Phycological Research 2019
12404
...........................................................................................................................................................................................
Phenotypic plasticity and morphological adaptation
of Halimeda opuntia (Bryopsidales, Chlorophyta) to light
intensity
Supattra Pongparadon,1 Sontaya Nooek2 and Anchana Prathep
1
Excellence Center for Biodiversity of Peninsula Thailand, Faculty of Science, Prince of Songkla University, Hat Yai,
Songkhla, Thailand and 2Seaweed and Seagrass Research Unit, Department of Biology, Faculty of Science, Prince of
Songkla University, Songkhla, Thailand
........................................................................................
ABSTRACT
Plasticity gives plants the ability to adapt their morphology
and growth to various environments. Environmental factors
can lead to changes in the growth forms of algae. Halimeda
opuntia was used as a model organism to determine the
effects of highly irradiated and shade conditions in the field
and its responses to low light conditions in the laboratory,
which are known to be some of the main factors driving its
morphological plasticity. To investigate the morphological and
anatomical characteristics of the species, a total of 61 charac-
teristics and 54 samples from Thailand, Japan and Indonesia
were examined. In addition, we also tested the response under
different light conditions in the laboratory. A phylogenetic tree
was constructed using the tufA marker to confirm the species
identification. We found that the thallus forms, branching pat-
terns and segment shapes and sizes responded to the differ-
ent light conditions. A brittle thallus network was found under
shade conditions with low light intensity, while a compact
thallus form was found under highly irradiated conditions. The
H. opuntia segment shape was reniform under highly irradi-
ated conditions, but its segments were adapted to be deeply
trilobed in the lower thallus and tripartite shape in the upper
thallus under shade conditions. The segment size also
adapted; the segments in shaded areas were longer than those
in sunny areas, which might have helped them increase their
thallus height to ensure exposure to the high light habitat.
This morphological variation and plasticity expanded our
understanding of the range of H. opuntia plasticity present in
the field and could help explain why this species is common
and successfully colonizes various habitats worldwide.
Key words: adaptation, light irradiance, morphological varia-
tions, phenotypic plasticity, species boundary.
........................................................................................
INTRODUCTION
Phenotypic plasticity is the ability of an individual organism to
respond to changes in environmental conditions by changing
its morphology or physiology (Schmalhausen 1949; Bradshaw
1965; Schlichting 1986). Phenotypic adaptation is a neces-
sity for sessile organisms such as plant, which allows them to
continually adapt to the conditions of their local environ-
ments, for example, light intensity, temperature, depth and
nutrients. These factors can influence the morphology, shoot
© 2019 Japanese Society of Phycology
doi: 10.1111/pre.
1,2
*
elongation, leaf shape, growth rate, flowering times and pho-
tosynthesis rate of plants.
The genus Halimeda is an interesting model organism as it
is well studied taxonomically. However, most sections within
the genus Halimeda (e.g., Opuntia J. Agardh ex De Toni,
Halimeda J. Agardh ex De Toni and Rhipsalis J. Agardh ex De
Toni) have high morphological plasticity and show morpholog-
ical variations that appear to correlate with habitats
(Verbruggen & Kooistra 2004; Verbruggen 2005; Verbruggen
et al. 2005a; Pongparadon et al. 2015). Halimeda
section Opuntia is also known to be morphologically plastic
(Verbruggen et al. 2006; Dijoux et al. 2012; Pongparadon
et al. 2015), and this section includes Halimeda opuntia,
Halimeda distorta and Halimeda hederacea. Pongparadon
et al. (2015) reported that H. opuntia populations in Thai
waters were genetically identical but showed morphological
variations among populations as their segment shapes differed
between shallow and deep water. In addition, Littler and Lit-
tler (2000) and Kooistra and Verbruggen (2005) reported that
H. opuntia had compact reniform segments in wave-exposed
areas and tripartite segments in sheltered lagoons.
This morphological plasticity could be a key prerequisite
for the potential adaptation and survival of populations in het-
erogeneous habitats. H. opuntia, which is commonly found in
both the Atlantic and Indo-Pacific Oceans and is one of the
most common and widespread species in both regions
(Guiry & Guiry 2013), was the focus of this study. H. opuntia
is distributed in various habitats, e.g., sandy beaches, reef
flats and subtidal reef areas, while molecular data indicate
that morphological plasticity occurs within species
(Pongparadon et al. 2015). We hypothesize that this wide dis-
tribution and high abundance of H. opuntia populations may
be related to their potential for morphological plasticity among
populations. A series of morphometric measurements and the
chloroplast-encoded tufA gene were used to explore the spe-
cies plasticity of H. opuntia. We examined (i) the environmen-
tal factors that could influence H. opuntia morphological and
anatomical variations in the field, (ii) the H. opuntia morpho-
logical and anatomical variations between highly irradiated
.........................................................................................
*To whom correspondence should be addressed.
Email: anchana.p@psu.ac.th
Communicating Editor: Tatyana Klochkova
Received 22 November 2018; accepted 29 July 2019.
2 S. Pongparadon et al.
and shade conditions, and (iii) the morphological and anatom-
ical plasticity of H. opuntia that occurred in response to differ-
ent light irradiation levels under laboratory conditions.
MATERIALS AND METHODS
Sample collections and environmental
factors
Halimeda opuntia samples were collected throughout most of
its distributional range in Thai waters, and some samples were
