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Phytochemistry 86 (2013) 4456
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Fatty acid proling of tropical marine macroalgae: An analysis

from chemotaxonomic and nutritional perspectives
Puja Kumari, A.J. Bijo, Vaibhav A. Mantri, C.R.K. Reddy , Bhavanath Jha
Discipline of Marine Biotechnology and Ecology, CSIR Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat 364002, India
a r t i c l e
i n f o
Article history:
Received 4 May 2012
Received in revised form 18 October 2012
Available online 17 November 2012
Keywords:
Macroalgae
Lipids
Fatty acids
PUFAs
n6/n3 Ratio
Chemotaxonomy
Endosymbiosis
a b s t r a c t
The lipid and fatty acid (FA) compositions for 100 marine macroalgae were determined and discussed
from the context of chemotaxonomic and nutritional perspectives. In general, the lipid contents in macroalgae were low (2.320 mg/g fr. wt.) but with substantially high amounts of nutritionally important
polyunsaturated fatty acids (PUFAs) such as LA, ALA, STA, AA, EPA and DHA, that ranged from 10% to
70% of TFAs. More than 90% of the species showed nutritionally benecial n6/n3 ratio (0.1:13.6:1)
(p 6 0.001). A closer look at the FA data revealed characteristic chemotaxonomic features with C18 PUFAs
(LA, ALA and STA) being higher in Chlorophyta, C20 PUFAs (AA and EPA) in Rhodophyta while Phaeophyta
depicted evenly distribution of C18 and C20 PUFAs. The ability of macroalgae to produce long-chain
PUFAs could be attributed to the coupling of chloroplastic FA desaturase enzyme system from a photosynthetic endosymbiont to the FA desaturase/elongase enzyme system of a non-photosynthetic eukaryotic protist host. Further, the principal component analysis segregated the three macroalgal groups with a
marked distinction of different genera, families and orders, Hierarchical cluster analyses substantiated
the phylogenetic relationships of all orders investigated except for those red algal taxa belonging to Gigartinales, Ceramiales, Halymeniales and Rhodymeniales for which increased sampling effort is required
to infer a conclusion. Also, the groups deduced from FA compositions were congruent with the clades
inferred from nuclear and plastid genome sequences. This study further indicates that FA signatures
could be employed as a valid chemotaxonomic tool to differentiate macroalgae at higher taxonomic levels such as family and orders.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Benthic marine macroalgae, commonly known as seaweeds are
multicellular photosynthetic organisms with considerable potentials for using as a source of bioactive compounds of immense
pharmaceutical and nutraceutical importance. They are rich
sources of nutritionally benecial components such as proteins,
carbohydrates, polyunsaturated fatty acids (PUFAs), antioxidants,
minerals, dietary bers and vitamins (Chandini et al., 2008;
Mohamed et al., 2011) and are thus consumed as functional foods.
There are 250 macroalgal species commercially utilized worldwide, of which 150 are consumed as human food (Barrow, 2007).
The macroalgal species, in general, are low in lipids and contain
15% on dry wt. basis. Nevertheless, the nutritionally important
C18 and C20 PUFAs including n3 PUFAs are present in substantially
high amounts with anti-inammatory, anti-thrombotic and antiarrhythmic responses (Kumari et al., 2010; Gillies et al., 2011).
The n-3 PUFAs are of particular importance as they cannot be
Corresponding author. Tel.: +91 278 256 5801x614; fax: +91 278 2567562/
2566970.
E-mail address: crk@csmcri.org (C.R.K. Reddy).
0031-9422/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.10.015
synthesized by humans and are thus obtained only through dietary

sources.
Fatty acids (FAs) being metabolites of conserved acetyl-CoA
pathway have been extensively studied from the context of chemotaxonomic perspectives in higher plants (Mongrand et al.,
2001, 2005; Dussert et al., 2008), cyanobacteria (Shukla et al.,
2011), bacteria (Malviya et al., 2011; Nez-Cardona, 2012), microalgae (Dunstan et al., 2005; Lang et al., 2011) and fungi (Mishra
et al., 2010). Further, Dunstan et al. (2005) deciphered the evolutionary relationship between the FA composition of Rhodophyceaen and Cryptophyceaen microalgae and the endosymbiotic
theory. According to the endosymbiotic theory, the micro- and
macroalgae of both Chlorophyceae and Rhodophyceae have been
originated from primary endosymbiosis of photosynthetic cyanobacteria and eukaryotic host while Phaeophyta diverged from red
algae via secondary (or tertiary) endosymbiosis along with other
chlorophyll c bearing algae such as Cryptophytes, Haptophytes,
diatoms, Dinoagellates and non-photosynthetic Apicomplexans,
ciliates and oomycetes, forming the super-group Stramenopiles
at the base of the tree of life (Baldauf et al., 2000; Baldauf, 2008;
Archibald, 2009, 2012; Dorrell and Smith, 2011; Baurain et al.,
2012; De Clerck et al., 2012; Green, 2010; Woehle et al., 2012).
P. Kumari et al. / Phytochemistry 86 (2013) 4456
But, there is no consensus among the researchers regarding the

number of secondary endosymbiosis and whether the endosymbiotic event involved different red algae and/or different heterotrophic hosts including the number of plastid losses in algae
containing chlorophyll c (Green, 2010; Archibald, 2012; Baurain
et al., 2012; De Clerck et al., 2012). Further, these endosymbiont
cyanobacteria that exhibited the capability of synthesizing C18
n-3 and n-6 PUFAs, nally resulted into double membrane chloroplasts in green and red algae, the main sites of FA biosynthesis in
photosynthetic eukaryotes (Sperling et al., 2003). Similarly, the
red algal endosymbiont formed 34 membrane bound plastids in
stramenopiles. Numerous studies and available EST databases of
algae have revealed that the evolution of green, red and brown algae has undergone multiple endosymbiotic and horizontal gene
transfer of plastidial and nuclear genomes, gene duplication and
losses, including those of FA metabolism (Domergue et al., 2003;
Ryall et al., 2003; Bowler et al., 2008; John et al., 2008; Le Corguill
et al., 2009; Michel et al., 2010; Coelho et al., 2012; Chan et al.,
2012; Chen and Smith, 2012; Cock et al., 2010; Dittami et al.,
2012). Further, it is well known that de novo FA biosynthesis takes
place in chloroplast up to C16:0/C18:0/C18:1, after which these
FAs are attached to phosphatidylcholine (PC). They are then either
desaturated and utilized for the synthesis of glycerolipids of chloroplast membrane or transported to ER for further chain elongation
and desaturation and thereafter utilized for the synthesis of membrane lipids, storage lipids or transported back to the chloroplast
for membrane biosynthesis (Ohlrogg and Browse, 1995; Uttaro,
2006; Joyard et al., 2010; Huerlimann and Heimann, 2012). As cyanobacteria can produce FAs only up to C18 (Liu et al., 2010; Iliev
et al., 2011), Dunstan et al. (2005) hypothesized that Rhodophytes
inherited the capability of synthesizing C20 PUFAs from the coupling of elongase and desaturase enzyme system of the eukaryotic
host with the photosynthetic n-3 C18 PUFA-producing prokaryote
(cyanobacteria) during the primary endosymbiosis. Similarly, the
secondary endosymbiosis of the photosynthetic n-3 C20 PUFAproducing eukaryote and the eukaryotic host capable of further
chain elongation and desaturation resulted in the de novo production of C22 PUFAs along with C20 PUFAs in Cryptophytes. However,
a few studies are available where FA composition of macroalgae
has been investigated in correlation to the endosymbiotic history.
