Phycological Research 2017; 65: 136–145
12172
...........................................................................................................................................................................................
High morpho-anatomical variability in Halimeda macroloba
(Bryopsidales, Chlorophyta) in Thai waters
Supattra Pongparadon,1 Giuseppe C. Zuccarello2 and Anchana Prathep
1
Seaweed and Seagrass Research Unit, Faculty of Science, Department of Biology, Excellence Center for Biodiversity of
Peninsula Thailand, Prince of Songkla University, Songkhla, Thailand, 2School of Biological Sciences, Victoria University of
Wellington, Wellington, New Zealand
........................................................................................
SUMMARY
Halimeda macroloba is a common widespread and morpholog-
ically variable species in the Indo-Pacific Ocean. A series of
morphometric data (16 morphological and 46 anatomical vari-
ables) and the chloroplast-encoded tufA gene were examined
to explore the morphological and genetic variations within the
species and to better determine species boundaries in H.
macroloba in Thai waters. Moreover, the environmental condi-
tions, which may affect morphological variations in H. macro-
loba, especially of Haplotype I were examined. Our results
showed that H. macroloba has more morpho-anatomical varia-
bility and broader species boundaries than previously thought
in several characters: holdfast type, segment shape and size,
node height with differences in pore size, the presence of two
additional patterns of peripheral utricle as seen in surface
view, and additional layers of utricles (up to seven layers).
Additionally, two new variable characteristics have been
reported for the first time, i.e. shape and margin of peripheral
utricles. Water depth and light correlated with thallus mor-
phology, larger thalli and segments with a greater number of
utricle layers were associated with subtidal plants, while smal-
ler thalli and segments were found in the intertidal or high
light intensity areas. The high plasticity of this species may
explain why in the Indo-Pacific Ocean this species is common
and dominant.
Key words: adaptation, haplotype network, Indo-Pacific
Ocean, phenotypic plasticity, species boundary, tufA.
........................................................................................
INTRODUCTION
Organisms can respond to their environment through changing
many aspects of their phenotypes, a phenomenon known as
phenotypic plasticity (Fernandez-Lopez 1998). These plastic
responses could be changes in morphology, anatomy, physiol-
ogy, development, growth, life history and reproductive timing
(Agrawal et al. 1999; Sultan 2000; Young et al. 2003). Plas-
tic responses can contribute to a species ability to survive in
variable habitats (Sultan 1998) and allow for a greater range
of habitats to be exploited. Plasticity had been reported in
land plants and many animals (Sultan 1995). Algae also
respond to changed environments by changing their morphol-
ogies that may be adaptive in the new environments (Sultan
1995; Lande 2009; Flukes et al. 2015). Plasticity in a spe-
cies’ defining characters can also lead to problems in taxo-
nomic circumscription.
doi: 10.1111/pre.
1
*
The genus Halimeda is a particularly interesting subject as
it is well studied taxonomically, cryptic species are known,
and phenotypic plasticity of defined morphological characters
can be studied. Species within some sections of Halimeda
(e.g. section Opuntia J. Agardh ex De Toni, section Halimeda
J. Agardh ex De Toni and section Rhipsalis J. Agardh ex De
Toni) exhibit high morphological plasticity (Verbruggen &
Kooistra 2004; Verbruggen et al. 2005a, 2006). The morpho-
logical range of some of these species can overlap, making
identification difficult. For example, in the section Rhipsalis,
H. incrassata (J. Ellis) J.V. Lamouroux was composed of three
pseudo-cryptic entities, two of which were described as new
species; H. kanaloana Vroom and H. heteromorpha N00 Yeurt
(Verbruggen et al. 2006). In addition, other Halimeda species
show morphological variations that appear to correlate with
habitats (Verbruggen 2005). There are many environmental
factors that affect morphology such as substrate type, depth,
light intensity and wave action (Gilmartin 1960; Wyrtki 1961;
Hillis-Colinvaux 1980; Kaandorp 1999; Morton & Blackmore
2001; Latypov 2003). For example, H. heteromorpha found
in sheltered fore-reef slopes and deeper water exhibit larger
thalli and bigger trilobed segments than plants found in other
environments (Verbruggen et al. 2006).
Even though a lot of research has been done on different
