Phycologia

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A comparative study of Sargassum species from

the Yucatan peninsula coast: morphological and
chemical characterisation

Luis Alberto Rosado-Espinosa, Yolanda Freile-Pelegrín, Emanuel Hernández-

Nuñez & Daniel Robledo

To cite this article: Luis Alberto Rosado-Espinosa, Yolanda Freile-Pelegrín, Emanuel Hernández-
Nuñez & Daniel Robledo (2020): A comparative study of Sargassum species from the Yucatan
peninsula coast: morphological and chemical characterisation, Phycologia

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PHYCOLOGIA
https://doi.org/10.1080/00318884.2020.1738194

A comparative study of Sargassum species from the Yucatan peninsula coast:

morphological and chemical characterisation
LUIS ALBERTO ROSADO-ESPINOSA, YOLANDA FREILE-PELEGRÍN, EMANUEL HERNÁNDEZ-NUÑEZ, AND DANIEL ROBLEDO
Department of Marine Resources, CINVESTAV Unidad Mérida, Mérida, Yucatán, CP 97310, México

ABSTRACT ARTICLE HISTORY

Several species of Sargassum have been reported from the Yucatan peninsula coast, many of which Received 12 April 2019
end up as beach cast material, accumulating on the coast during colder months. The phenotypic Accepted 28 February 2020
plasticity of Sargassum makes specimen identification difficult. Two approaches were used in the Published online 30 April
2020
current study to corroborate identification of the most abundant species: traditional taxonomic
characters, and chemical characterisation. Fresh material from eight species of Sargassum was col- KEYWORDS
lected on the Yucatan coast. Specimens were identified using the following morphological features: Alginate; Chemotaxonomy;
cryptostomata, blade shape and size, and the shape of floating vesicles. Two cell wall polysaccharides, Fucoidan; Sargassum;
alginate and fucoidan, were extracted, quantified, and characterised from five of the eight species Ubiquitous compounds
collected. Organic extracts were prepared and GC–MS analyses were performed on the three most
abundant species, looking for species-specific compounds. Our analysis identified different chemical
constituents depending on the solvent used. In the dichloromethane fraction, hexanoic acid, 2-ethyl
was obtained from S. buxifolium whereas benzyl benzoate and epistephamiersine were found in
S. hystrix. For the latter species, cholesta-4,6-dien-3-ol, (á)- was found in the hexane fraction, whereas
2,2ʹ-ethyldenebis(4,6- di-tert-butylphenol) and 2-pentadecanone, 6,10,14-trimethyl- were present in
S. fluitans. We discuss the applicability and usefulness of this method to assist taxonomic identification
of Sargassum species.

INTRODUCTION observed, due to a response to climatic and environmental

The genus Sargassum C.Agardh has the highest morphological
complexity in the class Phaeophyceae and the greatest number
of species in the order Fucales (Phillips 1995), with 536
recognised species distributed in almost all ocean basins
(Guiry & Guiry 2018). Twelve species of Sargassum have
been reported on Yucatan coasts (Díaz-Martín & Espinoza-
Ávalos 2000), some of which come ashore as drift material
deposited on beaches during cold months (November to
February). The morphology of Sargassum species displays
great phenotypic plasticity that makes identification between
benthic and pelagic species challenging, since changes in
environmental conditions, and the presence of ambiguous
characters within the same individual, or even in different
growth stages, are common (Kilar et al. 1992).
Information regarding the species richness of this genus
in the Gulf of Mexico and the Atlantic is scarce or is limited
to the American (i.e. USA) coast of the Gulf of Mexico
(Phillips & Fredericq 2000). Shores of Yucatan Peninsula,
inside the Gulf of Mexico, and the Caribbean Sea share
several species (Littler & Littler 2000; Taylor 1960).
Attempts have been made to verify taxonomic identity
based on traditional morphological characters such as axis
development, blade shape, vesicles, and receptacles; however,
variability of these features between individuals of the same
species, or even within the same individual, has been
conditions (Abbot et al. 1988). Moreover, the use of
molecular markers such as ITS, psaA, chloroplast partial
RuBisCO operon, rbcL, COI, cox3, 23S-tRNA Val spacer,
and partial mt23S (Camacho et al. 2015; Draisma et al.
2010; Mattio et al. 2015; Phillips & Fredericq 2000; Stiger
et al. 2003) does not resolve the identity of Atlantic species,
suggesting the presence of Sargassum section Sargassum, but
not defining the species present (Camacho et al. 2015;
Mattio et al. 2015).
The chemical composition of seaweed species can be
used as a taxonomic tool to distinguish species according
to the type of metabolite or even phycocolloid structure.
Chemical composition has been evaluated for several spe-
cies of Sargassum in relation to amino acid composition,
polyunsaturated fatty acids, pigments, terpenes, and sterols
(Chen et al. 2016; Khotimchenko 1991; Liu et al. 2009;
Soon-Ae & Lee 1988). These results show specific com-
pounds or differential content among species. In addition,
specific variation in alginate content from Sargassum spe-
cies has been described (Table 1). However, these values
may vary depending on the season specimens are collected
(Camacho & Hernández-Carmona 2012; Davis et al. 2004;
Ragaza & Hurtado 1999). Therefore, it is important to
evaluate populations from the same season for chemotaxo-
nomic analysis.