Understanding the FA proles in the light of endosymbiotic evolution of different lineages of macroalgae could be an important
prerogative for gaining insights into the algal lipid metabolism.
Furthermore, FA compositions of numerous macroalgae have
been reported world-wide for their nutritional potential but their
chemotaxonomic implications gained importance only in the last
decade. Recently, Galloway et al. (2012) reported FA signatures
of 40 temperate macrophytes (both seaweeds and sea grasses)
from San Juan Archipelago, USA. But the diversity of macroalgal
species investigated for FA composition is still low, approximated
to be <200 (Gosch et al., 2012) which is minuscule against total
estimated species of 9255, to date (Guiry and Guiry, 2012).
In our previous study, the FA proles of 27 macroalgal species
have been reported from Gujarat coast, India (Kumari et al.,
2010) but the study was limited to the understanding of chemotaxonomic relationships among few commercially important macroalgae. In the present study, we analyzed total lipid (TL) and FA
compositions of 100 macroalgae belonging to 46 different genera,
30 families and 18 orders encompassing samples from northern
and central west coast of India. An attempt was made to understand their chemotaxonomic relationships at different taxonomic
levels using multivariate principal component and hierarchical
cluster analysis and compared with the available phylogenetic
data. Furthermore, the results were also correlated with the endosymbiotic origin of three extant macroalgal lineages besides mapping the algal species rich in nutritionally essential n-3 PUFAs that
45
could possibly be cultivated for utilizing in various food and nutraceutical formulations.
2. Results and discussion
2.1. Lipids
The total lipid (TL) contents investigated in this study varied
signicantly (p 6 0.001) among different algal species (Table 1).
The brown algae showed the highest TL contents (5.720.1 mg/g
fr. wt.), followed by green algae (3.120 mg/g fr. wt.) and red algae
(2.312.2 mg/g fr. wt.). The Dictyotales and Bryopsidales (except
Caulerpa microphysa, Caulerpa racemosa v. occidentalis) had higher
TL contents that corroborate with the recent ndings of Gosch
et al. (2012). Further, Ulva spp. (except Ulva erecta), Monostroma
oxyspermum and Sargassum cinereum also showed high contents
of TL (1020 mg/g fr. wt.) in the present study. The variations observed between different species of the same genus was more
likely to be due to the inter-specic/intra-generic variations rather
than the geographical and environmental conditions as apparent
from the minor variations found in the environmental parameters
for the studied collection sites. Also, there was no trend observed
between TL contents of macroalgal species belonging to the same
genus and collection sites. Further, the lipid contents of the present
study were lower than those reported for the same or related species (such as Acrosiphonia sp., Cystoseira sp., Laurencia sp., Polysiphonia sp. and Scytosiphon sp.) from Caspian Sea (Dembitsky
et al., 1993), Sea of Japan (Khotimchenko, 1998), Bohai Sea (Li
et al., 2002) and Seribu Island, Indonesia (Santoso et al., 2006).
However, the lipid contents of C. racemosa, Caulerpa sertularioides,
Cladophoropsis javanica, S. cinereum, Padina spp. and Ulva spp. reported either similar or 1.22.0-fold higher values with an exception of U. erecta that showed equal content to the sibling species
of Ulva intestinalis studied from Bohai Sea (Li et al., 2002). These
variations could be attributed to the interplay of inter- or intraspecic variations along with the spatiotemporal variations in
environmental parameters across the world (Chandini et al., 2008).
Moreover, it is evident from this study that macroalgae contained lipids as high as 2% on fresh weight basis that diminishes
their prospects for biodiesel production. On the contrary, microalgae especially Chlorella, Botryococcus, Chaetoceros and Phaeodactylum are promising sources of biodiesel as they contain lipids
more than 40% on dry weight basis (Becker, 2007; Ryckebosch
et al., 2012; Yang et al., 2012). However, recently Gosch et al.
(2012) studied the macroalgae of genus Dictyota, Spatoglossum,
Derbesia and Caulerpa for lipids and reported a range from 10% to
12% on dry wt. basis that is quite comparable with those reported
for several microalgal species such as Tetraselmis, Rhodomonas,
Scendesmus and a few strains of Skeletonema and Isochrysis (Huerlimann et al., 2010; Mata et al., 2010), and thus emphasized the
need for considering macroalgae as a promising resource for production of oil-based bioproducts.
2.2. Fatty acid composition
2.2.1. Chlorophyta
The FA compositions of 33 species belonging to the orders of
Ulvales, Ulotrichales, Bryopsidales, Siphonocladales and Cladophorales are presented in Table 2 and Supplementary Table 1. The green
algal samples showed higher contents of unsaturated fatty acids
(UFAs) with the exception of Ulva lactuca, C. racemosa v. occidentalis,
C. racemosa v. corneyphora, Codium dwarkense, Bryopsis plumosa and
M. oxyspermum which had 1.11.4-fold higher contents of saturated
fatty acids (SFAs) (p 6 0.001). The PUFA contents in them ranged
between 28% (U. lactuca) and 71% (Caulerpa veravalensis) of total
46
Table 1
Total lipid content of macroalgal samples (expressed as means S.D., n = 3). The lipid
values for different algae were signicant at p 6 0.001.
S.No.
1
2
3
4
5
6
7
8
9
10
11
12
Species
TL (mg/g fr. wt.)