Halimeda sections and species groups (e.g. Kooistra et al.
2002; Verbruggen et al. 2006) in the levels of morphological
variation and species boundaries, many species are not fully
explored. H. macroloba Decaisne is found in the Indo-Pacific
Ocean, and is one of the most common and widespread spe-
cies in the region (Guiry & Guiry 2016). H. macroloba is dis-
tributed around Thai waters, in many different habitats and
surprisingly it is the only Halimeda species that occurs in the
Gulf of Thailand (Pongparadon et al. 2015). It is also known
to be morphologically plastic (Verbruggen et al. 2006; Dijoux
et al. 2012; Pongparadon & Prathep 2013; Pongparadon
et al. 2015). This high morphological plasticity makes species
determination sometime difficult and the species could be
misidentified.
This study focuses on H. macroloba in Thai waters. A
series of morphometric measurements and the chloroplast-
encoded tufA gene are used to explore and address the
.........................................................................................
*To whom correspondence should be addressed.
Email: anchana.p@psu.ac.th
Communicating Editor: Showe-Mei Lin
Received 7 April 2016; accepted 19 December 2016.
© 2017 Japanese Society of Phycology
Morphological variability in H.macroloba
following questions: Are there morphological and genetic dif-
ferences in H. macroloba among populations along the coast-
line of Thailand? Is there morphological differentiation within
each chloroplast haplotype of H. macroloba? What environ-
mental factors affect the morphology of H. macroloba?
MATERIALS AND METHODS
Samples collections
Halimeda macroloba samples were collected by snorkeling
and diving from 29 populations throughout most of their dis-
tributional range in Thai waters (Appendix S1). Visual esti-
mates were used to define and score the environmental
variables: six substrata types (mud, muddy sand, sand, sand
with coral rubble, coral rubble, rock and dead coral), two
levels of depth (intertidal and subtidal), two degrees of wave
action (shelter and exposed) and two levels of light intensity
(low and high light intensity) (Fig. 1, Table 1). Light intensity
was measured in some sites using an Onset-Hobo data logger
(Onset Computer Corporation, Contoocook, NH, USA). Three
to five specimens from each population, a total of 141 sam-
ples, were preserved on herbarium sheets for morphological
and anatomical observations. Small apical fragments of each
specimen were preserved either in 95% ethanol or quickly
Fig. 1. The location of Halimeda macroloba populations in Thai
Waters. (1) Ao Tang Khen (TK), Phuket. (2). Ko Kra Dan (KD),
Trang. (3) Ao Ta Loa Pa Lean (TLP), Satun. (4). Ko Lidee Yai
(LDY), Satun. (5) Ko Lidee Lek (LDL), Satun; (6) Ko Tha Rai (TR),
Nakon Sri Thummarat. (7) Ko Tan (TAN), Surat Thani. (8) Ko Mud
Sum (MS), Surat Thani. (9) Ko Rab (RAB), Surat Thani. (10) Ko
Pi Tak (PT), Chumporn. (11) Had Bo Moa (BM), Chumporn and
(12) Samae San (SS), Chonburi.
© 2017 Japanese Society of Phycology
137
dried in silica gel for molecular analysis. The specimen num-
ber of each sample in silica gel could be linked with a voucher
specimen. All voucher specimens were deposited in the
Prince Maha Chakri Sirindhorn Natural History Museum at
Prince of Songkla University (PSU).
Morphological and anatomical observations
The morphological and anatomical structures were examined
following Hillis-Colinvaux (1980) and Verbruggen et al.
(2005a,b) with some modifications. To decalcify the algal tis-
sue for anatomical observations, segments were incubated in
10% concentrated HCl at room temperature. Morphological
and anatomical characters were measured from segments in
the middle zone of the thallus (Verbruggen et al. 2005a),
measuring three randomly chosen segments from each speci-
men. In each segment, ten replicates of all characters were
randomly measured. The morphological and anatomical char-
acters examined are shown in Appendix S2. Some characters
that had not been previously studied (peripheral utricle shape
and utricle margin type) were also examined.
Molecular analyses
TufA has been proposed as a DNA barcoding marker for the
Ulvophyceae, including Halimeda (Kooistra et al. 2002; Ver-
bruggen 2005; Saunders & Kucera 2010; Dijoux et al.
2012). The molecular procedures for the chloroplast tufA fol-
lowed Pongparadon et al. (2015). Additionally, tufA
sequences of H. macroloba included all available published
sequences (AM049960, JN644690, HM140244 and
JN644693). Statistical parsimony was used to create a haplo-
type network using TCS 1.21 (Clement et al. 2000) to calcu-
late the minimum number of mutational steps by which the
sequences could be joined with 95% confidence.