CONTACT Daniel Robledo daniel.robledo@cinvestav.mx

Supplemental data for this article can be accessed on the publisher’s website.
© 2020 International Phycological Society
2 Phycologia

Table 1. Alginate yields extracted from representative Sargassum species in Kützing, S. natans (Linnaeus) Gaillon, S. ramifolium
different locations.
Kützing, and S. vulgare C.Agardh. A two-way ANOVA test
Yield (% was performed to analyse and compare the morphological
Species Location dw) Reference
characters among species, particularly the stable ones such as
S. aquifolium Indonesia 39.0 Setyawidati et al. (2018) blade width and blade length.
S. asperifolium Egypt 28.3 Larsen et al. (2003)
Biological material for taxonomic identification was stored
S. bacularia Malaysia 26.7 Chee et al. (2011)
in 4% formalin, and semi-permanent microscopic slides were
S. binderi Malaysia 38.7 Chee et al. (2011)
prepared from blades and vesicles using glycerol gelatin with
S. buxifolium Mexico 36.2 This report
solidified phenol. Specimens were deposited in the Alfredo
S. carpophyllum Philippines 32 Ragaza & Hurtado (1999)
Barrera Marin Herbarium (UADY).
S. conoides Malaysia 41.4 Chee et al. (2011)
S. cymosum Colombia 15.9 Camacho & Hernández-Carmona
Different analyses were conducted, based on amount of
(2012) biomass collected from identified species and the required
S. cymosum Brazil 28.8 Mafra. & Cunha (2004) biomass for each type of analysis. C:N ratio was determined
S. dentifolium Egypt 12.4 Larsen et al. (2003) in all specimens. Species with the highest collected biomass
S. filipendula Brazil 17.2 Betagnolli et al. (2014) were S. buxifolium, S. hystrix and S. fluitans, to which
S. filipendula Mexico 17.4 García-Ríos et al. (2012) chemical composition using GC–MS was evaluated.
S. filipendula México 44.2 This report Alginate and fucoidan content were analysed for the afore-
S. fluitans Cuba 24.5 Davis et al. (2004) mentioned species, along with S. filipendula and
S. fluitans Mexico 34.6 This report S. ramifolium.
S. hystrix Mexico 48.1 This report Alginate and fucoidans were extracted from dry samples
S. ilicifolium Philippines 23 Ragaza & Hurtado (1999) (48 h at 60 °C). Dried samples were ground in a commercial
S. latifolium Egypt 32.6 Larsen et al. (2003) mill and stored in labelled plastic bottles.
S. mangarevence Tahiti 9.3 Zubia et al. (2008) Fresh samples of collected material were drained and
S. muticum Morocco 25.6 Atouani et al. (2016)
blotted to remove excess water, placed in glass containers,
S. oligocystum Australia 20.5 Davis et al. (2004)
and frozen at –12° C for 2 days for chemical analysis. Frozen
S. ramifolium Mexico 43.5 This report
material was freeze-dried at 134 × 10−3 mbar, –44° C, using
S. siliquosum Philippines 41 Ragaza & Hurtado (1999)
a Labconco freeze-dry system/FreeZone® 4.5 (Kansas City,
S. siliquosum Malaysia 49.9 Chee et al. (2011)
Missouri, USA). Freeze-dried material was ground in
S. sp. Colombia 21.6 Camacho & Hernández-Carmona
(2012) a commercial mill and stored in labelled plastic bottles for
S. vulgare Brazil 16.9 Torres et al. (2007) later use.

