Received: 15 December 2020 | Accepted: 7 May 2021

DOI: 10.1111/1365-2745.13710

RESEARCH ARTICLE

Coordination of leaf economics traits within the family of the

world's fastest growing plants (Lemnaceae)

Hidehiro Ishizawa1,2 | Yusuke Onoda3 | Kaoru Kitajima3 | Masashi Kuroda1,4 |

Daisuke Inoue1 | Michihiko Ike1

1
Division of Sustainable Energy and
Environmental Engineering, Graduate Abstract
School of Engineering, Osaka University, 1. The duckweed family (Lemnaceae) is a group of free-­floating aquatic plants with
Suita, Japan
2 bodies consisting of single floating fronds that multiply clonally. Although they are
Research Institute of Green Science
and Technology, Shizuoka University, known to have the fastest relative growth rate (RGR) among higher plants, their
Hamamatsu, Japan
functional trait coordination in relation to within-­family variation of RGR is poorly
3
Division of Forest and Biomaterials Science,
Graduate School of Agriculture, Kyoto
understood.
University, Kyoto, Japan 2. We tested how duckweed species fit within the trait covariation patterns known
4
Faculty of Social and Environmental as the world-­wide leaf economics spectrum (LES). To this end, several functional
Studies, Tokoha University, Shizuoka, Japan
traits were evaluated for 15 duckweed species, and their covariation patterns
Correspondence were compared with those in the global database of plant functional traits.
Hidehiro Ishizawa
Email: ishizawahidehiro@gmail.com 3. As a group, duckweeds exhibited the most acquisitive suite of traits, with ex-
tremely small leaf mass per area (LMA), short life span and high mass-­based pho-
Funding information
Japan Society for the Promotion of Science, tosynthetic rate (Amass). These LES traits showed a tight correlation with RGR,
Grant/Award Number: JP18J10181 and corroborating our hypothesis that acquisitive leaf resource economics underpins
JP20J00210; Advanced Low Carbon
Technology Research and Development their extremely high RGR. However, unlike other higher plants, LMA showed weak
Program, Grant/Award Number: association with leaf life span and Amass within duckweed family. We also found
JPMJAL1108
a unique positive correlation between duckweed LMA and area-­based photosyn-
Handling Editor: Peter Vesk thetic rates, an indication that their LMA represents different functional signifi-
cance compared to typical higher plants.
4. Synthesis. Duckweeds, the world's fastest growing plants, mostly follow the
world-­wide LES and locate at its extreme end. The slight deviation from the LES
highlights that duckweeds experience some physical and chemical constraints not
faced by other higher plants.

KEYWORDS

aquatic plant ecology, duckweed, functional trait, leaf economics spectrum, leaf life span, leaf
mass per area, photosynthesis, relative growth rate

1 | I NTRO D U C TI O N
Lemnaceae is a monocot family consisting of five genera (Spirodela,
Landoltia, Lemna, Wolffiella and Wolffia) and 36 species of free-­
floating aquatic plants commonly called duckweeds, which distribute
globally except for polar regions (Bog et al., 2019; Landolt, 1986).
These plants are known for their simple body structure comprised of
a small leaf-­like organ called ‘frond’ with the size of 0.5 to 15 mm di-
ameter (Hillman, 1961). Spirodela, Landoltia and Lemna species have
up to a few tiny roots hanging down from the frond, while Wolffella

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ISHIZAWA et al.
and Wolffia have no roots. This extremely reduced body design,
minimizing non-­photosynthetic organs, presumably reflects natural
selection for rapid multiplication via vegetative budding (Kutschera
& Niklas, 2015). Indeed, the world's fastest relative growth rates
(RGRs) have been reported from duckweeds, which could double
their biomass within 1 to 2 days under optimal conditions (Sree,
Sudakaran, et al., 2015; Ziegler et al., 2014). These characteristics
make them ideal in low-­cost water treatment systems for removal
of excess nutrients from municipal, agricultural and industrial waste-
waters (Alaerts et al., 1996; Oron et al., 1988; Ziegler et al., 2016).
Also, due to their ability to rapidly produce biomass with high starch
and protein content, they are excellent as a renewable source of
biofuel, animal feeds and human food products (Appenroth et al.,
2017, 2018; Cheng & Stomp, 2009; Cui & Cheng, 2015). Because
their small and simple morphology make them an ideal model organ-
ism for experimental purposes, duckweeds have also been used in
several biochemical and ecological studies (Baldi et al., 1991; Hart
et al., 2019; Laird & Barks, 2018; Muranaka & Oyama, 2016; Xu
et al., 2018).
Despite the practical importance of duckweed species, the eco-
physiological basis underlying the super-­fast growth has not been
explained beyond a general notion that as a group they occupy the
extreme end of the scaling relationship of biomass production ver-
sus body size across higher plants (Kutschera & Niklas, 2015; Niklas
& Enquist, 2001). Several comparative studies have shown that
there are considerable interspecific and intraspecific variations in
biomass productivity, chemical composition and water remediation
capacity among duckweed species (Barbosa Neto et al., 2019; Sree,
Sudakaran, et al., 2015; Toyama et al., 2018; Ziegler et al., 2014). Yet,
the ecophysiological reasons underpinning these variations have
hardly been explored.
The trait-­based approach, which allows researchers to infer why
and how plant species differ in their functional or adaptive strate-
gies (Garnier & Navas, 2012; Kunstler et al., 2016), should be extend-
able to aquatic plants including duckweed species (Pan et al., 2019).
During the last few decades, databases on plant functional traits have
become world-­wide in coverage. The compilation of the data from
tens of thousands of woody and herbaceous species demonstrates
globally convergent patterns of trait covariations (Díaz et al., 2016).
In particular, the leaf economics spectrum (LES), that is, the tight co-
variation of leaf life span, leaf mass per area (LMA), leaf nitrogen
content (Nmass) and photosynthetic rate per leaf dry mass (Amass)
put forward as a synthesis by Reich et al. (1992, 1997, 1999) and
Wright et al. (2004), is a robust pattern that has been demonstrated
in multiple biomes. The LES illustrates why and how photosynthetic
capacity varies among species along a single axis of resource-­use
strategy from the ‘acquisitive’ strategy (represented by short lived,
nutrient rich, fast photosynthetic and cheaply constructed leaves)
to the ‘conservative’ strategy (with opposite set of leaf traits). Many
studies have shown that there are significant trait covariations even
within taxonomic groups, which are in general consistent with the
world-­wide LES (Hayes et al., 2019; Martin et al., 2017; Mason &
Donovan, 2015; Xiong & Flexas, 2018).
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Journal of Ecology | 2951
Although LES traits cannot be simply translated into whole-­
plant traits such as RGR (Poorter et al., 2014; Sartori et al., 2019;
Shipley, 2006), we hypothesized that duckweeds occupy the most
acquisitive end of the LES among higher plants. This is partially
supported by the scattered reports on low LMA, high Nmass and
high Amass of some duckweed species (Appenroth et al., 2018; Pan
et al., 2020; Paolacci, Harrison, et al., 2018; Wedge & Burris, 1982).
However, to date, no comprehensive studies have reported other
LES traits (e.g. leaf life span) and RGR for duckweed and other free-­
floating macrophytes.
At the same time, we predict significant deviations from the
typical LES in the within-­family pattern of duckweed trait syn-
drome. In the well-­k nown LES framework, the relative investments
to photosynthetic components (e.g. proteins) and structural com-
ponents (e.g. cell walls) increase and decrease, respectively, with
the reduction of LMA, underpinning the tight covariation of LMA,
N mass , A mass and life span (Onoda et al., 2017). However, there must
be an ultimate limit in reducing cell wall fraction. If duckweeds
have reduced cell wall fraction thanks to floating lifestyle, that
is, no need to hold leaves against gravity (Appenroth et al., 2018;
Blazey & McClure, 1968; Soda et al., 2015), there might be lit-
tle room for further reduction of structural investments because
terrestrial fast growing herbaceous species already have a small
cell wall fraction (<10%, Onoda et al., 2017). In addition, while
the LES predicts that N mass increases with decreasing LMA, there
should be some upper limit of N mass . This is because of the need
for essential components and metabolites other than protein, 16%
of which is N (i.e. N mass of 10% is equivalent to the protein con-
tent of c. 63%). Such restrictions on available fraction of biomass
would be especially severe for duckweeds, as their frond must
also store energy (nonstructural carbohydrates including starch)
and function as a reproductive organ. A possible outcome of these
limitations is the departure from the typical LES, in the direction
to weaken the association between LMA to other LES traits.
Exploring these aspects provides not only practical knowledge
on the duckweed functioning, but also basic ecophysiological in-
sights towards understanding the limit of the acquisitive strategy
achievable by higher plants.
Here, we ask two research questions. What position do duck-
weed species occupy within the world-­wide LES of higher plants? Do
duckweed functional traits exhibit covariation patterns consistent
to other higher plants? To answer these questions, we collected and
analysed functional traits of 15 duckweed species covering a wide
range of morphological variation in the duckweed family. A total of
13 traits, including central LES traits, RGR and other traits express-
ing their life-­history strategy (e.g. offspring number and starch accu-
mulation), were measured under standardized laboratory condition.
We then compared the duckweed trait syndromes with those in a
global database of plant functional traits (Global Plant Trait Network,
GLOPNET, Wright et al., 2004). The results show that duckweeds
possess the most acquisitive strategy among higher plants, with sig-
nificant departures from the typical LES in trait covariations within
the family.
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2952 | Journal of Ecology ISHIZAWA et al.