Data analyses
SPSS for Window 13.0 (SPSS, Chicago, IL, USA) was used
for statistical analyses. One-way analysis of variance (ANOVA)
was used to test significant differences in all morphological
and anatomical characters of H. macroloba among the three
haplotypes and within Haplotype I. Normality of data was
examined using Kolmogorov–Smirnov prior ANOVA. The
homoscedasticity was tested using Levene’s test to examine
variance of treatments. One-Way ANOVA was used if data
were homoscedastic and Welch ANOVA was used if data were
heteroscedastic. In addition, Tukey’s post-hoc test was used
Table 1. Environmental and habitat variables measured in sam-
ples sites
Environmental Categories/units
variables
Substrata 0 (mud), 1 (muddy sand), 2 (sand), 3 (sand with
coral rubble), 4 (coral rubble), 5 (rock),
6 (dead coral)
Depth 0 (intertidal; exposed and <5 m), 1 (subtidal;
submerged and >5 m)
Wave action 0 (Shelter), 1(Exposed)
Light 0 (low light; < 800 μmol photons m−2 s−1),
1(high light; > 800 μmol photons m−2 s−1)
138
to test the significance of morpho-anatomical differentiation
among haplotypes and within the most dominant haplotype.
Box plot maps were used to present the data if there were sig-
nificant differences. If the data could not be transformed to
meet the assumption of normality, non-parametric Kruskal–
Wallis was used to test significant differences in morphologi-
cal and anatomical characters. In addition, Canonical Corre-
spondence Analysis (CCA) and Monte Carlo permutation tests,
were performed using PC-ORD 5 software (Mccune & Grace
2002), to test the relationship between morpho-anatomical
characters and environmental conditions.
RESULTS
Genetic variation
All Thai samples sequenced were genetically identified as
H. macroloba. TufA showed four haplotypes in the Indo-
Pacific (Fig. 2). Haplotype I was the most common in
Thailand and had the widest distribution (Tanzania to New
Caledonia). Haplotype I was directly connected to all three
other haplotypes in the network analysis. In the lower Anda-
man Sea, there was another haplotype present, which differed
by 2 bp from Haplotype I to Haplotype II. Haplotype III dif-
fered by 3 bp from Haplotype I and was found in the middle
of the Gulf of Thailand. Haplotype IV was from Australia.
Morphological and anatomical variations
among haplotypes
A total of 16 morphological and 46 anatomical characters
of Thai Halimeda samples were examined and compared
S. Pongparadon et al.
among haplotypes. There were significant differences in four
characters among haplotypes (Fig. 3, Table 2): diameter of
1 utricle in surface view (Fig. 3A), 1 utricle maximum
width (Fig. 3B), 1 utricle height (Fig. 3C), and node height
(Fig. 3D). These characters in samples of Haplotype II
(n = 5) and Haplotype III (n = 5) are non-overlapping with
Haplotype I (n = 131) and smaller than those found in Hap-
lotype I. Most H. macroloba populations were Haplotype I,
thus further analyses and examinations were focused mainly
in this group.
Morphology and anatomy within haplotype I
Halimeda macroloba showed high morphological and anatomi-
cal variability within Haplotype I. New variable characteristics
have been reported for the first time and species boundaries
were extended in several characters.
Additional characteristic patterns
Surface pattern and diameter in surface view of peripheral
utricles. The peripheral utricle in surface view showed four
patterns: irregular circular, regular circular, rounded polygonal
and rounded polygonal with a circular middle (Fig. 4a–d).
Moreover, there were significant differences in diameter of
peripheral utricles in surface view (Fig. 4a, Table 3). SP
510 had the widest surface diameter of peripheral utricles
(47.9  1.1 μm), and SP 22 had the narrowest peripheral
utricles (26.5  0.3 μm).
Shape, margin and size of peripheral utricles. This is the first
report of variation in peripheral utricle shape and margins.
Peripheral utricle shape displayed two patterns: cone-shaped
and jar-shaped (Fig. 4e,f ). The margin of peripheral utricles
Fig. 2. Statistical parsimony net-
work of four tufA haplotypes (I–IV)
of H. macroloba and their distribu-
tion in the Indian and Pacific
coasts. In the network tree, line
and black circle indicate one
mutational step and missing hap-
lotype, respectively. The size and
number of each circle in the net-
work tree are proportional and
number of specimens detected.
Gray color indicates the Indo-
Pacific countries where H. macro-
loba was reported, and pie charts
represent the location of four hap-
lotypes. [Color figure can be
viewed at wileyonlinelibrary.com]
© 2017 Japanese Society of Phycology
Morphological variability in H.macroloba 139