collected from Makassar, Indonesia, and Ishigaki, Japan
(Appendix S1 in the Supporting Information). Three speci-
mens from each location for a total of 54 samples were pre-
served on herbarium sheets for morphological and anatomical
studies, and the apical segments were preserved in silica gel
for molecular study. Furthermore, to test the morphological
variations in H. opuntia under different light intensities, col-
lections were conducted under two completely different natu-
ral light conditions: (i) highly irradiated and (ii) shade. Light
intensity was measured under the water using an Onset-Hobo
data logger (Onset Computer Corporation, Ontoocook, NH,
USA) during a sunny day when low tide occurred between
10 am and 2 pm. Moreover, the environmental variables on
habitat types, zonation and depth were also recorded (Appen-
dix S2 in the Supporting Information). Visual estimates were
used to define the habitat variables. (i) For the highly irradi-
ated conditions, the light intensity was greater than 900 μmol
photons m−2 s−1 (the average was 1024.83  193.755 μmol
photons m−2 s−1), which occurred at a sandy beach (2–3 m
depth), reef flat in the intertidal zone (2–3 m depth) or shal-
low subtidal reef flat zone with clear water (3–5 m depth).
Samples were attached to coral rubble, rock and dead coral in
the sand. (ii) For the shade condition, the light intensity was
less than 100 μmol photons m−2 s−1 (the average was
54.54  3.36 μmol photons m−2 s−1) and mostly occurred at
the reef slope in the subtidal zone (5–10 m depth) or under
rock or coral in both the intertidal (2–3 m depth) and subtidal
(5–10 m depth) zones. All samples were preserved on herbar-
ium sheets for the morpho-anatomical study, and a few apical
and non-calcified segments were cut and preserved in silica
gel for molecular study.
Molecular and phylogenetic inference
Molecular analysis was used for identification and confirma-
tion of H. opuntia species. The DNA was extracted from all
samples preserved in silica gel following the methods in
Thomson & Henry (1995). The PCR procedure was carried out
following the methods in Pongparadon et al. (2015). The data
set, including published tufA sequences of H. opuntia and
other Halimeda species from GenBank (Appendix S3 in the
Supporting Information), was aligned and manually edited in
BioEdit (Hall 1999). Pseudocodium okinawaense and
Tydemania expeditionis sequences were designated as out-
groups. Phylogenetic analyzes were implemented using maxi-
mum likelihood (ML) and Bayesian inference (BI). Before the
ML and BI analyzes, the best-fit model of nucleotide substitu-
tion was selected using jMODELTEST 2.1.7 (Posada 2008;
Darriba et al. 2012). The ML tree was constructed using
RAxML (Stamatakis 2006) with the G + I model. Support for
branches was obtained from 1000 bootstrap replications. BI
analysis was performed using MrBayes v.3.2.1 (Ronquist &
Huelsenbeck 2003), with a random starting tree run for
3 000 000 generations, sampling tree every 1000 genera-
tions and a burn-in of 300 trees.
Morphological and anatomical measurements
For all 54 sequenced samples, the morphological and ana-
tomical structures were examined following Hillis-Colinvaux
(1980), Verbruggen et al. (2005a, 2005b, 2005c) and
Pongparadon et al. (2015) with some modifications. To decal-
cify the algal tissue for anatomical observations, segments
were incubated in 5–10% w/w concentrated HCl at room tem-
perature. Morphological and anatomical characters were mea-
sured from ten randomly selected coupled segments from
each upper and lower part with the exception of deviant seg-
ments: a basal and apical segment (Verbruggen et al. 2005a).
In each segment, ten replicates of all characteristics were ran-
domly measured; 26 morphological and 35 anatomical char-
acteristics were examined, as shown in Appendix S4 in the
Supporting Information.
Effects of light irradiances on H. opuntia in
the laboratory
The response of H. opuntia was evaluated after the samples
were transferred from highly irradiated habitats to a low-light
aquarium tank. A total of 30 samples of H. opuntia specimens
were collected from Tangkhen Bay, Phuket Province, the
Andaman Sea, Thailand. Thalli were compact forms with reni-
form segment shapes. The samples were associated with
coral, dead coral or rock in the shallow subtidal zone under
highly irradiated conditions (light intensity was greater than
900 μmol photons m−2 s−1), and the water temperature was
27–28 C. This area is a sheltered area with low wave exposure
and an average depth of 2 m at the highest tide. The samples
were transferred to the aquarium facility at the Phuket Marine
Biological Center (PMBC), Phuket Province, Thailand. The
samples were washed with filtered seawater and cleaned of
debris, associated algae and any epiphytes. Before experimen-
tation, ten segments from each upper and lower thallus part
were examined for all morpho-anatomical characteristics, as
described in Appendix S4 in the Supporting Information.
Some parts were used to examine the percentage of CaCO3
content by drying in an oven at 65 C to a constant dry weight,
and the calcified thallus dry weight (mg dry weight thallus−1)
was recorded. After weighing, the calcified thallus was
decalcified with 5–10% w/w concentrated HCl at room tem-
perature until bubbling ceased (Vroom et al. 2003), and the
samples were rinsed with fresh water to remove the traces of
acid. The decalcified thalli were placed in a drying oven at
65 C to a constant dry weight and then weighed to measure
their tissue dry weight (mg dry weight thallus−1). The percent-
age of CaCO3 content was calculated from the calcified thal-
lus dry weight and the tissue dry weight. The remaining thalli
with individual sizes of 10 cm width × 10 cm height were
placed in a shaded aquarium tank at the aquarium facility
© 2019 Japanese Society of Phycology
Plasticity of Halimeda opuntia
with natural low light conditions (the average was
75.32  26.02 μmol photons m−2 s−1). Each sample was
separated into small buckets, tagged with the thalli number
and placed in an aquarium tank with the dimensions of 1.2 m
width × 2.0 m length × 0.5 m height. The average seawater