2 | M ATE R I A L S A N D M E TH O DS subfamily Lemnoideae; Figure 1). These species were selected to

cover a wide range of the morphological and phylogenetic varia-
Abbreviations are summarized in Table 1. tions in Lemnaceae, except for the species that possess submerged
fronds (e.g. Lemna trisulca and Wolffiella gladiata). We obtained
plant materials that had been maintained in controlled condi-
2.1 | Plant materials tions for many years in several research institutions, including the
Institut für Integrative Biologie, ETH Zurich (Zurich, Switzerland),
In this study, we used 15 duckweed species encompassing all Graduate School of Science, Kyoto University (Kyoto, Japan), J
five genera of the duckweed family (Lemnaceae, also classified as Craig Venter Institute (Maryland, USA), Rutgers Duckweed Stock
Cooperative (New Jersey, USA), Graduate School of Environmental
TA B L E 1 A list of abbreviations used in this study Sciences, Hokkaido University (Hokkaido, Japan), Graduate Faculty
of Interdisciplinary Research, University of Yamanashi (Yamanashi,
Abbreviation Definition Units
Japan) and Graduate School of Engineering, Osaka University
Aarea Light-­saturated photosynthetic µmol CO2 m−2 s−1 (Osaka, Japan). For species identification and phylogenetic analy-
rate per area
ses, we obtained the DNA sequences of the plastid atpF–­atpH in-
Amass Light-­saturated photosynthetic µmol CO2 g−1 s−1 tergenic region of these duckweed clones from previous reports
rate per dry mass
(Borisjuk et al., 2018; Ishizawa et al., 2017; Muranaka et al., 2015;
Chl a/b Chlorophyll a/b ratio Wang et al., 2010). For two duckweed clones without accessible
Chlmass Chlorophyll content mg/g sequences, additional sequencing was performed according to the
GLOPNET Global plant trait network method described by Wang et al. (2010). Table S1 provides more
LES Leaf economics spectrum details of the origins and gene accession numbers of the plant ma-
LMA Leaf mass per area g/m 2 terials used in the present study.

LMR Leaf mass ratio g/g

NAR Net assimilation rate g m−2 day−1
2.2 | Plant growth conditions
Nmass Nitrogen content %
Narea Nitrogen content per area g/m2
All duckweed samples were surface sterilized with 0.5%–­2% sodium
RGR Relative growth rate day−1
hypochlorite supplemented with 0.02% Triton X100 for 1 to 5 min be-
SLA Specific leaf area m2/g
fore cultivating aseptically in half-­strength Schenk and Hildebrandt
SMA Standardized major axis medium (1/2 SH medium; 1,250 mg/L KNO3, 150 mg/L NH4H2PO 4,
[Correction added on 23 June 2021, after first online publication: SLA 97.7 mg/L MgSO 4, 75.5 mg/L CaCl2, 7.5 mg/L FeSO 4·7H2O, 5 mg/L
unit has been changed from (m2/g–­1) to (m2/g).] MnSO 4·H2O, 2.5 mg/L B(OH)3, 0.5 mg/L ZnSO 4·7H2O, 0.5 mg/L KI,

F I G U R E 1 Duckweed species used in this study. (a) Appearances of the duckweeds. Abbreviation and clone number of each species are shown
in parentheses. All images are shown at the same scale. (b) Phylogenetic tree of the duckweeds based on atpF–­atpH intergenic sequences. The
phylogenetic tree was drawn using a BioNJ method with clustalW v2.1. The sequence of Pistia stratiotes (NC_048522) was used for outgroup rooting

ISHIZAWA et al.
0.1 mg/L CuSO 4, 0.05 mg/L CoCl2·6H2O, 0.05 mg/L Na2MoO 4·2H2O,
10 mg/L EDTA·2Na; pH 5.8) containing 1 g/L of 2-­(N-­morpholino)
ethanesulfonic acid. The 1/2 SH medium was used in previous works
that compared the maximum growth rate of duckweed species (Sree,
Sudakaran, et al., 2015; Ziegler et al., 2014) and is regarded as one
of the best media for optimum duckweed growth (Yu et al., 2014).
The cultivation was done under the standard laboratory condition
in a growth chamber (28℃, photon flux of 80 µmol m−2 s−1, photo-
period of 16 hr: 8 hr, light: dark; LH-­220S, NK Systems). For trait
measurements, we first acclimated the plants to the aforementioned
conditions for at least a month, in order to ensure the constant plant
physiological status during cultivations (Datko et al., 1980). Then,
multiple fronds randomly picked from the 5-­
day pre-­
cultivation
2
flasks (approximately 7 cm altogether) were transplanted into a
500 ml flask containing 250 ml of the growth medium. The trans-
planted plants were grown for 5 days before harvesting for analyses,
until which time they could grow without overlapping each other
within the container. Unless otherwise indicated, each species was
grown in three replicate flasks and traits were measured indepen-
dently (n = 3).
2.3 | Measurements of duckweed functional traits
2.3.1 | Relative growth rate
During the 5-­day cultivation period, duckweed growth was monitored
by counting frond number at 24 hr intervals. Since all duckweed spe-
cies showed exponential growth throughout the duration of the ex-
periment (Figure S1), the potential growth rate of each species could
be adequately expressed by the RGR based on frond counts (frond
frond−1 day−1) as shown by Equation 1 below (Lasfar et al., 2007).
( ) ( )
ln FN5 − ln FN0
RGR = , (1)
5
where FN0 and FN5 represent the frond number before and after the
5-­day cultivation period respectively.
2.3.2 | Frond area, mass, leaf mass per area and net
assimilation rate
The average frond area of each duckweed species was determined
as follows. First, a sufficiently large number of duckweed fronds
(c. 150–­2,600), including both adult and young fronds, were col-
lected after the 5-­day cultivation period and floated on 25 ml of
white-­coloured melt agarose (1% agar, 1% skim milk) in a Petri dish.
Next, the solidified agarose gel was put upside down on a flat-­bed
scanner (GT-­S630, Epson), and a high-­resolution image (2400 dpi,
24-­bit colour) was obtained. Finally, the frond area was measured
using ImageJ software (ver. 1.52b) as previously described (Ishizawa
et al., 2019). Dry mass was also measured for a known number of
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Journal of Ecology | 2953
duckweed fronds (c. 160–­12,000) after drying in an oven at 100℃
for 24 hr. The LMA (g/m2) was then calculated as the average dry
mass per unit frond area.
Net assimilation rate (NAR; g m−2 d−1) was estimated according
to the following well-­known relationship (Hunt, 1982), assuming that
average frond area and mass of duckweeds were constant during the
5-­day cultivations.
RGR = NAR × LMR × SLA, (2)
where NAR represents the biomass increase rate per unit leaf area,
LMR is the leaf mass ratio (ratio of leaf mass to whole-­plant mass) and
SLA is the specific leaf area (leaf area per unit leaf mass). Because duck-
weeds have only trifle roots that account for less than a few per cent of
their biomass and that could be practically ignored (i.e. LMR = 1), and
because LMA (=1/SLA) is more commonly used in the LES, we simpli-
fied and rearranged Equation 2 to estimate NAR as follows.
RGR = NAR ×
1
. (3)
LMA
2.3.3 | Chlorophyll content and chlorophyll a/b ratio
For the evaluation of chlorophyll content (Chlmass; µg/g), a known
number of fronds (c. 20–­130) were immersed in 3 ml of 99.5% meth-
anol and pigments were extracted at 25℃ for 1 hr in dark. Then,
the absorbance of the extract at 650 nm (A650) and 665 nm (A665)
was measured with a spectrophotometer (UV-­1850, Shimazu). The
following equations (Grimme & Boardman, 1972) were used to cal-
culate the chlorophyll a and b content (µg/ml) and the chlorophyll
a/b ratio (Chl a/b).
Chlorophyll a content = 16.5 × A665 − 8.3 × A650 , (4)
Chlorophyll b content = 33.8 × A650 − 12.5 × A665 . (5)
2.3.4 | Starch and nitrogen content
The starch content of each duckweed species was analysed with a
total starch assay kit (Megazyme International). For the determina-
tion of Nmass, dried and ground plants were digested with sulfuric
acid and hydrogen peroxide (Matsunaga & Shiozaki, 1989) prior to
the determination of total nitrogen based on ammonium concentra-
tion using indophenol blue method according to standard methods
(American Public Health Association (APHA), 1995).
2.3.5 | Lifespan and offspring number
The average lifespan and offspring number of each duckweed
frond were determined by the method of Barks and Laird (2015).
In brief, duckweed colonies (groups of connected fronds) were
grown separately in the wells of 24-­well plates containing 2 ml of

