow homogenising variation among populations or low gene flow between populations due to
geographical barriers, limited long-distance pollination, and the ability to easily establish a
new population after colonisation. We propose that combined clonal and sexual propagation
PLOS ONE | https://doi.org/10.1371/journal.pone.0253255 June 23, 2021 15 / 22
PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
are drivers of the observed patterns of genetic variation and structure, together with the moun-
tain and river systems in northern Vietnam. Two populations sampled in more southern
regions of Vietnam were considerably less diverse and were most likely introduced. We sug-
gest a broader sampling of Musa balbisiana, as many regions with a similar suitable climate
have not been investigated yet as well as neighbouring countries also in northern Indo-Burma
(e.g. Laos, Myanmar). Moreover, a large scale phylogeographic study including populations
from the multiple countries of the natural distribution range of M. balbisiana and in regions
where M. balbisiana was introduced is necessary to further understand how this species has
evolved and to what extent this was caused by human interference.
Supporting information
S1 Fig. 3D AFC of SSR genotyping data. The marked population was clearly an outlier and
was removed from the study.
(TIF)
S2 Fig. Chromosome tracks showing the genomic location of the 16 nuclear SSR markers
retrieved from Wang et al. (2011) [64] and Rotchanapreeda et al. (2016) [65] in the refer-
ence genome sequence from a doubled-haploid accession of Musa balbisiana ‘Pisang Klu-
tuk Wulung’ (DH-PKW). Marker locations are labeled in blue by the marker ID.
(TIF)
S3 Fig. Results of the AMOVA for all included populations. Within population variance
(Within Pops), among population variance (Among Pops), and variance among regions are
presented. Regions include Northern Vietnam, Southern Vietnam, Central Vietnam, Western
China, Southern China, and Hainan.
(TIF)
S4 Fig. Principal coordinate analysis of wild populations of Musa balbisiana on the indi-
vidual level based on a Euclidean distance matrix. Each individual was coloured by popula-
tion.
(TIF)
S5 Fig. Output of StructureSelector for determining the optimal number of K. The optimal
number of K was determined for the full dataset (panels at the left hand side) and for native
Vietnamese populations (panels at the right hand side): (A) MedMed K and MaxMed K
through the Puechmaille method; (B), ΔK/K; (C), LnP(K)/K with K the number of clusters
assumed.
(TIF)
S1 Table. Overview of the 18 microsatellite markers used in this study. The underlined part
of the reverse sequences indicates the sequence of the primer tail: Q1 = TGTAAAACGACGGC
CAGT; Q2 = TAGGAGTGCAGCAAGCAT; Q3 = CACTGCTTAGAGCGATGC (Schuelke, 2000).
(DOCX)
S2 Table. Genomic coordinates of SSR markers. 16 nuclear markers with the corresponding
GenBank accession number of each sequence obtained from Wang et al. (2011) [64] and
Rotchanapreeda et al. (2016) [65] together with the genomic coordinates of their single best
BLAST hit.
(DOCX)
S3 Table. Determination of the optimal number of clusters. The optimal number of clusters
K was determined using MedMedK and MaxMedK according to the Puechmaille method,
PLOS ONE | https://doi.org/10.1371/journal.pone.0253255 June 23, 2021 16 / 22
PLOS ONE Genetic diversity and structure of Musa balbisiana populations in Vietnam
ΔK/K, and Mean LnP(K)/K. The number of clusters that were chosen for consecutive analyses
are indicated in bold.
(DOCX)
Acknowledgments
We are grateful to Wim Baert, Lynn Delgat, Annelies Heylen, Pieter Asselman, and Sander de
Backer for their technical laboratory work at Meise Botanic Garden. We would like to thank
the members of the research team of the Plant Resources Center of Vietnam that assisted in
the multiple collecting missions necessary to complete this work.
Author Contributions
Conceptualization: Arne Mertens, Dang Toan Vu, Loan Thi Le, Filip Vandelook, Steven B.
Janssens.
Data curation: Arne Mertens.
Formal analysis: Arne Mertens, Yves Bawin, Samuel Vanden Abeele.
Funding acquisition: Dang Toan Vu, Rony Swennen, Filip Vandelook, Steven B. Janssens.
Investigation: Arne Mertens, Yves Bawin, Samuel Vanden Abeele, Simon Kallow, Dang Toan
Vu, Loan Thi Le, Tuong Dang Vu, Bart Panis, Steven B. Janssens.
Methodology: Arne Mertens, Samuel Vanden Abeele.
Project administration: Dang Toan Vu, Rony Swennen, Filip Vandelook, Steven B. Janssens.
Resources: Arne Mertens, Yves Bawin, Simon Kallow, Dang Toan Vu, Loan Thi Le, Tuong
Dang Vu, Bart Panis, Steven B. Janssens.
Supervision: Rony Swennen, Steven B. Janssens.
Validation: Arne Mertens, Yves Bawin, Samuel Vanden Abeele, Simon Kallow, Dang Toan
Vu, Rony Swennen, Filip Vandelook, Steven B. Janssens.
Visualization: Arne Mertens.
Writing – original draft: Arne Mertens.
Writing – review & editing: Yves Bawin, Samuel Vanden Abeele, Simon Kallow, Dang Toan
Vu, Rony Swennen, Filip Vandelook, Bart Panis, Steven B. Janssens.
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