temperature was 27 C with filtered natural seawater that was
circulated for 24 h a day, which provided unlimited nutrients.
After 3 months, all samples were collected, and ten segments
from each upper and lower part were examined for morpho-
anatomical characters and percentage of CaCO3 content.
Furthermore, the response of H. opuntia was tested under
different light intensities, natural light (the average light
intensity was 307.78  14.18 μmol photons m−2 s−1), low-
light (the average light intensity was 39.37  2.34 μmol pho-
tons m−2 s−1) and high light (the average light intensity was
755.87  12.25 μmol photons m−2 s−1) conditions. Thirty
samples were collected from the intertidal zone of Tangkhen
Bay, Phuket Province, the Andaman Sea, Thailand, where the
samples were exposed to highly irradiated conditions at the
lowest tide (light intensity was greater than 900 μmol photons
m−2 s−1) and the average depth was 2–3 m at the highest
tide. Generally, all thalli were compact and had a reniform
segment shape; they were associated with coral, dead coral or
rock. The thalli with individual thallus sizes of approximately
10 cm width × 10 cm height were kept in a cool box and
transferred to the aquarium facility at Prince of Songkla Uni-
versity, Songkhla Province. The samples were washed with fil-
tered seawater and cleaned of debris, associated algae and
any epiphytes. Samples were separated into three sets and
placed in aquarium tanks under three different irradiation
treatments. Each sample was separated into small buckets
and placed in an aquarium tank with dimensions of 0.25 m
width × 0.5 m length × 0.3 m height, with filtered natural
seawater that was circulated for 24 h a day. The average sea-
water temperature was 27 C. After 2 months, all samples
were collected, and the morpho-anatomical characteristics of
ten segments from each upper and lower part were examined,
as previously described.
Data analyzes
Fifty-four specimens from each environmental condition
(highly irradiated vs shade habitats) and thirty specimens
from each experiment were used to evaluate the differences in
all morphological and anatomical characteristics of
H. opuntia between highly irradiated and shade habitats,
before and after the light shading experiment and among the
three different light irradiated experiments. One-way analysis
of variance (ANOVA) was used in R i386 3.3.4 (R Core Team,
2017). Box plot maps were used to present the data if there
were significant differences.
The environmental conditions (Appendix S2 in the
Supporting Information) and biological factors (Appendix S4
in the Supporting Information) were evaluated for significant
differences via canonical correspondence analysis (CCA), and
both a biological matrix (two conditions; highly irradiated and
shade habitats /54 samples) and an environmental matrix
(10 environmental variables/54 samples) were created. The
Monte Carlo permutation test was used and performed in R
i386 3.3.4 (R Core Team 2017).
© 2019 Japanese Society of Phycology
3
RESULTS
Phylogenetic, comparative morphology and
anatomy
All sequenced samples were genetically identified as H. opuntia,
which showed that all populations belonged to the same species
(Appendix S5 in the Supporting Information). Morphological vari-
ations were observed in the thallus form and segment shape of
H. opuntia, which consisted of two groups. The first group was a
compact and sprawling thallus, often with both vertical and hori-
zontal growth systems; the sprawls occurred over the substrate
with more than one rhizoidal holdfast, and the holdfast region
was not restricted; rhizoids occurred at intervals where the seg-
ment contacted the substratum. The thalli were bright green to
dark green when fresh and brownish-green to whitish when dried;
calcification was moderate to heavy, and branches were often
numerous (Fig. 1a,b). Segments were commonly reniform and
frequently ribbed (Fig. 1c,d). Both the upper and lower segments
were often markedly ribbed, and the ribs resulted from three bun-
dles of medullar siphons that connected the other three daughter
segments. The primary utricle pattern in the surface view was
rounded polygonal (Fig. 1e). The cortex generally contained three
to five layers of utricles (Fig. 1f). Nodal medullary filaments were
incompletely fused into two or three siphons (Fig. 1g,h). This
form was found on sandy beaches and reef flats in the intertidal
zone. The thalli were attached to rock, dead coral or coral rubble.
The second group was found on coral reefs in the subtidal
zone, growing under coral or rock. The thallus forms loosened
or appeared as a brittle network and had both vertical and hor-
izontal growth systems; the sprawls occurred over the sub-
strate with more than one rhizoidal holdfast, and the holdfast
region was not restricted. The rhizoids occurred at intervals
where the segment contacted the substratum (Fig. 2a,b). The
thalli were bright green to dark green when fresh and
brownish-green to whitish when dried. Calcification was mod-
erate to heavy. Branches were often numerous and mostly tri-
chotomous branching. The segment shape showed different
morphology from the base to the apex (Fig. 2c–h). Lower seg-
ments were often trilobed and tripartite, sometimes cylindrical
(Fig. 2c–e). The upper segments were tripartite and reniform
(Fig. 2f–h) and broad ovate at the apical segments. The outer-
most utricles generally remained attached after decalcification
and appeared as an irregular pattern of rounded polygons from
the surface view (Fig. 2i). The cortex generally contained
three to five layers of utricles (Fig. 2j). Nodal medullary fila-
ments were incompletely fused into two or three groups
(Fig. 2k,l). Both groups conformed to the descriptions of
H. opuntia.
Morphological and anatomical variations
between highly irradiated and shade
conditions
A total of 26 morphological and 35 anatomical characteristics
were examined and compared between highly irradiated and
shade conditions. The 54 thalli and 540 segments showed
differences in terms of form and size between the highly irra-
diated and shade conditions.
4 S. Pongparadon et al.