Chlorophyta
Ulvales
Ulva lactuca
Ulva fasciata
Ulva taeniata
Ulva pertusa
Ulva reticulata
Ulva beytensis
Ulva compressa
Ulva rigida
Ulva linza
Ulva exuosa
Ulva erecta
Ulva prolifera
15.7 0.8
13.7 0.8
16 1.0
14 4.0
10 2.0
20 2.0
9.2 1.0
16.3 1.4
16 0.8
13.8 1.3
7 0.2
14.7 0.2
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Bryopsidales
Caulerpa scalpelliformis
Caulerpa veravalensis
Caulerpa racemosa
Caulerpa racemosa v. corynephora
Caulerpa racemosa v. occidentalis
Caulerpa microphysa
Caulerpa verticillata
Caulerpa sertularioides
Codium dwarkense
Bryopsis pennata
Bryopsis plumosa
Trichosolen mucronatus
Udotea indica
Halimeda discoides
Halimeda tuna
28
Ulotrichales
Monostroma oxyspermum
17.3 1.2
29
30
31
Siphonocladales
Chamaedoris auriculata
Cladophoropsis javanica
Valoniopsis pachynema
8 2.0
10.6 1.3
6.1 0.4
32
33
Cladophorales
Chaetomorpha linum
Acrosiphonia orientalis
4.8 0.2
10.8 2.2
13.3 1.5
16 0.3
15 0.8
10.3 2.1
5.5 1.1
4.7 1.3
15.3 1.3
9.1 2.2
14 2.2
9.6 1.7
11.3 1.5
5.4 1.1
12 4.7
3.1 0.4
10 1.7
Phaeophyta
34
35
36
37
38
39
40
41
42
43
44
45
Dictyotales
Padina tetrastomatica
Padina gymnospora
Dictyopteris deliculata
Dictyota pinnatida
Dictyota bartayresiana
Dictyota dichotoma
Dictyota cervicornis
Dictyota ciliolata
Dictyota haukiana
Stoechospermum marginatum
Lobophora variegata
Spatoglossum asperum
46
47
48
49
50
51
52
53
54
55
Fucales
Sargassum tenerrimum
Sargassum johnstonii
Sargassum sp.
Sargassum carpophyllum
Sargassum plagiophyllum
Sargassum cinereum
Sargassum cinctum
Hormophysa cuneiformis
Cystoseira indica
Cystoseira trinodis
56
57
Ectocarpales
Hincksia mitchelliae
Scytosiphon lomentaria
58
59
Rhodophyta
Gracilariales
Gracilaria dura
Gracilaria salicornia
13.3 2.1
17.7 0.6
9.3 0.6
13.3 1.2
10 2.2
10.3 1.0
7.3 0.1
13.4 0.9
8.4 2.1
11.5 1.1
15 1.6
13.8 0.7
7.2 1.1
7.3 1.2
5.7 1.5
6.3 0.6
7.6 2.6
20 1.0
9.8 1.3
11 3.0
6.1 0.4
6.7 2.5
12.4 1.1
6.3 0.2
6.3 0.3
7.6 0.5
Table 1 (continued)
S.No.
60
61
62
63
64
65
Species
Gracilaria
Gracilaria
Gracilaria
Gracilaria
Gracilaria
Gracilaria
TL (mg/g fr. wt.)

textorii
corticata
corticata v. cylindrica
corticata v. folifera
debilis
verrucosa
7.3 1.2
8 2.0
5.2 1.1
5.7 0.6
2.9 0.2
9.7 0.6
66
67
68
69
70
71
Gigartinales
Sarconema scinaioides
Sarconema liforme
Hypnea valentiae
Hypnea musciformis
Hypnea spinella
Solieria robusta
4.3 0.8
9.8 0.7
5.8 0.8
7 0.12
7.8 0.7
9 0.6
72
73
74
75
76
Bangiales
Pyropia tenera
Pyropia yoezensis
Pyropia acanthophora
Pyropia acanthophora var. brasilensis
Pyropia sp.
7.7 0.5
7.0 0.2
7.3 0.3
7.3 0.5
7.3 0.4
77
78
79
80
81
82
83
Rhodymeniales
Rhodymenia sonderi
Coelarthrum muelleri
Botryocladia leptopoda
Botryocladia botryoides
Gastroclonium iyengarii
Champia parvula
Gelidiopsis variabilis
7.1 1.2
7.7 0.6
5.2 2.0
2.3 0.4
4.3 0.6
9.3 2.7
5.5 0.6
84
Gelidiales
Gelidiella acerosa
6.7 1
85
86
87
88
Halymeniales
Cryptonemia undulata
Halymenia porphyraeformis
Grateloupia indica
Grateloupia licina
11.3 1.6
10 1.7
6.4 0.1
5 0.9
89
90
91
92
93
94
95
96
97
98
Ceramiales
Odonthalia veravalensis
Acanthophora specifera
Acanthophora nayadiformis
Laurencia cruciata
Laurencia obstusa
Laurencia papillosa
Laurencia majusculus
Laurencia sp.
Polysiphonia ferulacea
Grifthsia corallinoides
11.4 4.4
6.8 0.5
10.4 1.1
8.2 1.2
5.4 0.4
9.3 0.5
8 1.5
6.6 1.8
9.6 2.8
4.2 1.3
Corallinales
Jania rubens
Scinaia monoliformis
12.2 1.4
5.2 0.4
99
100
FAs (TFAs). The major FAs detected in Chlorophyta members were

myristic (C14:0), palmitic (C16:0), stearic (C18:0), palmitoleic
(C16:1, n-9), oleic (C18:1, n-9), linoleic (C18:2, n-6; LA), a-linolenic
(18:3, n-3; ALA) and stearidonic acid (C18:4, n-3; STA) that collectively contributed to 58.788.9% of TFAs pool. Interestingly, no trans
FAs were encountered in any green algae that increase the risk of
cardiovascular diseases (Ferreri and Chatgilialoglu, 2005). This
study revealed that all the species exhibited the characteristic prole of C18 > C20 PUFAs (Khotimchenko et al., 2002; Li et al., 2002;
Kumari et al., 2010) except Halimeda tuna. The ratio of different
C18 PUFAs (LA, ALA and STA) further revealed that the levels of
ALA exceeded the contents of LA and STA taken together in all the
species except those of Trichosolen, Halimeda, Cladophoropsis,
Valoniopsis, Chaetomorpha and Acrosiphonia.
Moreover, members of the same genus exhibited similar FA patterns but differed signicantly in their individual FA contents
(p 6 0.001). All the members of Ulvales had characteristic FA proles of high palmitic, oleic, C18 PUFAs with ALA > LA (29.8 times)
and low C20 PUFAs as illustrated earlier for different Ulva spp.