Fig. 3. Whisker plots of signifi-

cantly different characters of H.
macroloba among three haplotypes.
Boxes represent the 25% and 75%
quartiles; whiskers represent the
lowest and highest data points. A
horizontal line in a box represents
mean  SE. (A) Diameter of 1 utri-
cle height in surface view. (B) 1
utricle maximum width. (C) 1 utri-
cle height. (D) Node height. Lower
case letters indicates statistical dif-
ferences (P < 0.05) among
populations.

Table 2. Morphological and anatomical characters of Halimeda Extended H. macroloba species boundaries
macroloba showing significant difference among the four
Thallus appearances. Overall thallus morphology of all sam-
haplotypes
ples was a single plane or fan-shaped, but thallus form dif-
Mean  SD
fered among populations (Appendix S3). Thallus
morphologies changed with environmental conditions, and
Characters Haplotype 1 Haplotype 2 Haplotype 3 thallus size varied in different tidal zones: small thalli (smal-
Diameter of 1 utricle 43.1  9.1 a
10.2  1.3 b
9.1  1.1b
ler than 10 cm) inhabiting the intertidal areas (Appendix
1 utricle maximum 43.0  8.9a 10.1  0.4 10.0  1.3b
b S3a–d) and large thalli (larger than 10 cm) were from subti-
width dal areas (Appendix S3e, f ). Moreover, specimens located
1 utricle height 89.6  19.8a 18.6  1.8b 20.6  3.0b in exposed habitats had short branches (less than three seg-
Node height 290.0  7.3a 71.4  2.3b 33.3  2.2b ments per branch) while long branches (more than three
a,b segments per branch) were found in extremely shel-
The different letters indicate significant difference.
tered bays.

Holdfast types. Most specimens had a bulbous holdfast that

also differed and had three forms: entire distal margin, thick
crowned margin and thin crowned margin (Fig. 4e–g). The
height and maximum width of peripheral utricle were signifi-
cantly different (Fig. 5b,c, Table 3). Peripheral utricle height
of SP 20 was the greatest (127.2  0.8 μm) and of SP
518 was the shortest (68.7  3.0 μm). The widest peripheral
utricle was in SP 370 (54.3  5.2 μm) and the narrowest
was in SP 521 (31.4  1.0 μm).
Number of utricle layers. The number of utricle layers varied
from four to seven layers (Fig. 4h–k), with six and seven layers
reported for the first time in this species. The layer numbers
were significantly different among populations (Fig. 5d,
Table 3). The maximum number of utricle layers was seven
layers, found in the specimens SP 13 and SP 509, but most
specimens had four or five layers.
anchored in sand, sand with coral rubble and muddy sand
substrates (Appendix S4a), except some specimens which
had modified epilithic holdfast or a loose rhizoidal holdfast on
dead coral substrata covered by a thin layer of sand
(Appendix S4b).
Segment appearance and size. Halimeda macroloba showed
several segment forms: reniform, discoid, elliptical-discoid,
broad ovate, obovate, broad obovate, cuneate and sub-
cylindrical (Appendix S5). The segment margins were either
entire distal margin, shallowly lobed, medium lobed or deeply
lobed (Appendix S6). The basal segments had two forms: sub-
cylindrical and cuneate, while most apical segments were ren-
iform. Segment size also varied in samples. There was signifi-
cant difference in segment length (Fig. 6a, Table 3). The
largest segments were in SP 510 (2.6  0.07 cm), and SP