2954 | Journal of Ecology
1/2 SH medium. For Spirodela polyrhiza, which has especially large
fronds, 6-­well plates with 10 ml of medium were used instead. Since
the life span and offspring number of duckweed fronds decrease
with increased birth order from ancestral fronds (Wangermann &
Ashby, 1951; Barks & Laird, 2015, 2016), we first obtained duck-
weed colonies containing fronds whose both birth order and paren-
tal birth order are one. This was done by the daily inspections and
removal of old colonies upon separation. After obtaining fronds that
met our criteria, we observed the fronds at 24 hr intervals through-
out their lifetime, and their life span and offspring number (number
of fronds released from an individual frond) were recorded (n = 6).
Here frond life span was defined as the dates from its birth (detach-
ment from the parent frond) to the loss of clonal reproduction (re-
lease of the final daughter frond; Barks & Laird, 2015). To prevent
nutrient shortages, the medium was replaced every day, and new
colonies detached from the observed individuals were discarded at
daily inspections.
2.3.6 | Photosynthetic measurement
We measured light-­
saturated photosynthetic rate of each duck-
weed species using a closed-­path CO2/H2O analyser (LI-­7000, Li-­
cor, Lincoln). After the 5-­day cultivation period, a known number
of fronds (c. 40–­3,000) were transplanted to a 100 ml glass vial
(⌀3.8 cm) containing 90 ml of 1/2 SH medium to cover the water
surface to form a single layer without fronds overlapping each other.
The vial was then put in the growth chamber at 28℃, and stand-
ard gas (0.05% CO2, 20% O2, 79.95% N2) was continuously passed
through the headspace of the vial at a flow rate of 100 ml/min.
Light was supplied by a LED panel that provided a photon flux of
700 µmol m−2 s−1. The system continuously recorded the CO2 con-
centration before and after passing through the vial, and values were
taken after stabilization (n = 2). Then, the net photosynthetic rate
per unit area (A area) and per unit mass (Amass) was calculated from the
frond area or frond mass of duckweeds used in the analysis.
2.4 | Statistical analysis
Data analysis was performed using R v.3.5.1 (http://www.r-­proje​
ct.org). For each measured trait, the coefficient of variation was
calculated as the ratio of the standard deviation to the mean. The
phylogenetic signal of each trait was evaluated based on the K value
of Blomberg et al. (2003) with the phytools package. A value of K = 1
represents the strongest phylogenetic signal possible, as it indi-
cates that the evolutionary changes in a trait can be explained by
Brownian motion; K = 0 indicates no phylogenetic signal (Blomberg
et al., 2003). Phylogenetic distances, computed by clustalW v2.1
based on plastid atpF–­atpH intergenic sequences, were used to
estimate the phylogenetic tree among the studied taxa. For each
trait, we used the fitdistrplus package of r to check whether the
variable exhibited normal or log-­normal distribution according to the
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ISHIZAWA et al.
log-­likelihood test, and traits that better fitted a log-­normal distribu-
tion were log-­transformed before further analyses.
For bivariate analysis, pairwise Pearson's correlation and stan-
dardized major axis (SMA) regression were calculated using the
corrr and smatr r packages respectively. To evaluate the relation-
ships between certain traits (LMA, life span, A mass, A area and Nmass),
we compared the results with the GLOPNET dataset, which con-
tains functional trait data from more than 2,500 terrestrial woody
and herbaceous plant species (Wright et al., 2004). The nitrogen
content per area (Narea) was also calculated as the product of Nmass
and LMA and included in the comparative analysis. The difference
of two SMA slopes was tested using the ‘sma’ function of the smatr
r package.
For multivariate analysis, principal component analysis (PCA)
was performed in order to identify major axes of interspecific trait
variation. We also tested whether the ordination of 15 duckweeds
in multivariate space is related to their genus-­level classification by
permutation multivariate analysis of variance (PERMANOVA). Both
PCA and PERMANOVA were performed based on Bray–­Cutis dis-
similarity using the vegan package of r.
3 | R E S U LT S
3.1 | Interspecific trait variation
Each of the measured traits showed considerable interspecific
variation among the 15 duckweed species (Table 2; Figure S2). The
traits represent duckweed morphology (frond area, frond mass and
LMA) showed especially large interspecific variation (up to 86.7-­,
56.6-­ and 5.6-­fold respectively). Notably, the RGR of Wolffiella
repanda (0.597, i.e., a doubling time of 27.9 hr) was close to the
highest for angiosperm, previously reported from Wolffia micro-
scopica (doubling time of 29.3 hr, Sree, Sudakaran, et al., 2015; or
approximately 24 hr, Appenroth et al., 2018). This duckweed clone
also showed the smallest LMA reported for any higher plant, except
for a submerged aquatic macrophyte species (e.g. Myriophyllum
sp. with an LMA of 2.8 g/m2; Gerber & Les, 1994). Duckweed life
span was generally very short (11.0–­32.2 days) and close to the
floating leaves of other aquatic plants (Kikuzawa & Ackerly, 1999;
Tsuchiya, 1991). Six traits (frond area, frond mass, LMA, NAR, off-
spring number and A area) exhibited significant phylogenetic signals
(Blomberg et al.'s, 2003, K > 0.538, p < 0.05), but the other seven
traits had much weaker and non-­significant phylogenetic signals
(Table 2).
3.2 | Pairwise trait correlations
For the 13 traits, we could examine 78 pairwise correlations, of
which 32 were significant (Figure 2; Table S2). Duckweed species
with larger frond area and mass had longer frond life span, yielding
more daughter fronds within their lifetime (r = 0.54–­0.67, p < 0.05;
 13652745, 2021, 8, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.13710 by Nat Prov Indonesia, Wiley Online Library on [25/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ISHIZAWA et al. Journal of Ecology | 2955

TA B L E 2 Variation in duckweed traits (n = 15)

Log-­likelihood values Descriptive statistics

Phylogenetic
Normal Log-­normal Mean Range CV (%) signal (K)

RGR [frond frond−1 day−1] 18.0 20.2 0.377 0.313–­0.597 19.3 0.057
2
Frond area [cm ] 23.1 34.3 0.040 0.002–­0.209 129.2 0.838*
Frond mass [mg] 23.2 33.5 0.042 0.004–­0.203 121.6 0.722*
LMA [g/m2] −46.8 −46.0 13.0 4.39–­24.6 42.2 0.545*
−2 −1
NAR [g m day ] −27.6 −26.1 4.64 2.62–­8.04 32.9 0.804**
Chlmass [mg/g] −41.0 −38.7 18.5 13.2–­3 0.6 20.0 0.054
Chl a/b 2.38 1.87 3.26 2.69–­3.67 6.34 0.161
Starch content [%] −45.6 −45.9 14.2 5.97–­22.2 35.6 0.242
Nmass [%] −44.8 −45.0 2.83 2.00–­3.94 17.2 0.047
Lifespan [day] −50.1 −50.2 21.7 11.0–­32.2 31.5 0.113
Offspring number −32.6 −33.1 13.3 8.83–­16.3 16.0 0.538*
−2 −1
Aarea [µmol CO2 m s ] −36.8 −37.6 15.2 10.0–­19.8 18.5 0.630*
Amass [µmol CO2 g−1 s−1] 1.3 0.2 1.31 0.81–­2.49 31.4 0.076

Note: Bold: best distribution fit model based on log-­likelihood value.