Fig. 1. Morphology and anatomy of plants growing in highly irradiated conditions. (a) and (b) Thallus characteristics. (c) Reniform shape of lower
segments. (d) Reniform shape of upper segments. (e) Surface pattern; rounded polygonal. (f) Utricle layers; three to four. (g) and (h) Node type;
completed fusion in two or three siphons. Scale bar: Fig. a-b = 1 cm, Fig. e-h = 50 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Thallus and segment forms upper segment was higher under the shade condition than under
the highly irradiated condition: 0.826  0.134 cm and
A compact thallus was found under highly irradiated conditions,
0.627  0.082 cm, respectively (P < 0.01, Fig. 3c, Table S1 in
while a lax thallus was found under shade conditions. Both the
the Supporting Information). The lower segments also showed
upper and lower segments in each environment also showed
significant size differences. The maximum width under the shade
shape differentiation in which the shape of the upper segment
condition was larger than that under the highly irradiated condi-
under highly irradiated conditions was reniform, while it was
tion: 0.848  0.017 cm and 0.736  0.015 cm, respectively
mostly tripartite with a stalk and sometimes reniform under shade
(P < 0.001, Fig. 3d, Table S1 in the Supporting Information).
conditions. The lower segment was reniform under highly irradi-
The height of the lower segment under the shade condition was
ated conditions, whereas it was deeply trilobed and tripartite and
also higher than that under the highly irradiated condition:
sometimes cylindrical under shade conditions (Table S1 in the
0.654  0.155 cm and 0.476  0.079 cm, respectively
Supporting Information).
(P < 0.001, Fig. 3e, Table S1 in the Supporting Information),
and the height:width ratio of the lower segments was higher
Segment size
under the shade conditions than under the highly irradiated con-
The maximum width and height of the upper segment were dition: 0.849  0.133 cm and 0.63  0.056 cm, respectively
affected by light intensity, and the segments under shade condi- (P < 0.001, Fig. 3f, Table S1 in the Supporting Information).
tions were higher (taller) than those under highly irradiated condi-
tions (P < 0.001, Fig. 3a-b, Table S1 in the Supporting
Information). The maximum width under the shade condition Relationship between morpho-anatomical
was larger than that under the highly irradiated condition:
variability and environmental factors
0.853  0.022 cm and 0.749  0.007 cm, respectively. The
average heights of the segments under the shade and highly irra- A possible correlation between morphological and anatomical
diated conditions were 0.717  0.162 cm and variations within H. opuntia species and environmental factors
0.521  0.168 cm, respectively. The height:width ratio of the was determined using CCA (Fig. 4; Table S2 in the Supporting