The purpose of the present study was to describe differ-
ences among Sargassum species found in the Yucatan
Peninsula. To confirm the identity of the most abundant
species found as beach cast biomass of Sargassum on
Yucatan coasts, two approaches were used: traditional analysis
of morphological characters and chemical composition.
MATERIAL AND METHODS
Biological material, pre-treatment and sample
processing
Sargassum species were collected immediately after an algal
stranding event during a cold front in November 2014 on the
Yucatan coast. Based on their morphological features, indi-
viduals of the same species were separated and weighed for
further analysis. Morphological features described for
Sargassum, commonly used in identification keys, were
used to identify species (Dawes & Mathieson 2008; Taylor
1960). These included: (1) presence or absence of cryptosto-
mata, (2) presence or absence of spines on central axis, (3)
shape and arrangement of flotation vesicles, (4) presence or
absence of apical spine in float vesicles, and (5) shape of
blades. According to Taylor (1960) & Dawes and Mathieson
(2008), material collected belonged to Sargassum buxifolium
(Chauvin) M.J.Wynne, S. hystrix J.Agardh, S. filipendula C.
Agardh, S. fluitans (Børgesen) Børgesen, S. furcatum
Chemical analyses
CARBON NITROGEN RATIO
Carbon–nitrogen ratio was determined using 10 mg (dry
mass) of each species. Samples were placed in tin capsules
and analysed using the combustion method with an Auto
analyser Flash EA 1112 Series-NC (Thermo Fisher Scientific,
Milan, Italy). Samples were analysed in triplicate for each
species.
STRUCTURAL CARBOHYDRATES, FUCOIDAN AND
ALGINATES
Sargassum filipendula, S. ramifolium, S. buxifolium,
S. hystrix and S. fluitans were analysed in triplicate for
alginate determination using the modified technique by
Hernández-Carmona et al. (2002); and for fucoidan using
the protocols of Tako et al. (2000) and Foley et al. (2011).
First, dried samples were sieved to remove the finer frac-
tion containing calcium carbonate remains of epiphytic
organisms, and to avoid neutralisation during the extrac-
tion phase. Ground dried Sargassum samples (10 g) were
dissolved in 200 ml of 0.05 M HCl and agitated for 3 h at
room temperature; after which, the sample was pressure
filtered. The solid fraction was recovered to obtain raw
alginates, and the first liquid fraction was retrieved to
obtain raw fucoidans.
The solid fraction was placed in a 250-ml beaker, with
90 ml of 0.1% formaldehyde for 30 min. Next, the sample
 Rosado-Espinosa et al.: A comparative study of Sargassum species from the Yucatan Peninsula 3

Figs 1–10. Morphological characteristics of Sargassum species analysed.

Fig. 1. Sargassum buxifolium. Scale bar = 5 cm.
Fig. 2. Sargassum natans. Scale bar = 5 cm.
Fig. 3. Sargassum hystrix. Scale bar = 5 cm.
Fig. 4. Sargassum buxifolium. Blades and vesicles. Upper: Morphological detail of basal and middle blades of thallus. Bottom: Morphological detail of apical blades
with receptacles and flotation vesicles. Scale bar = 1 cm.
Fig. 5. Sargassum natans. Blades and vesicles. Morphological details characteristic of species, blades with absent cryptostomata, and flotation vesicles with apical
spine. Scale bar = 1 cm.
Fig. 6. Sargassum hystrix. Morphological details of blades and vesicles. From left to right: three blades with scattered cryptostomata followed by five apical blades
with spherical flotation vesicles and receptacles. Scale bar = 1 cm.
Fig. 7. Sargassum fluitans. Assorted blades and vesicles; detail of blades with pronounced midrib and absence of cryptostomata. Scale bar = 1 cm.
Fig. 8. Sargassum filipendula. Blades and vesicles. Morphological detail of lanceolate blades with cryptostomata aligned to sides of pronounced midrib. Scale
bar = 1 cm.
Fig. 9. Sargassum ramifolium. Blades and vesicles. Blade morphology showing forked blades at various levels with cryptostomata aligned to midrib. Scale
bar = 1 cm.
Fig. 10. Sargassum furcatum. Blades and vesicles. Detail of lanceolate and sparingly furcated blades of apical blades with abundant spherical flotation vesicles.
Scale bar = 1 cm.
4 Phycologia