(Vaskovsky et al., 1996; Khotimchenko et al., 2002; Li et al.,
2002; Colombo et al., 2006; Yazici et al., 2007; Chakraborty and
Santra, 2008; Pereira et al., 2012). STA was found to be another
characteristic of Ulva spp. varying from 3.6% to 20% of TFAs and
such higher values were not observed for any other green macroalgal samples. Moreover, DHA in Ulva spp. has earlier only been reported from Yellow Sea (Vaskovsky et al., 1996), Black Sea and
Dardenelles (Yazici et al., 2007) in low amounts (0.20.6% of TFAs).
M. oxyspermum belonging to Ulotrichales recorded higher SFAs due
to higher stearic acid (C18:0), (17.4% of TFAs) but with lower PUFAs
in contrast with those values reported for Monostroma nitidum collected from Yamaguchi, Japan (Kaneniwa et al., 1998).
In contrast to Ulvales and Ulotrichales, the members of Bryopsidales, Siphonocladales and Cladophorales showed 26.4-fold
higher C20 PUFAs that accounted for 7.121.9% of TFAs. Such
higher C20 PUFAs contents for the taxa of these three orders have
also been reported in previous studies (Khotimchenko, 1993,
2003; Carbelleira et al., 1999; Goecke et al., 2010). C. dwarkense
showed relatively higher SFAs due to high palmitic (43% of TFAs)
and myristic acid (11.1% of TFAs) with low MUFAs and PUFAs
with minor DHA (1.3% of TFAs). These values were comparatively
higher than those reported for Codium elongatum (0.2% of TFAs)
and Codium tomentosum (1.1% of TFAs) (Khotimchenko, 2003;
Xu et al., 1998; Goecke et al., 2010). The taxa belonging to the
genus of Bryopsis and Halimeda showed FA patterns similar to
those recorded earlier for the same or related species (Vaskovsky
et al., 1996; Carbelleira et al., 1999). A few deviations were observed in the FA composition of Chaetomorpha spp. where LA
was reported as the predominant PUFA while ALA and STA together accounted to less than 2% of TFAs (Khotimchenko, 1993;
Khotimchenko et al., 2002). However, in the present investigation,
LA content was 1.2-fold higher than ALA and STA, which together
contributed to 13.2% of TFAs (p 6 0.001, Table 2). Similar variations in FA proles of Chaetomorpha spp. were reported by Yazici
et al. (2007) from Black Sea and Dardenelles.
2.2.2. Rhodophyta
The FA compositions of 43 taxa belonging to the orders of Gracilariales, Gigratinales, Bangiales, Rhodymeniales, Gelidiales, Halymeniales, Ceramiales and Corallinales are presented in Table 2
and Supplementary Table 1. Of the 43 species, 24 species had higher UFAs while 19 had higher SFAs with UFA/SFA ratio varying from
1.0 (Rhodymenia sonderii, Coelarthrum muelleri, Grateloupia indica,
Acanthophora spp. and Laurencia papillosa) to 2.2 (Gracilaria corticata). Only 13 species showed higher PUFAs with 65.6 2.5% of
TFAs being the highest in G. corticata followed by 60.4 2.3% in
Pyropia tenera, due to higher AA and EPA contents respectively.
All the species showed characteristic red algal proles of higher
C20 PUFAs with the exception of Sarconema liforme, Gastroclonium
iyengarii and Scinaia monoliformis. This nding is in agreement
with the earlier studies wherein C20 PUFAs (AA and EPA) were recorded as the dominant fraction of FAs in Rhodophytes (Khotimchenko et al., 2002; Li et al., 2002; Kumari et al., 2010). In
addition, red algae showed large variations in MUFAs and ranged
from 3.5% to 26.6% of TFAs. Myristic, palmitic, stearic, palmitoleic,
oleic, elaidic, dihomogammalinolenic (C20:3, n-6; DGLA), AA and
EPA were among the predominant FAs encountered and collectively accounted for 77.699.2% of TFAs. Further, FA proles obtained for the species of Gracilaria, Sarconema, Hypnea, Solieria,
Pyropia, Rhodymenia, Cryptonemia, Botryocladia, Champia, Gelidiopsis, Grateloupia, Halymenia, Odonthalia, Acanthophora, Polysiphonia,
Laurencia, Jania and Scinaia were consistent with those reported
in previous studies (Aknin et al., 1990; Levy et al., 1992; Vaskovsky
et al., 1996; Khotimchenko, 2005; Li et al., 2002; Colombo et al.,
47
2006; Chakraborty and Santra, 2008; Ortiz et al., 2010; Kumari

et al., 2010; Kendel et al., 2012; Pereira et al., 2012).
2.2.3. Phaeophyta
The FA compositions of 24 different brown algal species belonging to the three orders, Dictyotales, Fucales and Ectocarpales are
presented in Table 2 and Supplementary Table 1. The major FAs
encountered were myristic, palmitic, stearic, palmitoleic, oleic,
LA, STA, AA and EPA that collectively contributed to 80.192.2%
of TFAs as reported earlier for the members of this group (Bhaskar
and Miyashita, 2005; Khotimchenko et al., 2002; Li et al., 2002; Kumari et al., 2010). In contrast to green and red algae, both the polyenoic C18 and C20 FAs were predominant in these species and
varied from 23.6 8.6 to 58 4.0% of TFAs. This trend was a typical
character of Phaeophyta group that distinguishes them from rest of
the Chlorophyta and Rhodophyta members (Khotimchenko et al.,
2002; Li et al., 2002; Kumari et al., 2010). The comparative analysis
of C18 and C20 PUFAs distribution revealed that the members of
Phaeophyta could be divided into three groups. The rst group
comprised of Padina spp., Dictyota hauckiana, Cystoseira trinodis
and Hincksia mitchelliae that exhibited higher C18 PUFAs (1.1
2.2-fold) owing to higher STA contents. The second group included
Dictyota spp. (D. pinnatida, D. bartayresiana, D. dichotoma), Sargassum spp., Hormophysa cuneiformis, Cystoseira indica and Scytosiphon
lomentaria that exhibited higher C20 PUFAs than C18 PUFAs (due
to higher AA and EPA contents) while the third group consisted
of Dictyopteris deliculata, Dictyota crevicornis and Dictyota ciliolata
that recorded equal amounts of C18 and C20 PUFAs. The Phaeophyta members also reported higher MUFAs than both the Chlorophyta and Rhodophyta and accounted for 1951% of
unsaturation.
Further, the FA proles of Padina tetrastomatica, Stoechospermum marginatum and Spatoglossum asperum were consistent with
the reports of Bhaskar and Miyashita (2005) and Kumari et al.