Abbreviation: CV, coefficient of variation.
*p < 0.05.; **p < 0.01.

F I G U R E 2 Trait correlation network

among 15 duckweed species. Significant
(p < 0.05) positive and negative
correlations are represented by red and
blue lines respectively. Values on lines
represent the Pearson's correlation
coefficient (r). Refer to Table S2 for
detailed results of bivariate correlations

Figure 3a,b). These species tended to have lower starch content
(r = −0.696, p < 0.01) and NAR (r = −0.602, p < 0.05). There was
no significant relationship between frond area and LMA (r = −0.493,
p > 0.05; Figure 3c). NAR, which represents biomass gain rate
per unit frond area, was tightly correlated with A area (r = 0.819,
p < 0.001), with differences among three genera for which multi-
ple species were studied (Wolffia, Wolffiella and Lemna; Figure 3d).
Parallel to this, on mass basis, RGR was positively correlated with
Amass (r = 0.843, p < 0.001; Figure 3e). LMA was correlated positively
with A area and negatively with Amass, and the latter was correlated
with chemical constituents that contribute to photosynthesis, Nmass
and Chlmass (r = 0.547–­0.720, p < 0.05). Meanwhile, Aarea was posi-
tively correlated with the chlorophyll a/b ratio (r = 0.708, p < 0.01;
Figure 3f).
 13652745, 2021, 8, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.13710 by Nat Prov Indonesia, Wiley Online Library on [25/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2956 | Journal of Ecology ISHIZAWA et al.

F I G U R E 3 Relationships among the functional traits of 15 duckweed species. Bivariate relationships between frond area and frond mass
(a), frond area and life span (b), frond area and LMA (c), Aarea and NAR (d), Amass and RGR (e), and Aarea and Chl a/b (f) are shown. Different
coloured plots correspond to different duckweed genera. Refer to Figure 1 for abbreviations of duckweed species

3.3 | Principal components of trait variation
In the PCA of the 13 traits, the first and second principal compo-
nents together accounted for 69.8% of the total variance (Figure 4),
while the third axis accounted for additional 14.6% (Figure S2). The
first component (PC1) was explained largely by strong negative load-
ing of LMA, Aarea, NAR and Chl a/b, and positive loading of Amass,
Nmass, Chlmass and RGR. The differences among the three genera
with multiple species (Lemna, Wolffiella and Wolffia) were obvious
along this axis (high PC1 scores for Wolffiella, negative PC1 scores
for Wolffia and Lemna species in the middle). The starch content also
showed weak negative loading to the PC1, suggesting that species
with high area-­based productivity tended to store surplus carbon
gain for future use. The second component (PC2) was largely ex-
plained by the positive contribution of size (frond area and frond
mass) and demographic traits (life span and offspring number). PC2
reflected large within-­genus variations, especially in Wolffiella and
Lemna, with large overlap among the genera. The ordination of the
15 species corresponded moderately to their genus-­level classifica-
tions (PERMANOVA, F = 1.970, p = 0.043). Wolffia species, which
have particularly small and rootless bodies, showed distinct trait
sets compared with species in the other genera (PERMANOVA,
F = 4.079, p = 0.009).
3.4 | Comparison with the GLOPNET dataset
In Figure 5, the key traits that contribute to the LES for the 15 duck-
weed species are shown in the world-­wide LES trait space. As we
expected, duckweeds as a group occupied the low LMA end of the
LES, extending the spectrum in the direction of lower LMA, higher
Amass (Figure 5a) and shorter life span (Figure 5b). However, the slope
angle within the duckweed family was significantly smaller for Amass
versus LMA compared to the GLOPNET data (p < 0.001; Figure 5a).
In other words, the amount of Amass increase with a unit decrease in
LMA was less than what would be expected from extrapolation of
the LES. Unlike other higher plants, the correlation between life span
and LMA was not significant for duckweeds. Duckweeds extended
the GLOPNET correlation between Narea and LMA, with a signifi-
cantly different slope within duckweeds (p = 0.004; Figure 5c). The
Nmass of duckweeds was lower for a given LMA as expected from LES
(Figure 5d). There was a clear positive correlation between LMA and

ISHIZAWA et al.
F I G U R E 4 Principal component analysis showing multivariate
trait covariations in duckweed species. Arrows represent the
loadings of individual traits on PC1 and PC2. Refer to Figure 1 for
abbreviations of duckweed species
Aarea in the duckweed family, whereas such an association was not
observed in the GLOPNET (Figure 5e).
4 | D I S CU S S I O N
4.1 | General patterns in duckweed trait variations
As predicted, the 15 duckweed species showed very high RGR along
with the traits that indicate acquisitive strategies (i.e. low LMA,
short life span, high Nmass and high Amass), extending the world-­wide
LES for higher plants (Figures 2, 4 and 5). Hence, despite their seem-
ingly unusual lifeform, in a broad sense, their functional traits fit
within what would be expected from the leaf resource economics
in higher plants. However, the trait syndrome of duckweeds is not
a mere extension of LES. For example, duckweeds had lower Nmass
than what would be expected from the LES (Figure 5d). This is not
surprising, since extrapolation from the GLOPNET trend predicts
unrealistic Nmass value for duckweed LMA (up to 110 mg/g, corre-
sponding to a protein content of c. 70%). Considering the presence
of cell walls, metabolites and starch (on average 14.2%; Table 2),
there would be insufficient room to accommodate such large
amounts of protein. This illustrates the fact that at the extreme end
of LES, duckweeds hit certain physical and chemical constraints not
faced by other higher plants. Pan et al. (2020) also reported similar
downshifts of Nmass for wetland plants with extremely low LMA,
suggesting that the linearity of LMA versus Nmass regression cannot
13652745, 2021, 8, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.13710 by Nat Prov Indonesia, Wiley Online Library on [25/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Journal of Ecology | 2957
hold at the low-­LMA end of the LES, probably due to the maximum
possible protein contents.
While several trait associations could be seen as an extension
of the world-­wide LES, there were some associations within the
duckweed family that differed from those seen in other higher
plants (Figure 2). Particularly notable differences included the lack
of correlation between life span and LMA (Figure 5b), and a positive
correlation between LMA and Aarea (Figure 5e). The association of
LMA to Amass was also weaker than those in the GLOPNET data-
set (Figure 5a), probably owing to the weak association of LMA and
Nmass (Figure 5d). These unique patterns are in conflict with the key
underpinning of the LES that leaf life span represents a trade-­off
between leaf construction cost (LMA) and payback speed (Amass),
and that interspecific variation in LMA reflects differences in in-
vestment in cell wall and leaf durability (Kitajima et al., 2012; Onoda
et al., 2011; Poorter et al., 2009; Westoby et al., 2002).
These departure of the trait correlations within duckweed fam-
ily from the world-­wide LES could be attributed to the lower limit
of structural investments. The models of Katabuchi et al. (2017)
and Osnas et al. (2018) partition LMA variations to variations
due to structural components (e.g. cell walls; contribute to leaf
life span) and variations due to photosynthetic components (e.g.
proteins and sugars; contribute to A area). The exceptionally low
LMA of duckweeds probably reflects their ultimate reduction of
structural components, as corroborated by the very low levels of
cellulose and lignin contents across duckweed species (Appenroth
et al., 2018; Blazey & McClure, 1968; Soda et al., 2015). Hence,
unlike other higher plants for which lower LMA is attained mainly
by the reduction of structural components (Onoda et al., 2017),
structural investments of duckweeds may not vary so much as
expected from the 5.5-­fold variation of their LMA. Instead, fur-
ther reduction of LMA among duckweed species might have been
attained by the reduction in photosynthetic components, as sug-
gested by the steeper positive correlation between LMA and Narea
(Figure 5c). These results imply that different mechanistic rela-
tionship of leaf functional traits exists at the lowest-­LMA zone of
the LES, where the reduction in structural investments may have
reached its limitations.
We also suspect that species differences in light interception un-
derpin the tight correlation between LMA and Aarea (Figure 5e). With
their extremely thin fronds that sometimes overlap with each other
to make multiple layers (Driever et al., 2005), thin individual duck-
weed fronds transmit a substantial proportion of radiation. Perhaps,
further reduction of LMA might become suboptimal in terms of
photosynthetic light use efficiency. This suggests that the ultimate
reduction of LMA, which would be essential for realizing high RGR,
is attained at the cost of light capture and carbon gain. Unlike most
land plants which are sessile and require some costs to increase hor-
izontal space (e.g. horizontal branches), floating duckweed fronds
can freely increase horizontal space without additional cost. Thus,
the disadvantage of decreased Aarea through the reduction of LMA
would be compensated by rapid occupation of a large area achiev-
able by high RGR.
 13652745, 2021, 8, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.13710 by Nat Prov Indonesia, Wiley Online Library on [25/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2958 | Journal of Ecology ISHIZAWA et al.