© 2019 Japanese Society of Phycology

Plasticity of Halimeda opuntia 5

Fig. 2. Morphology and anatomy of

plants growing in lowly irradiated
conditions. (a) and (b) Thallus char-
acteristics. (c-e) Shape of lower seg-
ments. (c) Cylindrical shape.
(d) Deeply trilobed shape.
(e) Tripartite shape. (f-h) Shape of
upper segments. (f) Deeply lobed tri-
partite shape. (g) Tripartite shape
(h) Reniform shape. (i) Surface pat-
tern; rounded polygonal. (j) Utricle
layers; three to four. (k and l) Node
type; completed fusion in two or three
siphons. Scale bar: Fig. a-b = 1 cm,
Fig. I-L = 50 μm. [Color figure can be
viewed at wileyonlinelibrary.com]

Fig. 3. Summary (whisker plots) of

the morphological characters
between highly irradiated and shade
conditions. The bars represent the
lowest and highest data points.
(a) Upper segment maximum width.
(b) Upper segment height.
(c) Height:width ratio of the upper
segment. (d) Lower segment maxi-
mum width. (e) Lower segment
height. (f) Height:width ratio of the
lower segment.

Information). There was a significant relationship between
morph-anatomical variability and environmental factors
(P < 0.001), and the eigenvalues of axes 1 and 2 were 0.172
and 0.0005, respectively. Thallus and segment forms were
positively correlated with low light intensity and habitat under
rock or coral and negatively correlated with the intertidal zone
that included sandy beaches, reef flats and shallow water.
The length and width of the segment and ratio were positively
correlated with the intertidal zone, whereas the length and
width were negatively correlated with low light intensity and
habitats under rock or coral. On the other hand, other charac-
teristics were not correlated with these environmental factors.

© 2019 Japanese Society of Phycology

6 S. Pongparadon et al.

Fig. 4. Characteristic distributions of H. opuntia among environmental variables based on the canonical correspondence analysis ordina-
tion. Labelled lines indicate the direction along which each variable changes most. Filled circles = highly irradiated samples, Open cir-
cles = shade samples and Black squares = characters. Abbreviations: THForm, thallus form; LoSgForm, Lower segment form; LoSgH,
Lower segment height; LoSgMW, Lower segment maximum width; LoSgRatio, Lower segment length over width ratio; UpSgForm, Upper
segment form; UpSgH, Upper segment height; UpSgMW, Upper segment maximum width; and UpSgRatio, Upper segment length over
width ratio.

Fig. 5. Morphology changes in

H. opuntia before and after the
experiment. (a) Thallus characteris-
tics before the experiment. (b and
c) Shape of the upper segments.
(d) and (e) Shape of the lower seg-
ments. (f) Thallus characteristics
after 3 months. (g-k) Shape of the
upper segments. (l-r) Shape of the
lower segments. Scale bar = 1 cm.
[Color figure can be viewed at
wileyonlinelibrary.com]

© 2019 Japanese Society of Phycology

Plasticity of Halimeda opuntia 7

Fig. 6. Summary (whisker plots)

of morphological characteristics
before and after the shading
experiments. The bars represent
the lowest and highest data
points. (a) Upper segment maxi-
mum width. (b) Upper segment
height. (c) Height:width ratio of
the upper segment. (d) Lower
segment maximum width.
(e) Lower segment height. (f)
Height:width ratio of the lower
segment.