(2010). Among the six Dictyota species studied, D. pinnatida, D.
bartayresiana and D. dichotoma recorded higher C20 PUFAs (AA
and EPA), D. crevicornis and D. ciliolata reported equal amounts
while D. hauckiana recorded 1.4-fold higher C18 PUFAs chiey
due to STA (22.4 0.6% of TFAs). Similar FA proles with equal
C18 and C20 PUFAs has been reported for D. crevicornis,
C18 > C20 PUFAs (1.6-fold) for D. dichotoma (Khotimchenko,
1995) and D. ceylinica (2.14-fold) (Chakraborty and Santra, 2008).
Fucales members had marginal variations and relatively lower
myristic acid contents (5.07.5% of TFAs) as compared with Dictyotales (4.624.7% of TFAs). They further showed lower STA contents
(0.98.8% of TFAs) except Sargassum carpophyllum and Sargassum
plagiophyllum recording 11.5% and 12.3% of TFAs respectively.
Although, all the seven Sargassum species differed signicantly in
their FA contents (p 6 0.001) (Table 2), they showed similar FA
proles with higher MUFAs, PUFAs (C20 > C18 PUFAs) and low
SFAs with the exception of S. carpophyllum. The FA proles reported for the species of Sargassum, Hormophysa and Cystoseira
were in congruence with the earlier reports from other regions.
(Xiang-Chun et al., 1995; Vaskovsky et al., 1996; Heiba et al.,
1997; Khotimchenko, 1998; Khotimchenko et al., 2002; Li et al.,
2002; Yazici et al., 2007).
To the best of our knowledge, the FA proles for Trichosloen
mucronatus, Chamaedoris auriculata and C. javanica belonging to
Chlorophyta, C. muelleri, G. iyengarii and Grifthsia corallinoides
belonging to Rhodophyta are reported for the rst time in the present study (Table 2).
2.3. Multivariate analysis and fatty acid phylogeny
Principal component analysis (PCA) of FA data matrix provided a
global statistical distinction between the three groups, Chlorophyta,
48
Table 2
Fatty acid groups (given in% of total fatty acid methyl esters; TFAs) and nutritional indices in different macroalgal species expressed as means SD (n = 3).
Macroalgae
SFAs
MUFAs
PUFAs
PUFA/SFA
n6/n3
U.I.
Ulva lactuca
Ulva fasciata
Ulva taeniata
Ulva pertusa
Ulva reticulata
Ulva beytensis
Ulva compressa
Ulva rigida
Ulva linza
Ulva exuosa
Ulva erecta
Ulva prolifera
Caulerpa scalpelliformis
Caulerpa veravalensis
Caulerpa racemosa
Caulerpa racemosa v. corynephora
Caulerpa racemosa v. occidentalis
Caulerpa microphysa
Caulerpa verticillata
Caulerpa sertularioides
Codium dwarkense
Bryopsis pennata
Bryopsis plumosa
Trichosolen mucronatus
Udotea indica
Halimeda discoides
Halimeda tuna
Monostroma oxyspermum
Chamaedoris auriculata
Cladophoropsis javanica
Valoniopsis pachynema
Chaetomorpha linum
Acrosiphonia orientalis
Padina tetrastomatica
Padina gymnospora
Dictyopteris deliculata
Dictyota pinnatida
Dictyota bartayresiana
Dictyota dichotoma
Dictyota cervicornis
Dictyota ciliolata
Dictyota haukiana
Stoechospermum marginatum
Lobophora variegata
Spatoglossum asperum
Sargassum tenerrimum
Sargassum johnstonii
Sargassum sp.
Sargassum carpophyllum
Sargassum plagiophyllum
Sargassum cinereum
Sargassum cinctum
Hormophysa cuneiformis
Cystoseira indica
Cystoseira trinodis
Hincksia mitchelliae
Scytosiphon lomentaria
Gracilaria dura
Gracilaria salicornia
Gracilaria textorii
Gracilaria corticata
Gracilaria corticata v. cylindrica
Gracilaria corticata v. folifera
Gracilaria debilis
Gracilaria verrucosa
Sarconema scinaioides
Sarconema liforme
Hypnea valentiae
Hypnea musciformis
Hypnea spinella
Solieria robusta
Pyropia tenera
Pyropia yoezensis
Pyropia acanthophora
59.9 2.3
29.6 2.1
46.8 7.1
35.5 2.3
48.8 5.9
32.3 0.8
37.6 0.6
37.8 5.0
38.4 9.9
37.3 0.5
39.2 1.5
39.1 0.9
39.6 1.0
27.1 1.0
33.4 3.1
58.2 6.4
n. d.
40.3 2.0
44 2.7
41.4 0.9
65.4 2.6
42.1 6.2
51.5 4.4
49.3 10.8
43.5 3.3
46.1 2.2
41.9 0.9
55.1 10
43.9 3.2
45.7 1.4
46.4 1.4
37.5 3.5
41.7 4.8
46.5 0.4
33.8 0.2
50.6 2.8
55.8 3.4
28.9 1.0
39.0 9.1
36.5 1.9
39.9 0.9
34.2 1.7
59.3 8.7
48.3 1.2
51.1 3.4
49.5 1.0
46.3 4.4
49.6 0.6
52.4 10.4
42.8 1.9
43.2 6.4
35.9 14.7
40.7 1.4
47.0 7.4
39.1 1.8
35.7 1.5
59.7 1.8
33.2 1.1
78.2 0.5
60.0 4.4
31 2.5
61 2.5
62.7 3.0
40.1 3.6
46.6 3.2
64.7 3.1
39.9 8.5
59.0 6.0
65.1 1.7
65.7 1.5
61.4 1.3
34.3 1.0
37.2 2.7
35.9 2.1
12.2 1.8
7.5 0.9
5.4 1.0
9.4 0.4
7.1 0.7
10.9 0.8
8.3 0.2
8.1 4.0
6.0 0.8
7.5 0.4
7.1 2.4
9.5 0.8
6.4 0.1
1.9 0.2
5.7 1.4
2.9 0.4
10.2 5
5.2 0.3
6.1 3.0
2.2 0.6
6.6 0.1
6.5 2.6
8.2 1.1
8.7 0.2
5.7 2.1
6.1 2.3
7.9 1.3
3.5 2.6
13.7 1.0
9.4 1.2
15.7 5.1
21.9 5.5
8.7 0.7
14.9 0.6
16.9 0.7
11 1.2
11.3 1.3
13.1 0.6
16.6 5.8
12.1 0.7
12.0 0.4
22.2 0.7
17.2 1.9
12.9 0.9
25.4 2.1
17.6 0.6
15.2 0.3
18.2 0.5
11.3 1.3
13.0 0.4
32.9 8.7
18.5 4.4
15.9 1.5
14.0 1.4
12.6 0.6
12.4 1.6
10.5 1.6
4.1 0.5
10.6 0.2
11.5 1.6
3.5 0.1
9.3 1.1
5.1 0.6
11.7 4.5
9.2 1.6
17.9 3.8
19.7 9.3
18.2 1.3
15 1.8
6.2 1.1
17.4 0.7
5.4 1.1
10.1 1.4
12.9 1.2
28.0 0.7
63.1 1.9
48 7.8
55.2 2.1
44.8 5.9
56.9 0.2
54.2 0.8
54.2 8.9
55.6 9.1
55.2 0.6
53.7 0.9
51.4 0.9
54 1.0
71.1 0.9
60.8 3.0
39.1 6.0
36.5 3
54.6 1.9
50 5.3
56.8 1.1
28 2.6
51.5 7.9
40.4 3.4
42.3 10.5
50.9 5.4
47.9 0.7
50.2 1.