F I G U R E 5 Placement of duckweed species in the world-­wide LES trait space. Relationships between LMA and Amass (a), LMA and life
span (b), LMA and Narea (c), LMA and Nmass (d), and LMA and Aarea (e) are shown. Coloured plots and grey plots represent the 15 duckweed
species (present study) and species in the GLOPNET dataset (Wright et al., 2004) respectively. Relationships among traits are represented by
a standardized major axis regression where correlations are significant (p < 0.05). Note that laboratory-­grown duckweeds were used for trait
measurement, whereas the GLOPNET compiles data from wild plants

4.2 | Implications for duckweed growth strategies
The most predominant axis of duckweed trait variation (PC1) rep-
resents the trade-­off between photosynthesis (A area, i.e., photosyn-
thetic productivity per unit leaf area) and growth rate (RGR, i.e.,
rapid population expansion or dispersal) among duckweed species
(Figure 4). Some key traits associated with PC1 (LMA, A area and
NAR) showed strong phylogenetic signals (Table 2), and the two
newly evolved genera (Wolffiella and Wolffia) occupied the op-
posite ends of this spectrum, suggesting evolutionary divergence
along this trade-­off axis at the genus level among duckweed spe-
cies. It is also notable that Chl a/b, an indicator of plant adaptation
to light and nutrient availabilities (Dale & Causton, 1992; Kitajima &
Hogan, 2003; Terashima & Hikosaka, 1995), was closely associated
with PC1. This means that it is likely that adaptation to contrast-
ing light and nutrient environments has led to the diversification of
duckweed growth strategies, which deserves further examination.
The PC2 might reflect the reproductive capacity of duck-
weeds, but its adaptive implication is not as clear as that of PC1
(Figure 4). Since species with high PC2 scores (Spirodela, Landoltia
and Lemna species) are more successful in broad geographical re-
gions (Tippery & Les, 2020), it is likely that PC2 reflects adaptive
differences to contrasting environmental conditions. One possi-
bility is stress tolerance, and literature examining duckweed re-
sistance to environmental stressors consistently shows higher
tolerance in larger species (Song et al., 2015; Sree, Adelmann,
et al., 2015; Yang et al., 2018). In light of these results, we propose
that duckweed trait syndromes can provide a comprehensive pic-
ture of their adaptive strategies analogous to the famous C-­S-­R
triangle theory (Grime, 1977), which may be characterized by high
photosynthesis (low PC1 score), rapid growth (high PC1 score) and
stress tolerance (high PC2 score).
Yet, as our study was performed using a single genotype per
species and under a single set of culture conditions, it is limited in
confirming these ecological aspects. The natural growing environ-
ments of duckweeds would be different from our culture conditions
in terms of light intensity and the presence of stress factors. Hence,
one remaining issue is to understand the functional trait coordination
of duckweeds under environmental gradients (Stewart et al., 2020;
Yin et al., 2015; Ziegler et al., 2019). Competition and biogeogra-
phy of duckweeds (e.g. Armitage & Jones, 2019; Paolacci, Harrison,
et al., 2018; Paolacci, Jansen, et al., 2018; Zhao et al., 2017) in rela-
tion to their trait variations would also be promising future research
topics.

ISHIZAWA et al.
4.3 | Biotechnological implications
As duckweeds are useful for wastewater treatment and bio-
mass production, much effort has been dedicated to develop-
ing efficient duckweed cultivation systems and screening for
productive species and strains (Ishizawa et al., 2017; Ishizawa
et al., 2020; Sree, Sudakaran, et al., 2015; Yin et al., 2015; Ziegler
et al., 2014). The majority of studies have utilized RGR or simi-
lar metrics to evaluate duckweed growth (Lasfar et al., 2007),
equating high RGR to high productivity. However, we note
that RGR was negatively correlated with biomass productivity
per unit frond area (A area and NAR; Figure 2). Duckweeds with
high RGR may be superior in terms of rapid occupation of water
surface, but they are not necessarily superior in term of carbon
assimilation once they fully occupy the available water surface.
Hence, RGR cannot simply represent duckweed biomass produc-
tivity per unit cultivation area in industrial setting (where duck-
weeds cover entire water surface and form multiple layers), when
comparing species/strains differing in LMA. Paolacci, Harrison,
et al. (2018) reported that LMA of L. minor and L. minuta strains
changed by approximately twofold depending on light intensity,
indicating that LMA can easily be altered by culture conditions,
even within the same duckweed species. Therefore, we strongly
recommend measuring both RGR and LMA (to estimate NAR with
Equation 3) in future works for the accurate evaluation of duck-
weed productivity.
AC K N OW L E D G E M E N T S
The authors express their gratitude to Ian J. Wright and Peter
B. Reich for allowing them to use the GLOPNET dataset in the
analysis. We also thank Tokitaka Oyama, Shogo Ito, Tomoaki
Muranaka, Minako Isoda, Masaaki Morikawa, Kazuhiro Mori,
Tadashi Toyama and international duckweed community for pro-
viding and maintaining plant materials. We also thank Takashi
Machimura and Maiko Sakaguchi for their assistance with pho-
tosynthetic measurement. This study was partly supported by
grants from Advanced Low Carbon Technology Research and
Development Program (ALCA) JPMJAL1108 of the Japan Science
and Technology Agency and Japan Society for the Promotion of
Science KAKENHI JP18J10181 and JP20J00210. The authors de-
clare no competing interests.
AU T H O R S ' C O N T R I B U T I O N S
H.I. planned the research, performed the experiments, analysed the
data and wrote the manuscript; Y.O. and K.K. contributed to the data
analyses, interpreted the results and revised the manuscript; M.K.,
D.I. and M.I. interpreted the results and revised the manuscript. All
authors contributed critically to the drafts and gave final approval
for publication.
PEER REVIEW
The peer review history for this article is available at https://publo​ns.​
com/publo​n/10.1111/1365-­2745.13710.
13652745, 2021, 8, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.13710 by Nat Prov Indonesia, Wiley Online Library on [25/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Journal of Ecology | 2959
DATA AVA I L A B I L I T Y S TAT E M E N T
Raw data associated with this article are available from Dryad Digital
Repository https://doi.org/10.5061/dryad.b2rbn​zsfd (Ishizawa et al.,​
2021).
ORCID
Hidehiro Ishizawa https://orcid.org/0000-0003-0026-6039
Yusuke Onoda https://orcid.org/0000-0001-6245-2342
Kaoru Kitajima https://orcid.org/0000-0001-6822-8536
Masashi Kuroda https://orcid.org/0000-0002-8183-2997
Daisuke Inoue https://orcid.org/0000-0002-9216-8198
Michihiko Ike https://orcid.org/0000-0001-6237-8103
REFERENCES
Alaerts, G. J., Mahbubar, M. D. R., & Kelderman, P. (1996). Performance anal-
ysis of a full-­scale duckweed-­covered sewage lagoon. Water Research,
30(4), 843–­852. https://doi.org/10.1016/0043-­1354(95)00234​- ­0
American Public Health Association (APHA). (1995). Standard methods for
the examination of water and wastewater. APHA.
Appenroth, K. J., Sree, K. S., Bog, M., Ecker, J., Seeliger, C., Böhm, V.,
Lorkowski, S., Sommer, K., Vetter, W., Tolzin-­Banasch, K., Kirmse,
R., Leiterer, M., Dawczynski, C., Liebisch, G., & Jahreis, G. (2018).
Nutritional value of the duckweed species of the genus Wolffia
(Lemnaceae) as human food. Frontiers in Chemistry, 29, 483. https://
doi.org/10.3389/fchem.2018.00483
Appenroth, K. J., Sree, K. S., Böhm, V., Hammann, S., Vetter, W., Leiterer,
M., & Jahreis, G. (2017). Nutritional value of duckweeds (Lemnaceae)
as human food. Food Chemistry, 217(15), 266–­273. https://doi.
org/10.1016/j.foodc​hem.2016.08.116
Armitage, D. W., & Jones, S. E. (2019). Negative frequency-­dependent
growth underlies the stable coexistence of two cosmopolitan aquatic
plants. Ecology, 100, e02567. https://doi.org/10.1002/ecy.2657
Baldi, B. G., Maher, B. R., Slovin, J. P., & Cohen, J. D. (1991). Stable isotope
labeling, in vivo, of d-­and l-­tryptophan pools in Lemna gibba and the
low incorporation of label into indole-­3–­acetic acid. Plant Physiology,
95, 1203–­1208. https://doi.org/10.1104/pp.95.4.1203
Barbosa Neto, A. G., Morais, M. B., Dutra, E. D., & Calsa Junior, T. (2019).
Biological diversity of Lemna aequinoctialis Welw. isolates influences
biomass production and wastewater phytoremediation. Bioresource
Technology Reports, 6, 251–­259. https://doi.org/10.1016/j.biteb.2019.​
03.011
Barks, P. M., & Laird, R. A. (2015). Senescence in duckweed: Age-­related
declines in survival, reproduction and offspring quality. Functional
Ecology, 29(4), 540–­548. https://doi.org/10.1111/1365-­2435.12359
Barks, P. M., & Laird, R. A. (2016). A multigenerational effect of parential
age on offspring size but not fitness in common duckweed (Lemna
minor). Journal of Evolutionary Biology, 29(4), 748–­756. https://doi.
org/10.1111/jeb.12823
Blazey, E. B., & McClure, J. W. (1968). The distribution and taxonomic sig-
nificance of lignin in the Lemnaceae. American Journal of Botany, 55,
1240–­1245. https://doi.org/10.1002/j.1537-­2197.1968.tb074​91.x
Blomberg, S. P., Garland Junior, T., & Ives, A. R. (2003). Testing for
phylogenetic signal in comparative data: Behavioral traits are
more labile. Evolution, 57(4), 717–­745. https://doi.org/10.1111/
j.0014-­3820.2003.tb002​85.x
Bog, M., Appenroth, K. J., & Sree, K. S. (2019). Duckweed (Lemnaceae):
Its molecular taxonomy. Frontiers in Sustainable Food Systems, 3, 117.
https://doi.org/10.3389/fsufs.2019.00117
Borisjuk, N., Peterson, A. A., Lv, J., Qu, G., Luo, Q., Shi, L., Chen, G.,
Kishchenko, O., Zhou, Y., & Shi, J. (2018). Structural and biochemical
properties of duckweed surface cuticle. Frontiers in Chemistry, 6, 317.
https://doi.org/10.3389/fchem.2018.00317