© 2017 Japanese Society of Phycology

140 S. Pongparadon et al.

Fig. 4. Variation in cortex zone. (a)–(d) Variation in surface pattern of peripheral utricle. (a) Irregular circular (specimen- SP 1).
(b) Regular circular (specimen- SP 520). (c) Rounded polygonal (specimen- SP 379). (d) Rounded polygonal with a circular middle (speci-
men- SP 509). (e)–(g) Variation in peripheral utricle shapes and margin. (e) Light microscopy and line drawing of cone-shaped peripheral
utricle (specimen- SP 520). (f ) Jar-shaped peripheral utricle with thick crowned margin (specimen- SP 379). (g) Jar-shaped peripheral utri-
cle with thin crowned margin (specimen- SP 509). (h)–(k) Variation in number of utricle layers. (h) Four layers (specimen- SP 1). (i). Five
layers (specimen- SP 509). (j) Six layers (specimen- SP 35). (k) Seven layers (specimen- SP 13). Dashed lines indicate utricle layer bound-
aries. Scale bar: a–d = 20 μm, e–k = 50 μm.
© 2017 Japanese Society of Phycology
Morphological variability in H.macroloba 141

Table 3. Morphological and anatomical characters of H. macroloba showing significant difference among Haplotype I specimens

Characters Transformation method Mean  SD F or H-ratio P-values


Diameter of 1 utricle at surface view (μm) Log (x + 1) 35.5  7.0 36.941 <0.001
1 utricle height (μm) Log (x + 1) 91.4  14.3 29.734 <0.001
Maximum width of 1 utricle (μm) 1/x 38.6  7.8 7.348 <0.001
Number of utricles layers† — 5.0  1.0 126.000‡ <0.001
Segment length (cm) Log (x + 1) 2.1  0.4 8.460 <0.001
Segment width (cm) Log (x + 1) 2.5  0.6 4.833 <0.001
Node height (μm)† — 184.7  14.3 114.199‡ <0.001
Diameter pores (μm) x2 37.3  14.3 29.487 <0.001
Medullary siphon diameter (μm)† — 75.8  39.9 111.532‡ <0.001
Secondary utricle heights (μm)† — 90.5  26.2 114.763‡ <0.001
Secondary utricle maximum width (μm)† — 34.3  11.8 110.547‡ <0.001
Tertiary utricle heights (μm)† — 233.1  62.5 76.032‡ <0.001

†
Kruskal–Wallis test was used because the data did not meet the assumptions of analysis of variance.
‡
Kruskal–Wallis H statistic.

Fig. 5. Whisker plots of signifi-

cantly different characters within H.
macroloba Haplotype I. (a). Diameter
of 1 utricle height in surface view.
(b) 1 utricle height . (c) Maximum
width of 1 utricle. (d) Number of
utricles layers. (a)–(c) Results of
one-way analysis of variance.
(d) Results of non-parameteric Krus-
kal–Wallis test. Population abbrevia-
tions found in Appendix S1. See the
captions in Figure 3 for other
information.

425 had the shortest (1.6  0.1 cm). There was also signifi-
cant difference in segment width (Fig. 6b, Table 3). Halimeda
macroloba SP 37 had the widest (3.7 0.2 cm), and SP
32 had the narrowest (1.6  0.1 cm). Segment shape was
also extremely variable among different environmental condi-
tions. Segments from the subtidal were larger than those from
the intertidal.
Although all samples showed similar node pattern, there was
significant difference in node height (Fig. 6c, Table 3). The
highest node height was in SP 37 (288.2  8.9 μm). The
lowest was in SP 513 (106.8  1.9 μm). The pore diameter
was also significantly different (Fig. 6d), largest in the SP
35 (55.2  1.9 μm).

Medullar siphons. Medullar siphons were significantly differ-

Node height and pore size. Medullar siphons adhere closely at ent in diameter (Fig. 6e, Table 3). The maximum diameter
nodes in a single unit, showing an adhesion belt with pores was in SP 37 (169.8  6.9 μm), whereas the minimum was
between parallel siphons of different sizes (Appendix S7a,b). in SP 511 (30.0  1.8 μm).

© 2017 Japanese Society of Phycology

142 S. Pongparadon et al.

Fig. 6. Whisker plots of signifi-

cantly difference morphological
character distribution within Hap-
lotype I. (a) Segment length.
(b) Segment width. (c) Node
height. (d) Diameter pores.
(e) Medullary siphon diameter. (f )
Secondary utricle heights.
(g) Secondary utricle maximum
width. (h) Tertiary utricle heights.
(a,b,d) results of one-way analysis
of variance. (c,e–h) Results of
non-parameteric Kruskal–Wallis
test. See the captions in Figures 3
and 5 for other information.