Table 2. Morphological characters of Sargassum species accumulated on the coast of Yucatan. Features follow published descriptions for Sargassum in the literature
(Dawes & Mathieson 2008; Littler & Littler 2000; Mattio & Payri 2011; Taylor 1960).
Blade lengtha Blade widtha
Species Main axis Blades (cm) (cm) Air vesicles Cryptostomata
S. buxifolium Cylindrical; smooth; Oval to oblong; smooth to slightly 23.1–34.5 6.7–8.4 Spherical; devoid of Scattered;
with short foliose lateral serrated margins; dark midrib in mature 28.80 a 7.61 thorns; abundant prominent and
branches blades (± 5.70) (± 0.85) and scattered marked on sides of
margin
S. hystrix Cylindrical smooth; Oblong to elliptic; serrated on their 6.0–24.7 4.5–9.0 Spherical; smooth Scattered;
shaft-shaped branches margins; marked midribs 15.40 b 6.81 surface; numerous inconspicuous;
in whorls (± 9.31) (± 2.28) scarcely visible
S. filipendula Cylindrical; smooth; Linear; long and narrow; basal blades 33.2–50.5 3.2–5.1 Spherical; rarely Abundant; oval,
tough; slender; sometimes bifurcated; serrated margins; 41.89 c 4.20 apiculate; abundant arranged parallel to
sometimes a shaft; prominent midrib (± 8.63) (± 0.92) central midrib;
branches without present in vesicles
blades, i.e. naked and stems
S. fluitans Without dominant axis; Narrow to lanceolate; thick and firm; 30.4–49.0 6.7–9.2 Round to oval; Absent
cylindrical; smooth; golden serrated margin; clear and diffuse 39.74 c & d 8.04 smooth surface;
yellow; dense, sparsely midrib (± 9.32) (± 1.25) rarely pedicellate;
branched axis numerous at base of
blades
S. natans Without dominant axis; Acute, linear and firm; 12.2–30.4 1.9–3.4 Ovoid; with tiny tip Not visible
cylindrical; smooth; prominent serrated margin; 21.34 b 2.71 thorn or foliar hook
clear coloured axis; midribs prominent or diffuse (± 9.12) (± 0.78) at apex
secondary branched at
base
S. vulgare Cylindrical; dark; shaft Narrowly lanceolate; firm; dark; NS NS Rounded, located Numerous and
with tiny spines sharply serrated margin or sub-entire near axes; lack pigmented,
below; prominent distinctive midrib terminal spine randomly
distributed
S. furcatum Axes sparingly and Lanceolate–linear to linear; dark brown; 17.0–26.5 2.2–3.8 Completely One row aligned
minutely muriculate; bifurcated near distal part of blade; 21.81 b 3.02 spherical; small in with midrib
cylindrical dentate to smooth margin; midrib (± 4.74) (± 0.78) apical areas; occur in
distinctive to costate clusters
S. ramifolium Thin main axes Lanceolate and furcate near middle of 22.8–36.5 1.8–3.4 Completely One row aligned
cylindrical; very short; blade; furcated at second and third level 29.71 a 2.65 spherical; smaller in with margin
sparingly laterally in same blade; slightly serrated; (± 6.85) (± 0.79) apical than basal
branched distinctive costate midrib; very dark area; occur in
brown clusters
Range, mean, and standard deviation indicated (n ≥ 8). Different letters indicate statistical difference between blade lengths. NS, data not shown.