41.6 7.4
42.6 3.4
45.1 1.3
38 6.4
40.7 3.3
49.8 5.6
38.7 1.0
49.3 0.9
38.7 3.1
33.0 2.3
58.0 4.0
44.4 14.9
51.4 2.7
48.2 0.6
43.7 1.9
23.6 8.6
39.2 1.9
23.7 1.5
33 1.4
38.5 4.2
32.4 0.2
36.4 9.1
44.7 2.2
24.1 2.5
45.7 10.5
43.5 0.7
38.9 8.6
48.4 1.7
51.9 3.0
29.8 0.2
62.8 1.2
11.5 0.8
28.6 2.9
65.6 2.5
29.8 2.8
32.3 3.4
48.4 3.4
44.3 4.1
17.6 0.8
40.4 2.0
23.2 5.2
20 2.7
28.2 2.1
21.3 2.1
60.4 2.3
52.9 2.8
51.3 3/1
0.47 0.03
2.14 0.2
1.06 0.3
1.56 0.15
0.94 0.22
1.76 0.05
1.44 0.04
1.47 0.44
1.54 0.56
1.48 0.03
1.37 0.03
1.31 0.05
1.36 0.06
2.63 0.13
1.84 0.24
0.68 0.17
0.68 0.05
1.36 0.12
1.14 0.18
1.38 0.06
0.43 0.06
1.26 0.36
0.79 0.13
0.91 0.37
1.18 0.21
1.04 0.05
1.20 0.06
0.79 0.27
0.98 0.15
0.99 0.05
0.82 0.16
1.09 0.11
1.21 0.28
0.83 0.03
1.46 0.04
0.77 0.1
0.60 0.07
2.01 0.08
1.23 0.59
1.41 0.15
1.21 0.04
1.28 0.11
0.42 0.2
0.81 0.06
0.47 0.06
0.67 0.04
0.84 0.16
0.65 0.01
0.73 0.29
1.05 0.1
0.56 0.02
1.61 1.25
1.07 0.04
0.86 0.29
1.24 0.1
1.46 0.06
0.50 0.02
1.89 0.1
0.15 0.01
0.48 0.08
2.13 0.25
0.49 0.06
0.52 0.08
1.21 0.15
0.96 0.15
0.27 0.01
1.04 0.23
0.40 0.12
0.31 0.05
0.43 0.04
0.35 0.04
1.76 0.08
1.43 0.18
1.36 0.14
0.3 0.01
0.2 0.01
0.4 0.1
0.2 0.01
0.3 0.2
0.1 0.1
0.4 0.1
0.2 0.2
0.2 0.01
0.3 0.01
0.5 0.1
0.6 0.1
0.4 0.1
0.3 0.01
0.2 0.01
0.6 0.1
0.8 0.3
0.6 0.01
1.0 0.3
0.5 0.1
1.1 0.1
0.6 0.2
0.5 0.1
1.9 1.6
0.3 0.01
0.6 0.2
0.8 0.1
0.2 0.1
0.8 0.01
1.8 0.7
1.6 0.4
1.3 0.9
1.4 0.5
1.2 0.3
0.9 0.01
2.4 0.2
3.9 1.8
0.6 0.01
0.9 0.4
0.9 0.01
0.7 0.01
0.4 0.01
2.8 0.7
0.8 0.1
1.3 0.1
2.7 0.2
1.6 0.3
2.3 0.1
1.3 0.1
1.3 0.1
0.5 0.1
2.1 0.1
7.4 0.4
1.6 0.2
1.7 0.2
0.5 0.01
1.4 0.3
194.7 41
1
1
1
42 17.4
8.7 2.7
59.2 29
0.6 0.01
2.5 2.0
2.4 0.6
0.8 0.01
1.2 0.5
16 4.6
0.8 0.01
0.7 0.1
1.2 0.2
1.2 0.1
104.1 2.8
211.4 5.2
159.7 27
195 6.4
159.4 28
201.6 6.1
173.3 3.2
191.8 37
193 35
170 1.9
167 6.9
164.3 6.9
177.5 3
226 2.6
196.6 11
128.3 18
122.1 7
176.7 6.1
172.6 6
176.9 3
91 7.8
166 26
137 11.6
144.3 33
191.3 8.8
178 19.3
198.6 7.0
133.8 27
163 10
152 4.9
141.2 18
162.9 12
161.3 18
149.8 5.9
193.7 2.0
145.7 9.4
133.3 9.9
241.8 5.6
187.2 53
207 9.5
184.6 3.5
197.4 5.8
95.2 2.7
175.3 7.1
117.6 8.1
141 5.0
166 18.3
137.8 0.9
147 36.2
185 8.4
139 3.0
186.4 44
152.3 3.0
159 35.1
183.7 7.9
221 1.0
122.4 6.4
249 3.6
50.6 1.3
105.8 15.1
257 10
118.4 12.4
125.3 9.8
194.5 11.3
205.3 21
91.4 1.7
134.5 12.6
118.2 25.0
91.6 12.4
98.1 3.9
113.9 9.0
272 5.2
232 13.7
226 5.8
AI
1.1 0.06
0.4 0.03
0.7 0.2
0.4 0.05
0.8 0.15
0.4 0.01
0.5 0.01
0.5 0.09
0.6 0.25
0.5 0.02
0.6 0.03
0.5 0.02
0.5 0.02
0.4 0.02
0.5 0.08
1.2 0.3
1.0 0.13
0.5 0.05
0.7 0.05
0.6 0.02
1.6 0.22
0.6 0.17
0.9 0.12
0.9 0.41
0.7 0.09
0.7 0.12
0.6 0.02
0.8 0.26
0.7 0.11
0.7 0.09
0.8 0.08
0.5 0.05
0.7 0.14
0.8 0.12
0.4 0.01
0.9 0.08
1.1 0.2
0.3 0.02
0.6 0.25
0.4 0.06
0.6 0.01
0.5 0.03
1.4 0.42
0.8 0.05
1.0 0.13
0.9 0.01
0.7 0.15
0.8 0.03
0.9 0.31
0.6 0.05
0.7 0.2
0.5 0.33
0.5 0.02
0.8 0.26
0.6 0.04
0.5 0.02
0.2 0.01
0.4 0.03
2.9 0.14
1.3 0.21
0.4 0.05
1.3 0.14
1.5 0.15
0.6 0.1
0.8 0.1
1.4 0.2
0.4 0.07
1.4 0.35
1.7 0.12
1.6 0.15
1.2 0.08
0.4 0.03
0.4 0.05
0.5 0.03
TI
0.8 0.03
0.2 0.02
0.4 0.11
0.2 0.02
0.4 0.17
0.2 0.01
0.3 0.01
0.3 0.11
0.3 0.12
0.3 0.01
0.3 0.01
0.3 0.01
0.3 0.01
0.2 0.01
0.2 0.02
0.6 0.12
0.7 0.1
0.3 0.03
0.4 0.11