2960 | Journal of Ecology
Cheng, J. J., & Stomp, A. M. (2009). Growing duckweed to recover nutri-
ents from wastewaters and for production of fuel ethanol and animal
feed. Clean –­Soil, Air, Water, 37(1), 17–­26. https://doi.org/10.1002/
clen.20080 ​0210
Cui, W., & Cheng, J. J. (2015). Growing duckweed for biofuel production:
A review. Plant Biology, 17, 16–­23. https://doi.org/10.1111/plb.12216
Dale, M. P., & Causton, D. R. (1992). Use of the chlorophyll a/b ratio as a
bioassay for the light environment of a plant. Functional Ecology, 6(2),
190–­196. https://doi.org/10.2307/2389754
Datko, A. H., Mudd, S. H., & Giovanelli, J. (1980). Lemna paucisostata
Hegelm. 6746: Life cycle and characterization of the colony types
in a population. Plant Physiology, 65(5), 913–­ 923. https://doi.
org/10.1104/pp.65.5.913
Díaz, S., Kattge, J., Cornelissen, J. H. C., Wright, I. J., Lavorel, S., Dray, S.,
Reu, B., Kleyer, M., Wirth, C., Colin Prentice, I., Garnier, E., Bönisch,
G., Westoby, M., Poorter, H., Reich, P. B., Moles, A. T., Dickie, J.,
Gillison, A. N., Zanne, A. E., … Gorné, L. D. (2016). The global spec-
trum of plant form and function. Nature, 529, 167–­171. https://doi.
org/10.1038/natur​e16489
Driever, S. M., van Nes, E. H., & Roijackers, R. M. M. (2005). Growth
limitation of Lemna minor due to high plant density. Aquatic Botany,
81(3), 245–­251. https://doi.org/10.1016/j.aquab​ot.2004.12.002
Garnier, E., & Navas, M. L. (2012). A trait-­based approach to comparative
functional plant ecology: Concepts, methods and applications for
agroecology. A review. Agronomy and Sustainable Development, 32,
365–­399. https://doi.org/10.1007/s1359​3-­011-­0 036-­y
Gerber, D. T., & Les, D. H. (1994). Comparison of leaf morphology among
submersed species of Myriophyllum (Haloragaceae) from different
habitats and geographical distributions. American Journal of Botany,
81(8), 973–­979. https://doi.org/10.2307/2445290
Grime, J. P. (1977). Evidence for the existence of three primary strategies
in plants and its relevance to ecological and evolutionary theory. The
American Naturalist, 111, 1169–­1194. Retrieved from https://www.
journ​als.uchic​ago.edu/doi/10.1086/283244
Grimme, L. H., & Boardman, N. K. (1972). Photochemical activities of
a particle fraction P1 obtained from the green alga Chlorella fusca.
Biochemical and Biophysical Research Communications, 49(6), 1617–­
1623. https://doi.org/10.1016/0006-­291X(72)90527​-­X
Hart, S. P., Turcotte, M. M., & Levine, J. M. (2019). Effects of rapid evo-
lution on species coexistence. Proceedings of the National Academy of
Sciences of the United States of America, 116(6), 2112–­2117. https://
doi.org/10.6084/m9.figsh​are.75990​95.v1
Hayes, F. J., Buchanan, S. W., Coleman, B., Gordon, A. M., Reich, P. B.,
Thevathasan, N. V., Wright, I. J., & Martin, A. R. (2019). Intraspecific
variation in soy across the leaf economics spectrum. Annals of Botany,
123, 107–­120. https://doi.org/10.1093/aob/mcy147
Hillman, W. S. (1961). The Lemnaceae, or duckweeds: A review of the
descriptive and experimental literature. The Botanical Reviews, 27(2),
221–­287. https://doi.org/10.1007/BF028​60083
Hunt, R. (1982). Plant growth curves: A functional approach to plant growth
analysis. Edward Arnold.
Ishizawa, H., Kuroda, M., Morikawa, M., & Ike, M. (2017). Evaluation
of environmental bacterial communities as a factor affecting the
growth of duckweed Lemna minor. Biotechnology for Biofuels, 10, 62.
https://doi.org/10.1186/s1306​8-­017-­0746-­8
Ishizawa, H., Ogata, Y., Hachiya, Y., Tokura, K., Kuroda, M., Inoue, D.,
Toyama, T., Tanaka, Y., Mori, K., Morikawa, M., & Ike, M. (2020).
Enhanced biomass production and nutrient removal capacity of
duckweed via two-­ step cultivation process with a plant growth-­
promoting bacterium, Acinetobacter calcoaceticus P23. Chemosphere,
238, 124682. https://doi.org/10.1016/j.chemo​sphere.2019.124682
Ishizawa, H., Onoda, Y., Kitajima, K., Kuroda, M., Inoue, D., & Ike, M.
(2021). Data from: Coordination of leaf economics traits within the
family of the world’s fastest growing plants (Lemnaceae). Dryad
Digital Repository, https://doi.org/10.5061/dryad.b2rbn​zsfd
13652745, 2021, 8, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.13710 by Nat Prov Indonesia, Wiley Online Library on [25/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ISHIZAWA et al.
Ishizawa, H., Tada, M., Kuroda, M., Inoue, D., & Ike, M. (2019).
Performance of plant growth-­ promoting bacterium of duckweed
under different kinds of abiotic stress factors. Biocatalysis and
Agricultural Biotechnology, 19, 101146. https://doi.org/10.1016/j.
bcab.2019.101146
Katabuchi, M., Kitajima, K., Wright, S. J., Van Bael, S. A., Osnas, J. L. D.,
& Lichstein, J. W. (2017). Decomposing leaf mass into photosyn-
thetic and structural components explains divergent patterns of trait
variation within and among plant species. bioRxiv. https://doi.org/​
10.1101/116855
Kikuzawa, K., & Ackerly, D. (1999). Significance of leaf longevity in
plants. Plant Species Biology, 14, 39–­45. https://doi.org/10.1046/​
j.1442-­1984.1999.00005.x
Kitajima, K., & Hogan, K. P. (2003). Increases of chlorophyll a/b ratios
during acclimation of tropical woody seedlings to nitrogen limitation
and high light. Plant, Cell and Environment, 26(6), 857–­865. https://
doi.org/10.1046/j.1365-­3 040.2003.01017.x
Kitajima, K., Llorens, A. M., Stefanescu, C., Timchenko, M. V., Lucas, P.
W., & Wright, S. J. (2012). How cellulose-­based leaf toughness and
lamina density contribute to long leaf lifespans of shade-­tolerant
species. New Phytologist, 195(3), 640–­652. https://doi.org/10.1111/​
j.1469-­8137.2012.04203.x
Kunstler, G., Falster, D., Coomes, D. A., Hui, F., Kooyman, R. M., Laughlin,
D. C., Poorter, L., Vanderwel, M., Vielledent, G., Wright, S. J., Aiba,
M., Baraloto, C., Caspersen, J., Cornelissen, J. H. C., Gourlet-­Fleury,
S., Hanewinkel, M., Herault, B., Kattge, J., Kurokawa, H., … Westoby,
M. (2016). Plant functional traits have globally consistent effects on
competition. Nature, 529, 204–­207. https://doi.org/10.1038/natur​e​
16476
Kutschera, U., & Niklas, K. J. (2015). Darwin-­Wallace demons: Survival
of the fastest in populations of duckweeds and the evolutionary his-
tory of an enigmatic group of angiosperms. Plant Biology, 17, 24–­32.
https://doi.org/10.1111/plb.12171
Laird, R. A., & Barks, P. M. (2018). Skimming the surface: Duckweed as a
model system in ecology and evolution. American Journal of Botany,
105(12), 1–­10. https://doi.org/10.1002/ajb2.1194
Landolt, E. (1986). Biosystematic investigations in the family of duckweed
(Lemnaceae) (Vol. 2). Geobotanischen Inst ETH.
Lasfar, S., Monette, F., Millette, L., & Azzouz, A. (2007). Intrinsic growth
rate: A new approach to evaluate the effects of temperature, pho-
toperiod and phosphorus-­ nitrogen concentrations on duckweed
growth under controlled eutrophication. Water Research, 41(11),
2333–­2340. https://doi.org/10.1016/j.watres.2007.01.059
Martin, A. R., Rapidel, B., Roupsard, O., Van den Meersche, K., Melo
Virginio Filho, E., Barrios, M., & Isaac, M. E. (2017). Intraspecific
trait variation across multiple scales: The leaf economics spec-
trum in coffee. Functional Ecology, 31, 604–­612. https://doi.
org/10.1111/1365-­2435.12790
Mason, C. M., & Donovan, L. A. (2015). Evolution of the leaf economics
spectrum in harbs: Evidence from environmental divergences in leaf
physiology across Helianthus (Asteraceae). Evolution, 69(10), 2705–­
2720. https://doi.org/10.1111/evo.12768
Matsunaga, T., & Shiozaki, H. (1989). Sulfuric acid-­hydrogen peroxide
digestion for determination of total nitrogen in plant material con-
taining nitrate nitrogen. Japanese Journal of Soil Science and Plant
Nutrition, 60(5), 458–­460. https://doi.org/10.20710/​dojo.60.5_458
Muranaka, T., Okada, M., Yomo, J., Kubota, S., & Oyama, T. (2015).
Characterization of circadian rhythms of various duckweeds. Plant
Biology, 17(s1), 66–­74. https://doi.org/10.1111/plb.12202
Muranaka, T., & Oyama, T. (2016). Heterogeneity of cellular circa-
dian clocks in intact plants and its correlation under light-­ dark
cycles. Science Advances, 2, e1600500https://doi.org/10.1126/
sciadv.1600500
Niklas, K. J., & Enquist, B. J. (2001). Invariant scaling relationships for in-
terspecific plant biomass production rates and body size. Proceedings