was filtered, 100 ml of distilled water was added, and pH
was adjusted to 4 using 0.5 M HCl. The mixture was stirred
for 15 min without vortex and filtered. The liquid fraction
was reserved to obtain the second fraction of fucoidans that
was mixed with the first fraction of fucoidans. The solid
fraction was mixed with distilled water (100 ml) while
stirring for 15 min, and filtered again to obtain the third
and last fraction for fucoidans, which was added to the
other two fractions. Distilled water (166 ml) was added to
the solid residue and the pH was raised to 10 by slowly
adding Na2CO3 while stirring. The mixture was kept for
2 h at 80 °C while stirring or until a paste was formed,
keeping the pH at 10, adding Na2CO3 when necessary. The
paste was diluted in water to a volume of 500 ml and
finally filtered. The viscous solution was poured into
500 ml of 100% ethanol and stirred with a glass rod.
Alginate was collected with the rod while stirring. Excess
alcohol was squeezed and the fibres dried at temperature
below 60 °C.
The three filtered fractions obtained were adjusted to pH
7 using NaOH 0.5 M. Once neutralised, 500 ml of 100%
ethanol was added while stirring slowly. The resulting liquid
was concentrated using a rotary evaporator (40 °C at
50 rpm) to a volume no larger than 50 ml (between 20 and
50 ml depending on the species). The concentrate was dia-
lysed in sealed membranes with one litre of distilled water
for 24 h. Subsequently, the concentrate was frozen and
lyophilised until the sample was completely dehydrated.
Phycocolloid yields of samples were calculated with the
following formula:
Yield (%) = total dry weight phycocolloid (g)/dry weight of
initial sample of algae (g) * 100.
The FT-IR spectra of alginates were recorded on
a PerkinElmer Frontier FT-IR spectrometer (Norwalk,
Connecticut, USA), at room temperature. Alginate films
were obtained by evaporation of an aqueous solution of
alginate extracts (0.2% w/v) and measured in the infrared
region between 5000 and 456 cm−1. A commercial Sigma
Alginate (Sigma-Aldrich, St. Louis, Missouri, USA) was used
for comparison.
The FT-IR spectra of fucoidans were measured using dried
fucoidan samples (5 mg) macerated in 195 mg of anhydrous
KBr using a mortar. IR spectra were recorded with a Thermo
Nicolet NEXUS 670FT-IR Spectrometer (East Lyme,
Connecticut, USA) at room temperature and measured in
 Rosado-Espinosa et al.: A comparative study of Sargassum species from the Yucatan Peninsula
Figs 11-12. Polysaccharides of Sargassum fluitans, S. hystrix, S. buxifolium,
S. ramifolium, and S. filipendula; letters indicate significant differences between
species.
Fig. 11. Alginate yield (% dry weight). Data analysed using one-way analysis
of variance (ANOVA) followed by Tukey’s test at 5% level (P < 0.05).
Fig. 12. Fucoidan yield (% dry weight). Data analysed using one-way analysis
of variance (ANOVA) followed by Tukey’s test at 5% level (P < 0.05) to
evaluate differences between samples.
the infrared region between 4000 and 400 cm−1. A total of 64
scans were averaged for each sample at 4 cm−1 resolution and
referenced against air.
GAS CHROMATOGRAPHY-MASS SPECTROMETRY
ANALYSIS
Seaweeds stored at –20 °C were milled using a commercial
blender. Subsequently, milled algae (10 g) was extracted using
dichloromethane: methanol (1:1; v/v) for 24 h, simultaneously
with maceration assisted by mechanical agitation at room
temperature. Supernatants were filtered and vacuum-
evaporated using a rotary evaporator to obtain a dry extract.
The extract was fractioned into three parts using hexane (no
polar compounds), dichloromethane (low polar compounds),
and acetone (moderate polar compounds) fractions on a silica
5
gel column (30 cm x 2.5 cm, 60 Å). Fractions were evaporated
until dry. Resulting samples were used for gas chromatogra-
phy and mass spectrophotometry to evaluate the compounds
in each species. Compound analysis was performed using
a TSQ Quantum XLS Ultra GC–MS System (Thermo
Scientific, Waltham, Massachusetts, USA) and controlled by
XcaliburTM software (Waltham, Massachusetts, USA).
Compounds were separated using an HP-5MS capillary col-
umn (30 m x 0.35 mm, i.d., 0.25 µm film thickness) (J&W
Scientific, Folson, California, USA). Mass range of GC–MS
was set between 50 and 500. The temperature programme was
set at 180 ºC for 1 min with an increase of 10 °C min−1 to 200
°C followed by 3 °C min−1 to 300 °C and hold for 10 min.
Injector temperature and transfer line were set at 250 °C and
280 °C, respectively. To identify compounds, mass spectra of
samples were compared with those of the NIST/EPA/NIH
Mass Spectral Library 2.0. Using these data, a principal com-
ponent analysis (PCA) was performed using The Unscrambler
X.10.3, to relate the retention time of the chemical com-
pounds in each species to each solvent. PCA analysis used
variable ‘species’ (S. buxifolium, S. fluitans, S. hystrix) against
the variable ‘solvent’ (hexane, dichloromethane and acetone)
to generate the corresponding dispersion diagrams.
RESULTS
Taxonomic identification
Eight species of Sargassum were identified according to com-
mon taxonomical characters (Figs 1–10, Table 2): pelagic
species identified were S. fluitans and S. natans; benthic spe-
cies with oblong laminae were S. buxifolium and S. hystrix;
benthic species with forked blades were S. ramifolium and
S. furcatum; and benthic species with elongated blades were
S. filipendula and S. vulgare, the latter for which only a small
piece of a damaged stem was found.
The two-way ANOVA performed on the morphological
characters did not show differences in blade width; however,
variation in blade length was found among species (Table 2).
The ANOVA performed using blade length–width ratio
showed the same differences among the previously described
individuals.
Chemical analysis
CARBON TO NITROGEN RATIO
C to N ratios were obtained for all species identified. Species
with the highest values were S. buxifolium, S. hystrix and
S. natans with ratios (mean ± s) of 11.8 ± 0.7, 11.4 ± 0.9, and
10.7 ± 0.8, respectively, while the lowest ratios corresponded
to S. vulgare (8.0 ± 0.5), S. furcatum (8.8 ± 0.8), S. filipendula
(8.9 ± 0.4), S. ramifolium (9.1 ± 0.5) and S. fluitans
(9.2 ± 0.5) with values below 10.
ALGINATES AND FUCOIDANS
Alginate extraction was performed in three phases, recover-
ing approximately 65% during the first extraction, 25% in
the second, and 10% in the final extraction. This was done
for all species except S. filipendula, which required only two
6 Phycologia