0.3 0.02
1.3 0.23
0.4 0.15
0.5 0.06
0.9 0.8
0.3 0.05
0.4 0.07
0.4 0.02
0.5 0.16
0.5 0.08
0.6 0.17
0.8 0.15
0.5 0.17
0.5 0.17
0.7 0.09
0.3 0.01
1.0 0.05
1.5 0.51
0.2 0.01
0.5 0.46
0.3 0.04
0.4 0.02
0.3 0.03
2.4 1.6
0.6 0.06
1.3 0.14
1.2 0.1
0.7 0.22
1.1 0.03
0.9 0.4
0.6 0.06
0.7 0.11
0.6 0.35
1.1 0.07
0.8 0.34
0.5 0.06
0.3 0.01
1.2 0.12
1.0 0.07
11.9 1.0
3.9 0.61
0.9 0.11
3.4.11
2.6 1.03
1.5 0.16
0.5 0.08
2.7 0.8
0.7 0.22
1.3 0.5
1.9 0.56
3.3 0.27
1.4 0.2
0.4 0.05
0.3 0.01
0.4 0.03
49

Table 2 (continued)
Macroalgae
SFAs
MUFAs
PUFAs
PUFA/SFA
Pyropia acanthophora var. brasilensis

Pyropia sp.
Rhodymenia sonderi
Coelarthrum muelleri
Botryocladia leptopoda
Botryocladia botryoides
Gastroclonium iyengarii
Champia parvula
Gelidiopsis variabilis
Gelidiella acerosa
Halymenia porphyraeformis
Grateloupia indica
Grateloupia licina
Odonthalia veravalensis
Acanthophora specifera
Acanthophora nayadiformis
Laurencia cruciata
Laurencia obstusa
Laurencia papillosa
Laurencia majusculus
Laurencia sp.
Polysiphonia ferulacea
Jania rubens
Scinia monoliformis
LSDa
41.2 5.0
37.7 1.1
49.1 2.2
50.4 6.2
56.6 2.3
47.7 0.5
57.1 4.7
59.7 1.5
47.4 4.74
44.6 1.8
32.9 2.4
39.2 5/0
49.9 0.7
47.2 3.0
46.3 0.9
51.2 3.0
49.7 4.9
50 3.8
60.7 1.4
48.4 3.4
62 9.9
74.5 7.7
51.5 1.4
52.5 2.8
65.8 5.8
65.6 5.4
11.9
9.2 3.8
9.4 2.1
8.0 0.8
16.6 8.3
15.9 0.9
26.6 0.7
9.9 1.7
17.4 1.6
13.3 1.7
11.7 2.1
25.3 1.3
8.1 2.5
13.1 1.0
10.8 0.8
13.7 4.1
12.1 1.3
4.5 0.8
11.7 2.1
11.7 0.3
14.7 2,1
13.7 3.9
15.1 1.7
14.2 1.3
20.0 2.3
13.1 2.8
19.3 6.9
7.1
49.6 8.2
53 3.0
42.9 1.6
33.6 2.0
27.7 2.4
25.8 0.3
33.3 3.0
23 1.0
39.4 6.4
43.8 3.2
41.9 1.4
52.7 4.2
37.2 1.2
42.2 3.2
40.1 3.5
36.7 3.2
46.1 4.1
38.9 5.4
27.8 1.7
37.0 15
24.4 7.2
10.6 0.4
34.4 1.5
27.6 2.2
21.2 2.4
15.2 1.6
11.8
1.23 0.32
1.46 0.12
0.87 0.07
0.67 0.05
0.49 0.06
0.54 0.01
0.59 0.1
0.39 0.01
0.84 0.21
0.98 0.06
1.28 0.13
1.37 0.27
0.74 0.03
0.90 0.13
0.86 0.07
0.72 0.1
0.94 0.18
0.79 0.16
0.46 0.04
0.77 0.08
0.41 0.18
0.14 0.01
0.67 0.04
0.53 0.06
0.32 0.03
0.23 0.01
0.45
n6/n3
1.3 0.2
1.4 0.1
88.2 9.4
5.7 0.8
1.7 0.2
3.6 0.7
5.1 1.4
1.0 0.1
0.8 0.5
0.6 0.1
18.8 6.2
1.7 0.7
1.9 0.2
0.5 0.2
0.9 0.3
0.6 0.1
0.9 0.2
1.8 0.1
1.2 0.3
1.1 0.2
1.6 0.3
1.7 0.1
0.5 0.1
1.1 0.2
2.9 0.5
1.1 0.01
17.5
U.I.
AI
TI
213.4 35.6
224 13.0
168 25.7
151 3.3
121 12.3
122 3.9
94 12.3
105 3.6
182 38
204 9.2
185.3 5.7
225 26.2
166.3 5.9
195.4 21.3
187 14.1
171 14.6
212.6 17.0
171.6 19.5
134 7.7
169.6 9.2
115.3 32.1
56.6 2.2
157.8 4.9
123 10.5
90.7 12.1
73.3 2.1
46.1
0.6 0.12
0.5 0.18
0.5 0.04
0.6 0.07
1.1 0.12
0.9 0.06
0.7 0.05
1.5 0.06
0.8 0.11
0.7 0.03
0.4 0.05
0.6 0.12
0.9 0.02
0.8 0.08
0.8 0.04
0.9 0.1
1.0 0.18
0.8 0.08
1.6 0.73
0.8 0.08
1.6 0.73
2.7 0.27
0.9 0.04
0.9 0.08
1.7 0.09
1.5 0.4
0.44
0.5 0.18
0.4 0.03
0.01 0.01
0.2 0.03
0.6 0.06
0.3 0.06
0.2 0.02
1.2 0.07
0.6 0.27
0.5 0.07
1.2 0.06
0.5 0.18
0.9 0.07
0.5 0.1
0.6 0.1
0.6 0.1
0.6 0.07
0.9 0.0
1.2 0.16
0.7 0.1
1.7 0.71
4.6 0.2
0.6 0.03
1.0 0.15
2.6 0.4
2.10.13
0.84
Abbreviations: SFAs: Saturated fatty acids, MUFAs: Monounsaturated fatty acids, PUFAs: Polyunsaturated fatty acids, U.I.: Unsaturation index, AI: Atherogenic index, TI:
Thrombogenic index.
a
LSD values obtained from ANOVA. The values in a column are signicantly different at p 6 0.001.