Plasticity response to light intensity
Before the transferring experiment, the thallus form was com-
pact and dense with hard and highly calcified reniform seg-
ments (Fig. 5a–e). After 3 months, the form and size of the
thallus and segment changed after the samples were trans-
ferred to the aquarium tank under low light conditions
(Fig. 5f–r). The form or size of the old segments did not
change, while the thalli and segment size, form and branching
of the new growth segments differed in the first month. The
thalli increased in height, and their forms changed from com-
pact thalli that usually exhibited trichotomous branching
(Fig. 5a) to loosely brittle networks of thalli that were mostly
arranged in a row and sometimes with dichotomous branching
(Fig. 5f). The new upper segment forms changed from reni-
form (Fig. 5b-c) to various deeply trilobed shapes, tripartite
and sometimes cylindrical (Fig. 5g–k). The segment size was
significantly different before and after the low light experi-
ment (P < 0.001, Table S3 in the Supporting Information).
The segment width decreased after growth under a low light
intensity (0.767  0.011 cm and 0.564  0.022 cm before
and after, respectively; Fig. 6a; Table S3 in the Supporting
Information), while the newly grown segment height increased
(0.632  0.016 cm; Fig. 6b; Table S3 in the Supporting
Information). Moreover, the height:width ratio after the experi-
ment was higher than that before the low light experiment
(1.312  0.088 cm and 0.597  0.012 cm, respectively;
Fig. 6c; Table S3 in the Supporting Information). The new
lower segment shape also changed from reniform and broad
tripartite (Fig. 6d,e) to cylindrical, deeply bilobed, deeply tri-
lobed and tripartite (Fig. 6l–r), and the segments were signifi-
cantly smaller in size (P < 0.001, Table S3 in the Supporting
Information). The average width of the newly grown segments
decreased from before to after the low light experiment
(0.767  0.011 cm and 0.564  0.022 cm, respectively;
Fig. 6d; Table S3 in the Supporting Information), while the
segment height increased (0.435  0.008 cm and
0.561  0.013 cm, respectively; Fig. 6e; Table S3 in the

Fig. 7. Summary (whisker

plots) of morphological charac-
teristics among natural, low and
high light conditions. Bars repre-
sent the lowest and highest data
points. (a) Upper segment maxi-
mum width. (b) Upper segment
height. (c) Height:width ratio of
the upper segment. (d) Lower
segment maximum width.
(e) Lower segment height. (f)
Height:width ratio of the lower
segment.