Figs 15-17. Bi-plot representation obtained from PCA analysis of the data matrix of compounds obtained after GC–MS (Table S3) indicating separation of exclusive
compounds found in Sargassum buxifolium, S. fluitans, and S. hystrix for the organic solvents used.
Fig. 15. Acetone extract.
Fig. 16. Dichloromethane extract.
Fig. 17. Hexane extract.

extractions to recover total alginate. Species with the highest
alginate yields were S. hystrix (48.1% ± 2.5%), followed by
S. filipendula (44.2% ± 6.2%), S. ramifolium (43.5% ± 2.1%),
and S. buxifolium (36.2% ± 0.8%). S. fluitans (34.6% ± 2.8%)
had the lowest yield (Fig. 11).
Crude fucoidan yields extracted from the liquid residues in
the alginate extractions are shown in Fig. 12. Highest fucoidan
yield was in S. fluitans (4.4% ± 0.48%) followed by
S. filipendula (3.2% ± 0.45%), S. buxifolium (2.7% ± 0.30%),
and S. ramifolium (2.4% ± 0.34%). S. hystrix (2.1% ± 0.11%)
had the lowest yield.
FT-IR SPECTRA FOR ALGINATES AND FUCOIDANS
Infrared spectra obtained for both alginates and fucoidans
from Sargassum species are shown in Figs 13, 14, respectively.
Characteristic alginate peaks were observed at 3429, 1630,
1428 and 1030 cm−1 wavelength, when compared with the
commercial Sigma sample. Peaks at 1030 and 1125 cm−1 were
 Rosado-Espinosa et al.: A comparative study of Sargassum species from the Yucatan Peninsula 7

Fig. 13. Infrared spectra of the alginates obtained from Sargassum buxifolium, S. filipendula, S. fluitans, S. hystrix, and S. ramifolium. Alginate (Alg.) from Sigma was
used as control.

Fig. 14. Infrared spectra of fucoidans obtained from Sargassum buxifolium, S. filipendula, S. fluitans, S. hystrix, and S. ramifolium.
8 Phycologia

Table 3. Exclusive chemical compounds found on Sargassum species analysed by GC–MS in this study.
Species Solvent Name Apex RT Start RT End RT Area % Area Height % Height Probability
S. buxifolium Dicloromethane Hexanoic acid, 2-ethyl- 9.52 9.4 9.5 42827470 0.06 14567265 0.12 87.0
(C8H16O2)
S. hystrix Hexane Cholesta-4,6-dien-3-ol, 34.75 34.7 34.8 77517424 0.26 24418964 0.34 93.0
(á)- (C27H44O)
S. hystrix Dicloromethane Benzyl Benzoate (BnBzO) 21.47 21.4 21.5 49619884 0.08 19125980 0.16 92.1
S. hystrix Dicloromethane Epistephamiersine 22.35 22.3 22.4 57567704 0.09 19965094 0.17 80.9
(C21 H27NO6)
S. fluitans Hexane 2-Pentadecanone, 22.14 22.1 22.2 109643588 0.24 34740850 0.29 89.1
6,10,14-trimethyl-
(C18H36O)
S. fluitans Hexane 2,2ʹ-Ethyldenebis(4,6-di- 29.86 29.8 29.9 23121236 0.05 7657151 0.06 92.6
tert-butylphenol)
CH3CH{C6H2[C(CH3)3]2
OH}2
RT, retention time in minutes; Area and Height in digital units.