ISHIZAWA et al.
of the National Academy of Sciences of the United States of America,
98(5), 2922–­2927. https://doi.org/10.1073/pnas.04159​0298
Onoda, Y., Westoby, M., Adler, P. B., Choong, A. M. F., Clissold,
F. J., Cornelissen, J. H. C., Díaz, S., Dominy, N. J., Elgart, A.,
Enrico, L., Fine, P. V. A., Howard, J. J., Jalili, A., Kitajima, K.,
Kurokawa, H., McArthur, C., Lucas, P. W., Markesteijn, L., Pérez-­
Harguindeguy, N., … Yamashita, N. (2011). Global patterns of leaf
mechanical properties. Ecology Letters, 14, 301–­312. https://doi.
org/10.1111/j.1461-­0248.2010.01582.x
Onoda, Y., Wright, I. J., Evans, J. R., Hikosaka, K., Kitajima, K.,
Niinemets, Ü., Poorter, H., Tosens, T., & Westoby, M. (2017).
Physiological and structural tradeoffs underlying the leaf eco-
nomics spectrum. New Phytologist, 214, 1447–­1463. https://doi.
org/10.1111/nph.14496
Oron, G., de Vegt, A., & Porath, D. (1988). Nitrogen removal and con-
version by duckweed grown on waste-­water. Water Research, 22(2),
179–­184. https://doi.org/10.1016/0043-­1354(88)90076​- ­0
Osnas, J. L. D., Katabuchi, M., Kitajima, K., Wright, S. J., Reich, P. B.,
Van Bael, S. A., Kraft, N. J. B., Samaniego, M. J., Pacala, S. W.,
& Lichstein, J. W. (2018). Divergent drivers of leaf trait variation
within species, among species, and among functional groups.
Proceedings of the National Academy of Sciences of the United
States of America, 115(21), 5480–­5 485. https://doi.org/10.1073/
pnas.18039​89115
Pan, Y., Cieraad, E., Armstrong, J., Armstrong, W., Clarkson, B. R.,
Colmer, T. D., Pedersen, O., Visser, E. J. W., Voesenek, L. A. C., &
van Bodegom, P. M. (2020). Global patterns of the leaf economics
spectrum in wetlands. Nature Communications, 11, 4519. https://doi.
org/10.1038/s4146​7-­020-­18354​-­3
Pan, Y., Cieraad, E., & van Bodegom, P. M. (2019). Are ecophysi-
ological adaptive traits decoupled from leaf economics traits
in wetlands? Functional Ecology, 33, 1202–­ 1210. https://doi.
org/10.1111/1365-­2435.13329
Paolacci, S., Harrison, S., & Jansen, M. A. K. (2018). The invasive duck-
weed Lemna minuta Knuth displays a different light utilization strat-
egy than native Lemna minor Linnaeus. Aquatic Botany, 146, 8–­14.
https://doi.org/10.1016/j.aquab​ot.2018.01.002
Paolacci, S., Jansen, M. A. K., & Harisson, S. (2018). Competition be-
tween Lemna minuta, Lemna minor, and Azolla filiculoides. Growing
fast or being steadfast? Frontiers in Chemistry, 6, 207. https://doi.
org/10.3389/fchem.2018.00207
Poorter, H., Lambers, H., & Evans, J. R. (2014). Trait correlation net-
works: A whole-­plant perspective on the recently criticized leaf
economic spectrum. New Phytologist, 201(2), 378–­382. https://doi.
org/10.1111/nph.12547
Poorter, H., Niinemets, Ü., Poorter, L., Wright, I. J., & Villar, R. (2009).
Causes and consequences of variation in leaf mass per area (LMA):
A meta-­analysis. New Phytologist, 182(3), 565–­ 588. https://doi.
org/10.1111/j.1469-­8137.2009.02830.x
Reich, P. B., Ellsworth, D. S., Walters, M. B., Vose, J., Gresham, C., Volin,
J., & Bowman, W. (1999). Generality of leaf traits relationships:
A test across six biomes. Ecology, 80(6), 1955–­ 1969. https://doi.
org/10.2307/176671
Reich, P. B., Walters, M. B., & Ellsworth, D. S. (1992). Leaf lifespan in
relation to leaf, plant and stand characteristics among diverse
ecosystems. Ecological Monographs, 62(3), 365–­ 392. https://doi.
org/10.2307/2937116
Reich, P. B., Walters, M. B., & Ellsworth, D. S. (1997). From tropics to
tundra: Global convergence in plant functioning. Proceedings of the
National Academy of Sciences of the United States of America, 94(25),
13730–­13734. https://doi.org/10.1073/pnas.94.25.13730
Sartori, K., Vasseur, F., Violle, C., Baron, E., Gerard, M., Rowe, N., Ayala-­
Garay, O., Christphe, A., Garcia de Jalón, L., Masclef, D., Harscouet, E.,
del Ray Granado, M., Chassagneux, A., Kazakou, E., & Vile, D. (2019).
Leaf economics and slow-­ fast adaptation across the geographic
13652745, 2021, 8, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.13710 by Nat Prov Indonesia, Wiley Online Library on [25/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Journal of Ecology | 2961
range of Arabidopsis thaliana. Scientific Reports, 9, 10758. https://doi.
org/10.1038/s4159​8-­019-­46878​-­2
Shipley, B. (2006). Net assimilation rate, specific leaf area and leaf mass
ratio: Which is most closely correlated with relative growth rate?
A meta-­analysis. Functional Ecology, 20(4), 565–­574. https://doi.
org/10.1111/j.1365-­2435.2006.01135.x
Soda, S., Ohchi, T., Piradee, J., Takai, Y., & Ike, M. (2015). Duckweed bio-
mass as a renewable biorefinery feedstock: Ethanol and succinate
production from Wolffia globosa. Biomass and Bioenergy, 81, 364–­
368. https://doi.org/10.1016/j.biomb​ioe.2015.07.020
Song, L., Vijver, M. G., & Peijnenburg, W. J. G. M. (2015). Comparative
toxicity of copper nanoparticles across three Lemnaceae species.
Science of the Total Environment, 518–­519, 217–­224. https://doi.
org/10.1016/j.scito​tenv.2015.02.079
Sree, K. S., Adelmann, K., Garcia, C., Lam, E., & Appenroth, K. J. (2015).
Natural variance in salt tolerance and induction of starch accumula-
tion in duckweeds. Planta, 241, 1395–­1404. https://doi.org/10.1007/
s0042​5-­015-­2264-­x
Sree, K. S., Sudakaran, S., & Appenroth, K. J. (2015). How fast can an-
giosperms grow? Species and clonal diversity of growth rates in the
genus Wolffia (Lemnaceae). Acta Physiologiae Plantarum, 37, 204.
https://doi.org/10.1007/s1173​8-­015-­1951-­3
Stewart, J. J., Adams, W. W., Escobar, C. M., López-­Pozo, M., & Demmig-­
Adams, B. (2020). Growth and essential carotenoid micronutrients in
Lemna gibba as a function of growth light intensity. Frontiers in Plant
Science, 11, 480. https://doi.org/10.3389/fpls.2020.00480
Terashima, I., & Hikosaka, K. (1995). Comparative ecophysiology of leaf
and canopy photosynthesis. Plant, Cell and Environment, 18(10),
1111–­1128. https://doi.org/10.1111/j.1365-­3 040.1995.tb006​23.x
Tippery, N. P., & Les, D. H. (2020). Tiny plants with enormous potential:
Phylogeny and evolution of duckweeds. In X. H. Cao, P. Fourounjian,
& W. Wang (Eds.), The duckweed genomes (pp. 19–­38). Springer Nature
Switzerland. https://doi.org/10.1111/j.1365-­3 040.1995.tb006​23.x
Toyama, T., Hanaoka, T., Tanaka, Y., Morikawa, M., & Mori, K. (2018).
Comprehensive evaluation of nitrogen removal rate and bio-
mass, ethanol, and methane production yields by combination of
four major duckweeds and three types of wastewater effluent.
Bioresource Technology, 250, 464–­473. https://doi.org/10.1016/j.
biort​ech.2017.11.054
Tsuchiya, T. (1991). Leaf life span of floating-­leaved plants. Vegetatio,
97(2), 149–­160. https://doi.org/10.1007/BF000​35388
Wang, W., Wu, Y., Yan, Y., Ermakova, M., Kerstetter, R., & Messing, J. (2010).
DNA barcoding of the Lemnaceae, a family of aquatic monocots. BMC
Plant Biology, 10, 205. https://doi.org/10.1186/1471-­2229-­10-­205
Wangermann, E., & Ashby, E. (1951). Studies in the morphogenesis of
leaves. VII. Part II. Correlative effects of fronds in Lemna minor. New
Phytologist, 50, 200–­209. https://doi.org/10.1111/j.1469-­8137.1951.
tb051​85.x
Wedge, R. M., & Burris, J. E. (1982). Effects of light and temperature on
duckweed photosynthesis. Aquatic Botany, 13, 133–­140. https://doi.
org/10.1016/0304-­3770(82)90047​-­X
Westoby, M., Falster, D. S., Moles, A. T., Vesk, P. A., & Wright, I. J. (2002).
Plant ecological strategies: Some leading dimensions of variation be-
tween species. Annual Review of Ecology and Systematics, 33, 125–­
159. https://doi.org/10.1146/annur​ev.ecols​ys.33.010802.150452
Wright, I. J., Reich, P. B., Westoby, M., Ackerly, D. D., Baruch, Z., Bongers,
F., Cavender-­Bares, J., Chapin, T., Cornelissen, J. H. C., Diemer, M.,
Flexas, J., Garnier, E., Groom, P. K., Gulias, J., Hikosaka, K., Lamont,
B. B., Lee, T., Lee, W., Lusk, C., … Villar, R. (2004). The worldwide leaf
economics spectrum. Nature, 428, 821–­827. https://doi.org/10.1038/
natur​e 02403
Xiong, D., & Flexas, J. (2018). Leaf economics spectrum in rice: Leaf an-
atomical, biochemical, and physiological trait trade-­offs. Journal of
Experimental Botany, 69(22), 5599–­ 5609. https://doi.org/10.1093/
jxb/ery322