© 2019 Japanese Society of Phycology

8 S. Pongparadon et al.
Fig. 8. Chloroplast movement adaptation in Halimeda species at
Tangkhen Bay, Phuket Province, the Andaman Sea, Thailand.
(a) H. opuntia. (b) H. macroloba. (c) H. borneensis. [Color figure
can be viewed at wileyonlinelibrary.com]
Supporting Information). In addition, the height:width ratio
was higher after growth than before growth under low light
intensity (1.330  0.070 cm and 0.562  0.012 cm,
respectively; Fig. 6f; Table S3 in the Supporting Information).
However, there were no significant differences in any of the
anatomical characteristics. Furthermore, the new growth seg-
ments were softer and poorly calcified. The percentage of
CaCO3 content in the segments before and after the experi-
ment was significantly different (P < 0.001, Table S3 in the
Supporting Information); the segments were less calcified
under low light conditions.
After 2 months, the different irradiated light experiments
showed the same patterns in which the new upper segments
showed different forms among the treatments: the new seg-
ments under both the natural and high light conditions had
the same reniform shape, while the new upper segments
under the low light conditions had deeply trilobed, tripartite
and sometimes cylindrical shapes. Similar segment size was
found among treatments (Fig. 7a–f; Table S4 in the
Supporting Information) except the new upper segment height
and new upper segment height: width ratio under the lower
light condition were significantly higher than those under the
other conditions (0.683  0.031 cm and 1.741  0.020 cm,
respectively; Fig. 7b,c).
DISCUSSION
Species boundaries and variations among
species
H. opuntia samples genetically constitute a monophyletic
taxon as the phylogenetic tree showed a well-supported (99%)
clade with all sequences, but the morphological characteris-
tics exhibited substantial variations. The samples in the cen-
tral Indo-Pacific Ocean showed low genetic variations. Our
morphologies corresponded with the phylogenetic morphol-
ogies with all morphs belonging to H. opuntia. H. opuntia
showed two morphological forms. The first morph with a
tightly packed thallus consisted of ribbed reniform segments
that grew in highly irradiated intertidal conditions in both the
intertidal and subtidal zones, including sandy beaches, coral
reef flats and reef slopes. This morph matched the classical
taxonomy in Barton (1901), Taylor (1950), and Hillis-
Colinvaux (1980) and was also recorded in wave-exposed
rocky surface areas (Littler & Littler 2000) and highly irradi-
ated conditions (Kooistra & Verbruggen 2005).
The second morph with a brittle network, ribbed tripartite
or reniform upper segments and deeply trilobed, tripartite and
cylindrical lower segments grew in shade conditions in coral
reefs in subtidal zones, e.g., under rocks or coral. This morph
was reported by Littler and Littler (2000) as having a brittle
network with tripartite segments in mangroves and sheltered
lagoons, and Kooistra and Verbruggen (2005) and El-Manawy
and Shafik (2008) reported that H. opuntia showed this form
in low light and shade aquarium conditions.
The thalli and segments in the second morph were similar
to those of Halimeda cf. distorta (morph 2b, Kooistra &
Verbruggen 2005), H. opuntia forma elongata (plate 41, Taylor
(1950)) and H. minima (the Andaman Sea morph,
Pongparadon et al. unpublished data). Although their morpho-
logical characters overlapped in terms of thallus and segment
shape and size, the anatomical characteristics of the primary
utricle shape and size and their habitat were clearly
© 2019 Japanese Society of Phycology
Plasticity of Halimeda opuntia
distinguished. Thus, using anatomical characteristics clearly
helped in identifying this species.
The variations within the H. opuntia species were low com-
pared to those in other Halimeda species. H. opuntia has only
two morphs and showed plasticity in only the morphological
variations in thallus and segment size and shape, while
Halimeda macroloba has more morpho-anatomical variability
that is affected by water depth and light in terms of several
characteristics, such as holdfast type, segment size and
shape, peripheral utricle patterns as seen from the surface
view, layers of utricles and shapes of the primary utricle
(Pongparadon et al. 2017). In addition, Halimeda
heteromorpha is another species that has high variation within
the species. This species is large and densely ramified in
extremely sheltered bays but has large, trilobed segments and
lax branching on sheltered fore-reef slopes (Verbruggen et al.
2006). These characteristics could also lead to mis-
identification as Halimeda incrassata or Halimeda
melanesica; however, the combination of anatomical charac-
teristics, ecological habitat and geographic distribution could
help resolve the identification issue.
Ecological factors and their morphological
plasticity and adaptations
Light intensity was the main factor driving H. opuntia morpho-
logical plasticity, while habitat type, depth and zonation were
less likely to influence morphology, such as thallus branching
pattern, segment form and size. The correlation of H. opuntia
with environmental factors is relatively low in comparison to
H. macroloba, which shows high eigenvalues and responses to
many environmental conditions, such as light intensity, depth
and substrate type (Pongparadon et al. 2017). A compact
thallus with ribbed reniform segments was found in highly
irradiated conditions, and in shade conditions, especially
under rocks or coral in the subtidal zone, the thalli changed
to lax branching, such as a network with deeply trilobed lower
segments and ribbed reniform or tripartite upper segments.
Most segments showed a stalk zone that led to a larger size
than the segments with lax branching and a higher thallus.
This increase in size with high and narrow segments possibly
occurred to increase the branch length to expose the organism
sunlight outside the shade zone. Moreover, the compact thal-
lus and smaller segment size of Halimeda in highly irradiated
conditions might help alleviate photoinhibition under the
extremely high light conditions of the intertidal zone and
exposed area. However, the population of H. opuntia changes
in response to other environmental parameters. This study
does not confirm the effects of wave exposure, herbivores or
nutrients, which might also affect the morphological plastic-
ity, thallus form, segment size, segment weight, number of
segments per thallus and CaCO3 content of H. opuntia.
This phenomenon also occurred when the high-irradiance
forms of H. opuntia were moved to a shaded aquarium, where
they adapted to brittle networks, such as those grown in
shaded zones or low light subtidal conditions. The morpholog-
ical adaptation of the species was confirmed by the changes
in its thallus forms and segment shapes and sizes when it was
cultured under a light intensity that was lower than that in