used to obtain the M:G ratio. Sargassum hystrix showed the
highest M:G value (62.3; Table S1).
The FT-IR spectra of fucoidans analysed have close struc-
tural similarity in all Sargassum species; however,
S. buxifolium and S. fluitans presented signals in the
2852 cm−1 band, most likely related to a weak signal for C:
H. In the other species, these signals were absent (Table S2).
GAS CHROMATOGRAPHY – MASS SPECTROMETRY
ANALYSIS
Results showed ubiquitous and unique compounds in the three
Sargassum species: S. buxifolium, S. hystrix, and S. fluitans. By
ordering retention times, peaks of most compounds found were
probed with probabilities of less than 80% certainty of the
molecules found in the digital library of the SigmaPlot Macro
Library. For these species, ubiquitous compounds were found,
including stigmasta-5,24-dien-3-ol (fucosterol), ϛ tocopherol,
and cholesterol (Table S3). Six unique compounds were identi-
fied from S. buxifolium, S. hystrix and S. fluitans (Table 3). In the
dichloromethane fraction, 2-ethylhexanoic acid was obtained
from S. buxifolium, whereas, benzyl benzoate and epistepha-
miersine were found in S. hystrix. In the hexane fraction cho-
lesta-4,6-dien-3-ol, (á) – was obtained from S. hystrix, whereas
2,2ʹ-ethylidenebis (4,6-di-tert-butylphenol) and 2-pentadeca-
none, 6,10,14-trimethyl- were found in S. fluitans.
Principal component analysis (PCA) showed a separation
of species compounds extracted with a specific solvent.
Species separation was obtained by chemical composition
using retention times of the compounds found in each solvent
(Figs 15–17). Cluster analysis performed independently for
each solvent resolved 100% of the variability: 55% in the
first axis (PC1) and 45% in the second axis (PC2). Retention
time values for chemical compounds present in each species
can be observed in the scatter diagram (Supplementary infor-
mation on PCA analysis).
DISCUSSION
Sargassum species from Yucatan coasts were separated by
the main morphological characters described as taxonomic
characters for each species. Similarities between species
were also observed. For example, similarities in blade
shapes were observed in S. filipendula and S. vulgare,
S. ramifolium and S. furcatum, and S. hystrix and
S. buxifolium; however, there were one or several other
characters that allowed us to distinguish and delimit spe-
cies. Thus, the presence of bifurcating basal blades in
S. filipendula was absent in S. vulgare. The later also had
small spines on the main axis as well as abundant scattered
and highly pigmented cryptostomata. Differences between
S. ramifolium and S. furcatum were mainly in the abun-
dance of secondary axes with high blade density, the abun-
dance of bifurcated blades in the thallus of S. ramifolium, as
well as the level of bifurcation, with more abundant sec-
ondary branches as well as thinner blades; unlike
S. furcatum which had higher blade width and less common
first level blade bifurcations (Moreira & Cabrera 2005).
Differences observed between S. hystrix and S. buxifolium
were the presence of blade undulations and abundant clus-
ters of flotation vesicles in the former. In relation to pelagic
species, several differences were observed between
S. fluitans and S. natans; whereas the only similarity was
the absence of cryptostomata in blades.
All species were collected on the same date and in the
same area; however, fucoidan and crude alginate content
show significant differences among species. Ragaza and
Hurtado (1999), Davis et al. (2004), and Camacho and
Hernández-Carmona (2012) reported seasonal variation in
alginates of different Sargassum species. Alginate is
a structural carbohydrate which allows thalli to be flexible,
giving them necessary elasticity to resist seasonal weather
changes in currents, and making the algae more resistant to
surf. In this regard, we found higher alginate content in
benthic species than in pelagic species; in contrast, pelagic
species had higher levels of fucoidans. Biological function of
fucoidans in brown algae is mainly related to zygote adhe-
sion (Goodner & Quatrano 1993; Kropf et al. 1988;
McCandless 1981); however, these species reproduce by frag-
mentation, although Moreira & Suárez (2002) reported sex-
ual reproduction in these species in Cuba. Nevertheless,
a possible reason for the high fucoidan content in
S. fluitans may be related to the affinity of ester-sulphate
 Rosado-Espinosa et al.: A comparative study of Sargassum species from the Yucatan Peninsula
groups with magnesium ions (Percival 1979), which are
highly hydrated, causing fronds of pelagic Sargassum to
retain water and therefore avoid desiccation. A different
proportion of mannuronic acid (M) to guluronic acid (G)
in Sargassum species based on infrared spectra area (A)
A1125/A1030 is shown in Table S1.
M:G ratio can be used as an indicator of some rheological
properties of alginates. Şen et al. (2010) showed that at
a higher M:G ratio, the viscosity of alginate could have
a pseudo-plastic, fluid behaviour; while at a lower ratio, it
has a Newtonian behaviour. Şen (2011) showed that free-
radical elimination properties of the alginate increase at
higher G:M ratio (e.g. 70:30).
According to Fenoradosoa et al. (2010) and Leal et al.