Relationship between morpho-anatomical
variability and environmental factors
A possible correlation between morphological variation within
Haplotype I and environmental factors was determined using
CCA. There was a significant relationship between morpho-
anatomical variability and environments (P < 0.001) and
eigenvalues of axes 1–3 were 0.591, 0.299 and 0.112,
respectively. The cumulative percent variance on the two first
axes of the general CCA explained 24.5%. Thallus size,
lengths and widths of segment and node height were posi-
tively correlated with depth and negatively correlated with
light. Length and width of segment and number of utricle
layers increased with increasing depth and decreased with

© 2017 Japanese Society of Phycology

Morphological variability in H.macroloba
Fig. 7. Character distribution of H. macroloba among environ-
mental variables based on the canonical correspondence analysis
ordination. Labeled lines indicate the direction along which each
variable changes most. The gradient is dominated by variation in
depth, wave and light.
lesser light intensity. Diameters of peripheral utricles in sur-
face view, pore size, length and width of peripheral utricle
were not correlated with these environmental factors (Fig. 7).
DISCUSSION
There is very little genetic variation in H. macroloba based on
tufA sequences, with four haplotypes in the Indo-Pacific. In
Thai waters, the haplotype diversity of populations along the
coast of the Andaman Sea and the Gulf of Thailand was gen-
erally low. Most morphological variants of Halimeda speci-
mens were associated with the main haplotype (Haplotype I),
which was widely distributed in the Indo-Pacific region and is
found both in the Andaman Sea and the Gulf of Thailand.
This low level of genetic variations of algae in Thai waters is
common, and previously reported in Padina boryana Thivy
(Wichachucherd et al. 2014) and Sargassum polycystum
C. Agardh (Kantachumpoo et al. 2013). Low genetic variation
is also reported in the common seagrass, Halophila ovalis (R.
Brown) J. D. Hooker (Nguyen et al. 2014). The Thai-Malay
peninsula has been suggested to be a major biogeographic
barrier, due to differences in sea surface circulation, between
the Andaman Sea and the Gulf of Thailand (Nguyen et al.
2014; Wichachucherd et al. 2014; Pongparadon et al.
2015). Haplotype II of H. macroloba was restricted to the
lower Andaman Sea possibly due to the prevailing currents in
the lower Andaman Sea that did not flow through to the Gulf
or mix with north Thailand waters. The Gulf of Thailand is a
semi-closed area and water is retained in the Gulf
(Pongparadon et al. 2015). This may explain the limited dis-
tribution of Haplotype III in the Gulf.
The lack of any apparent variations in this species in the
Indo-Pacific Ocean (e.g. similar haplotypes found in Thailand
and New Caledonia) could be due to the sample sizes in non-
Thai areas and/or the genetic marker used, which is too con-
served. More samples and other markers need to be investi-
gated to resolve this.
© 2017 Japanese Society of Phycology
143
Our results suggest that specimens with different plastid
haplotypes were morphologically different. To determine if
this is due to adaptation or haplotype-specific lineage varia-
tion needs further study. The collection of more samples from
other Indian and Pacific Ocean populations is needed to char-
acterize the morphological variation within haplotypes. The
use of more variable molecular markers (e.g. nuclear riboso-
mal ITS1-5.8S-ITS2 region, plastid UCP7 region (rps19-
rps3); Verbruggen 2005) may also provide more resolutions
and would provide a better understanding of phylogeographic
distribution of H. macroloba in the Indo-Pacific Ocean.
This study extends the morphological and anatomical vari-
ation found within H. macroloba. The holdfast type of
H. macroloba was proposed to be bulbous (Hillis-Colinvaux
1980), although a bulbous holdfast is observed in most
H. macroloba when growing in unconsolidated substrata, the
holdfast of samples growing on rock or dead coral substrata
formed a loose rhizoidal holdfast. Therefore, a bulbous hold-
fast cannot be a diagnostic character for H. macroloba. Con-
siderable size and shape variations can be observed in the
utricles. Utricle shape is diagnostic for some Halimeda spe-
cies and sections. For example, H. scabra M. Howe has a ter-
minal spine on primary utricles while H. macrophysa
Askenasy utricles are markedly inflated. H. macroloba has
inflated primary utricles (Barton 1901; Hillis-Colinvaux 1980;