nature. The thalli grew by increasing the segment height and
© 2019 Japanese Society of Phycology
9
changing the branching pattern. H. opuntia had varying mor-
phological plasticity in response to light levels. A previous
study on H. opuntia showed similar patterns in segment size
and shape. The populations in low light levels have loose
thalli, small segments and tri-partite segments (Kooistra &
Verbruggen 2005). H. opuntia segments from the inshore
patch reefs were large, and the thallus form was more upright
than that in the population from the offshore spur and groove
area (Yñiguez et al. 2010). Additionally, in other species,
such as H. macroloba, thallus and segment sizes are large in
subtidal zones and low light intensity conditions
(Pongparadon et al. 2015). Likewise, in H. tuna, the mature
segments from the deep and low light areas were wider and
longer than those in shallow and highly irradiated areas
(Vroom et al. 2003; Neustupa & Nemcova 2018).
Moreover, light environments in temperate coastal systems
have highly variable irradiance due to shading, depth gradi-
ents, water turbidity and sublittoral habitat and temporally
variable irradiance due to circadian and seasonal scales (Kirk
1994; Talarico & Maranzana 2000). Irradiance also affects
morphological plasticity. For example, the marine algae
Asparagopsis armata displays light-induced plasticity in terms
of size and shape, which is expressed as few branches
coupled with branch elongation under reduced irradiance
(Monro & Poore 2005). Moreover, algal morphology responds
to irradiance at the level of the individual thallus (Fischer
et al. 2004). Collado-Vides (2002) reported that the morpho-
logical variation among natural populations of the green alga
Caulerpa prolifera showed phalanx-type individuals in sunny
areas and guerrilla-type individuals in shaded ones.
The calcification rate and CaCO3 contents of Halimeda
were decreased under low light intensities (Castro-Sanguino
et al. 2016). H. opuntia had a low percentage of CaCO3 when
it grew under very low light conditions in the shading experi-
ment. The calcification process was activated after photosyn-
thesis began in the daytime when the seawater pH increased
in the intercellular spaces of its siphon (de Beer and Larkum
2001). Under low light conditions, the photosynthesis rate
and calcification rate in H. opuntia were than those under
high light conditions. This scenario has previously been dem-
onstrated in H. macroloba and Halimeda borneensis, where
high light intensities (500 and 900 μmol photons m−2 s−1)
rather than low light intensity (50 μmol photons m−2 s−1) were
the best conditions to promote photosynthesis and calcifica-
tion in Halimeda (Kaewsrikhaw et al. 2016; Prathep et al.
2018). In contrast, extremely high light irradiation affected
the photosynthesis rate and calcification rate by causing
photoinhibition and decreased photosynthetic and calcifica-
tion performance (Vroom et al. 2003).
When H. opuntia was exposed to a high light intensity dur-
ing low tide, it adapted its chloroplast movements; its chloro-
plast was withdrawn to the middle and lower parts of the
segment, and a white band appeared on its margin (Fig. 8a).
This chloroplast movement phenomenon also occurs in other
Halimeda species, such as H. macroloba and H. borneensis
(Fig. 8b,c). These chloroplast photo-movements are a
response to fluctuating light intensity that occurs in plants
such as angiosperms and algae. A chloroplast moves away
from the surface to the middle of the cell when exposed to
strong light intensity, and this process is called the chloro-
plast avoidance response. When chloroplasts are under low
10
light intensity, this process is called the chloroplast accumu-
lation response, which occurs when chloroplasts accumulate
along the cell wall (Sato & Kadota 2007). Interestingly, chlo-
roplast movement in Halimeda species also occurs at night,
when segments change from a dark green color to an almost
white color (Drew & Able 1990).
H. opuntia is a pantropical species that is widely distrib-
uted and common in many habitats, while some Halimeda
species are restricted to narrow regions and specific habitats,
such as H. minima, H. renschii and H. gigas, which are dis-
tributed in only the Indo-Pacific region and subtidal zone. The
ability of H. opuntia to adapt to different light environments
might contribute to its colonization success. This species can
survive in several habitats from sandy beaches in intertidal
zones to coral reefs in the subtidal area because they quickly
respond and change their thalli forms and segment shapes.
Moreover, the holdfast type of this species with several rhizoi-
dal axes is a special characteristic that adds to the success of
this species. This species can also produce multiple points of
a few rhizoids to attach to many kinds of substrates, such as
small rocks or coral, sand, dead coral and healthy coral. This
study on morphological plasticity, genetic variations and envi-
ronmental factors expanded our knowledge of the range of
plasticity and adaptation processes of organisms to various
habitats and environments.
ACKNOWLEDGMENTS
This work was supported by the Thailand Research Fund
(TRF) Grant for New Researcher, TRG6180010 and the Post-
doctoral Fellowship from Prince of Songkla University. The
additional field collections in Indonesia and Japan were
supported by the Japan Society for the Promotion of Science
(JSPS: JSPS Core-to-core RENSEA Seagrass and Seaweed
Taxonomy workshop). The aquarium facility was supported by
the Phuket Marine Biological Center (PMBC), Phuket Province
and Biology Department, Faculty of Science, Prince of Song-
kla University during the experiment.
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11
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Appendix S1. H. opuntia specimens used for morpho-
anatomical and genetic analysis in this study
Appendix S2. Environmental variables were measured in each
sampling sites.
Appendix S3. Lists of chloroplast tufAsequences were loaded
from Genbank.
Appendi S4. List of morphology and anatomy characters
Appendix S5. Maximum likelihood topology of tufA gene
sequences of Halimeda. Numbers at the nodes are maximum-
likelihood bootstrap percentages followed by Bayesian infer-
ence posterior probabilities
Table S1. Characteristic of H. opuntia between sunny and
shad conditions showing Mean  SE and results of one-way
analysis of variance.
Table S2. Canonical correspondence values and percent
explained variance associated with the first three axes for eco-
logical factors measured.
Table S3. Characteristic of H. opuntia between before and
after experiment under the low light intensity showing
Mean  SE and results of one-way analysis of variance.
Table S4. Characteristic of H. opuntia among experiment
under the natural, low and high light irradiated conditions
showing Mean  SE and results of one-way analysis of
variance.