(2008), the band at 3296 cm−1 corresponds to the
O-H stretching vibration of carbohydrates in alginates.
The bands at 2919 and 2852 cm−1 correspond to C-H,
indicating that the presence of CH2 and CH3 (1601 cm−1
was responsible for the C-6 group of fucose, galactose or
the C-H stretching pyranoid ring and OH flexural vibra-
tions, respectively, which was characterised by polysacchar-
ide absorption). The band at 1421 cm−1 indicated the
presence of organic sulphates. The band observed at
1247 cm−1 corresponded to C-O-C and indicated the pre-
sence of carbohydrate esters. In addition, the band
1601 cm−1 also indicates the COO of uronic acid. The
absorption band at 833 cm−1 reflects C=C-H and is based
on the presence of an α-type glycoside bond.
Characterisation of differences between Sargassum species
based on chemical composition was obtained by GC–MS
analysis. Unique or exclusive compounds were found for
the most abundant species in the hexane and dichloro-
methane fractions. Sargassum buxifolium was previously
considered a variety of S. hystrix until Wynne (2011) sepa-
rated them, based mainly on differences in reproduction.
The presence of unique compounds highlights these differ-
ences at chemical level. The affinity of the compounds for
each solvent indicates their nature; for S. buxifolium and
S. hystrix the compounds were found mainly in low-
polarity solvents, while in S. fluitans the ubiquitous com-
pounds were found in a non-polar solvent, separating each
of the species analysed.
Most chemotaxonomic studies are used to delimit algae at
family level, or for larger taxa, correlating groups of specific
compounds as markers (i.e. fatty acids) for various processes
including the origin and endosymbiosis relationships of
organisms (e.g. Kumari et al. 2013). However, there are few
studies using chemotaxonomy to identify unique compounds
for the delimitation of species. Le Lann et al. (2008) used
high-resolution, magic angle spinning nuclear magnetic reso-
nance (HR-MAS NMR) to discriminate species of Turbinaria
J.V.Lamouroux, in two regions of the Pacific Ocean. These
authors differentiated T. conoides and T. ornata, which are
indistinguishable based on morphology, using the aforemen-
tioned technique of grouping them by PCA according to their
composition, similar to the results shown in Figs 15–17 of this
study. Jégou et al. (2010) used two techniques: HR-MAS NMR
and LC/ESI-MSn (the latter homologous to GC–MS used in
the present work) to distinguish species of Cystoseira C.
9
Agardh collected on French shores of the Atlantic Ocean,
which presented up to three synonyms due to ambiguous mor-
phological characters. The intention of both studies was to
distinguish the algae at species rank using different techniques,
and not to look for differential compounds in each species of
Turbinaria or Cystoseira.
Sargassum species in the present study had some ubiqui-
tous compounds such as stigmasta-5,24-dien-3-ol (fucosterol),
ϛ tocopherol, and cholesterol; however, differential composi-
tion was found using GC–MS analysis, especially in hexane
and dichloromethane fractions. Sargassum buxifolium pre-
sented a fatty acid, hexanoic acid, 2-ethyl-, that was not
present in the other two species. Moody & Reddy (1978)
reported that consumption of this fatty acid has toxic effects.
Three unique compounds were found in S. hystrix: (1) benzyl
benzoate, an aromatic carboxylic acid found naturally in
plants, and formerly isolated from the bark of several trees
belonging to the genus Styrax (Valdez-Sepúlveda et al. 2015);
this compound is widely used as a miticide drug, being lethal
to Sarcoptes scabiei De Geer and therefore useful in the treat-
ment of scabies (WHO 2013); (2) epistephamiersine, an iso-
late compound from several types of plants, and considered to
be an alkaloid; and (3) a cholesterol derivative and
a differential lipid, 2-pentadecanone, 6,10,14-trimethyl-,
which was also found in S. fluitans. This polymer was origin-
ally isolated from Scolopendra subspinipes Leach (Yoon et al.
2006), and has antioxidant and radiation protecting capacity,
and is widely used in the industry as a polymer in plastic
production. Consequently, it is not possible to determine
whether this compound is produced by the algae or it is
absorbed by thalli during their passing through coastal waters.
Knowing the unique chemical compounds in each species
could prove to be a useful tool in taxonomic studies. It could
also provide an alternative to genetic studies when these
cannot provide accuracy at species level, and also supplement
traditional morphological and genetic studies. However, com-
plementary research is needed for the same species from other
geographic areas and different seasons to confirm the pre-
sence of the compounds reported here, to verify their useful-
ness as chemotaxonomic markers.
ACKNOWLEDGEMENTS
E. Caamal Fuentes and C. Chávez-Quintal provided skillful technical
assistance. Comments on Sargassum taxonomical characters by Dra.
E.F. Vázquez-Delfín are greatly acknowledged.
FUNDING
PhD scholarship Conacyt (334360) to L.A. Rosado-Espinosa and finan-
cial support from CONACYT PN-2015-01-575.
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