Verbruggen & Kooistra 2004) but in this study we found two
additional shapes of utricle margins, cone-shaped and jar-
shaped. In surface view, the appearance of peripheral utricles
depends on the degree of utricle attachment. Previous studies
suggested that the peripheral utricle pattern in surface view
was circular when the primary utricles are not attached to
each other (Hillis-Colinvaux 1980; Verbruggen 2005; Pong-
paradon & Prathep 2013; Pongparadon et al. 2015). In this
study, within the circular pattern two types were distin-
guished: irregular and regular depending on the primary utri-
cle arrangement. Additionally, we also found two more new
patterns: rounded polygonal when the utricles are loosening
attached, and rounded polygonal with a circular-center when
the attachment of utricles crowns is loose. Furthermore, the
number of utricle layers also differs among H. macroloba
populations. H. macroloba has three to five utricle layers in
previous reports (Barton 1901; Hillis-Colinvaux 1980; Ver-
bruggen & Kooistra 2004), and four to seven layers in this
study, with the maximum number of layers found in subtidal
populations. The nodal fusion pattern, all medullar siphons
adhering closely at nodes in a single unit (Barton 1901;
Hillis-Colinvaux 1980; Verbruggen & Kooistra 2004), is the
diagnostic character for H. macroloba and allows clear mor-
phological identification (Hillis-Colinvaux 1980; Kooistra
et al. 2002; Verbruggen & Kooistra 2004). Although all sam-
ples show this node pattern, with an adhesion belt with pores,
there was significant difference in node heights and pore
sizes. This study reports boarder nodal belt sizes
23.8–607.7 μm than previously (60–85 μm; Hillis-Colinvaux
1980). We concluded that the morphology of molecularly
identified H. macroloba is much more varied than previously
reported.
Morphological and anatomical characters of H. macroloba
varied with depth and light, as clearly shown in CCA. Thallus
size, segment size and number of utricle layers of
H. macroloba increased with increasing depth as well as
144
between subtidal versus intertidal plants. This leads to
increasing size and surface area of H. macroloba segments.
This is a common phenomenon observed in Halimeda. For
instance, H. tuna (J. Ellis & Solander) J.V.Lamouroux indivi-
duals with plants in deep water having broader segments than
in shallow water (Hillis-Colinvaux 1980; Vroom & Smith
2003; Pongparadon et al. 2015). These could be to capture
more light as suggested for H. opuntia (Linnaeus) J.-
V. Lamouroux (El-Manawy & Shafik 2008) and H. gigas W.-
R. Taylor, which also had larger segments in deeper water
(Pongparadon et al. 2015). Furthermore, H. macroloba also
showed differences in branching pattern when exposed to
increased wave action. In intertidal wave-exposed areas,
H. macroloba had smaller thalli and short branches while in
sheltered bays they had larger thalli and long branches. These
changes could be responses to these different environments.
In conclusion, H. macroloba in Thai waters has morpho-
anatomical variation broader than previously reported in sev-
eral characters, and environmental factors correlated with
morphological and anatomical characters, especially thallus
size, segment size and the number of utricle layers. In addi-
tion, genetic variation was low, in Thai H. macroloba
(Haplotype I) but the species showed high phenotypic plastic-
ity in Thailand. It is possible that H. macroloba is common
and dominant in many areas of the Indo-Pacific Ocean
because of its high plasticity and adaptation to different envi-
ronmental conditions. On the other hand, this high plasticity
also means that identification of H. macroloba may be diffi-
cult unless environment is also taken into consideration. Fur-
ther studies need to look at environmental conditions in
further detail, and transplantation or environment manipula-
tion will also be important to better understand
environmental-based morphological plasticity in
H. macroloba.
ACKNOWLEDGMENTS
This work was supported by the Higher Education Research
Promotion and National Research University Project of
Thailand, Office of the Higher Education Commission, a
scholarship for an overseas research study, PSU and Graduate
School, Prince of Songkla University, Thailand. We are appre-
ciative to Ms. Ratchanee Keawsrikaw for statistical analysis.
We also thank the Seaweed and Seagrass research unit of
Prince of Songkla University for help with field work and com-
ments throughout the study.
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