2962 | Journal of Ecology
Xu, S., Stapley, J., Gablenz, S., Boyer, J., Appenroth, K. J., Sree, K. S.,
Gershenzon, J., Widmer, A., & Huber, M. (2018). Low genetic varia-
tion is associated with low mutation rate in giant duckweed. Nature
Communications, 10, 1243. https://doi.org/10.1038/s4146​7-­019-­
09235​-­5
Yang, J., Li, G., Bishopp, A., Heenatigala, P. P. M., Hu, S., Chen, Y., Wu,
Z., Kumar, S., Duan, P., Yao, L., & How, H. (2018). A comparison of
growth on mercuric chloride for three Lemnaceae species reveals
difference in growth dynamics that effect their suitability for use
in either monitoring or remediating ecosystems contaminated with
mercury. Frontiers in Chemistry, 6, 112. https://doi.org/10.3389/
fchem.2018.00112
Yin, Y., Yu, C., Yu, L., Zhao, J., Sun, C., Ma, Y., & Zhou, G. (2015). The in-
fluence of light intensity and photoperiod on duckweed biomass and
starch accumulation for bioethanol production. Bioresource Technology,
187, 84–­90. https://doi.org/10.1016/j.biort​ech.2015.03.097
Yu, C., Sun, C., Yu, L., Zhu, M., Xu, H., Zhao, J., Ma, Y., & Zhou, G. (2014).
Comparative analysis of duckweed cultivation with sewage water and
SH media for production of fuel ethanol. PLoS ONE, 9, e115023(12),
https://doi.org/10.1371/journ​al.pone.0115023
Zhao, Z., Shi, H., Kang, X., Liu, C., Chen, L., Liang, X., & Jin, L. (2017).
Inter-­and intra-­specific competition of duckweed under multiple
heavy metal contaminated water. Aquatic Toxicology, 192, 216–­223.
https://doi.org/10.1016/j.aquat​ox.2017.09.023
Ziegler, P., Adelmann, K., Zimmer, S., Schimidt, C., & Appenroth, K. J.
(2014). Relative in vitro growth rates of duckweeds (Lemnaceae)
13652745, 2021, 8, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.13710 by Nat Prov Indonesia, Wiley Online Library on [25/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ISHIZAWA et al.
–­The most rapidly growing higher plants. Plant Biology, 17(s1), 33–­
41. https://doi.org/10.1111/plb.12184
Ziegler, P., Sree, K. S., & Appenroth, K. J. (2016). Duckweeds for water
remediation and toxicity testing. Toxicology and Environmental
Chemistry, 98(10), 1127–­1154. https://doi.org/10.1080/02772​
248.2015.1094701
Ziegler, P., Sree, K. S., & Appenroth, K. J. (2019). Duckweed biomarkers
for identifying toxic water contaminants? Environmental Science and
Pollution Research, 26, 14797–­14822. https://doi.org/10.1007/s1135​
6-­018-­3 427-­7
S U P P O R T I N G I N FO R M AT I O N
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Supporting Information section.
How to cite this article: Ishizawa, H., Onoda, Y., Kitajima, K.,
Kuroda, M., Inoue, D., & Ike, M. (2021). Coordination of leaf
economics traits within the family of the world's fastest
growing plants (Lemnaceae). Journal of Ecology, 109,
2950–­2962. https://doi.org/10.1111/1365-­2745.13710