Rhodophyta and Phaeophyta although it explained only 36% of the

variations (PC1 21% and PC2 15%) revealing many correlations of
taxonomic implications. Axis I separated the Chlorophyta members
(group I) from Rhodophyta (group II) while Phaeophyta members
(group III) were positioned intermediate between these two, as evident from the PCA bi-plot (Fig. 1). The most discriminant variables
along the axis I were STA, GLA, LA, ALA, DHA, AA, EPA and elaidic
acid while along axis II were oleic, myristic, palmitic, 11,14,17eicosatrienoic acid (ETA) and DGLA. All the species belonging to
the same genus were clustered together with a few exceptions of
U. lactuca (due to higher loadings of stearic acid), S. cinereum, Hypnea musciformis (due to DGLA) and C. trinodis (due to LA, STA and
GLA). A large number of outliers were detected in this model, Ulva
exuosa, C. racemosa and Caulerpa veravalensis owing to the higher
loadings of ALA, Padina gymnospora, H. mitchelliae and S. liforme
due to STA, D. ciliolata, D. bartayresiana due to ETA, S. marginatum,
S. asperum, G. corticata, P. tenera, G. iyengarii and Gracilaria salicornia
due to oleic, myristic, AA, EPA, LA and palmitic acids respectively.
Further, bi-plot of FA groups data matrix successfully explained
80% of the variances (PC1 48% and PC2 32%), (Fig. 2). This indicated the vast diversity present in FA data matrix in macroalgae;
when PCA was applied on individual FAs the model could explain
only 36% of the variations satisfactorily, but when they were clustered together in groups, FA trends were observed to be conserved
within the respective phyla. Similar observations were deciphered
by Kumari et al. (2010) and Galloway et al. (2012) who demonstrated that the comparisons made at ranks of order and family explained less variation inherent in the macroalgal FAs as compared to
the phyla level.
Moreover, the dendrogram generated from Ward hierarchical
clustering to assess the phylogenetic relationship between different macroalgal species has grouped them into nine clusters
(Fig. 3). The groups deduced from FA compositions were congruent
with the clades inferred from their nuclear and plastid genomes for
the genus Ulva, Bryopsis, Halimeda, Sargassum, Dictyota and Gracilaria, (Caisov et al., 2011; Duan et al., 2012; Verbruggen et al.,
2009; Silberfeld et al., 2010; Tronholm et al., 2010; Pareek et al.,

2010). Further, Ulva spp. and Monostroma sp. were closely related
to each other forming Ulvales-Ulotrichales clade identical to the
one obtained from molecular data (Leliaert et al., 2012). Caulerpa,
Bryopsis, Halimeda, Codium and Trichosolen belonging to the same
order Bryopsidales were grouped together and their inter-relationship were in agreement with the multi-locus-time calibrated phylogeny of siphonaceous green algae based on rbcL, tufA, atpB, 18S
rRNA gene sequences (Lam and Zechman, 2006; Verbruggen
et al., 2009). Similarly, Padina, Dictyota, Dictyopteris, Stoechospermum, Spatoglossum belonging to Dictyotales (except P. tetrastomatica) and Sargassum (except S. cinereum), Hormophysa and Cystoseira
belonging to Fucales were grouped together in agreement with the
multi-marker phylogeny deduced by Silberfeld et al. (2010). Similar trends were observed for Rhodophyta where FA trends remained conserved up to orders except for Gigartinales,
Halymeniales, Ceramiales and Rhodymeniales. The algal species
belonging to the families of Cystocloniaceae, Solieriaceae, Rhodymeniaceae, Champiaceae, Lomentariaceae, Halymeniaceae and
Rhodomelaceae were grouped separately. This deviation could be
due to inadequate taxon sampling and further efforts including
extensive sampling may give a better resolution. Similar clustering
is also shown in a two dimensional plot where the correlation
between different species is more illustrative (Fig. 4). Our study
corroborates with the ndings of Lang et al. (2011), Galloway
et al. (2012) and Gosch et al. (2012) that FA distribution
reects phylogenetic relationships among phyla, classes and orders
as seen in the genomic and molecular phylogenies at higher ranks
in algae. However, higher variations in FA contents at the levels
of genus and species pose difculty in discriminating species as
observed in the present study especially in the case of red
macroalgae.
The production of C18 and C20 PUFAs in green and red macroalgae could be attributed to their primary endosymbiotic origin of
engulfment of C18 PUFA producing photosynthetic cyanobacteria
by eukaryotic host capable of further elongation and desaturation
50
Fig. 1. Bi-plot representation of macroalgal samples obtained from PCA analysis of FA data matrix, with rst two principal components. The macroalgal species are indexed
from 1 to 100 according to Table 1.
Fig. 2. Bi-plot of macroalgal samples, obtained by PCA analysis of FA groups namely, SFA, MUFA, PUFA, C18 PUFA, C20 PUFA, n-3 PUFA and n-6 PUFA, with rst two principal
components. The macroalgal species are indexed from 1 to 100 according to Table 1.
de novo similar to the green and red microalgae as explained by

Dunstan et al. (2005) since it is noteworthy to note that cyanobacteria contain FAs only up to the chain length of 18-carbon (Liu
et al., 2010; Iliev et al., 2011). Phaeophyta members that originated
from secondary endosymbiosis of red algae and an eukaryotic host
further capable of elongation and desaturation of FAs de novo produce C18 and C20 PUFAs along with minor amounts of C22 PUFAs
(C22:4, n-6) such as in Dictyota spp. (<0.4% of TFAs, not included in
present analysis). Similarly, Cryptophytes, Haptophytes, Dinoagellates, other Heterokont algae and Apicomplexans belonging to
the red algal lineage that diverged along with brown algae during
secondary endosymbiosis also produce C22 PUFAs (Dunstan et al.,
2005; Leblond et al., 2005; Lang et al., 2011). Some of the species
belonging to these groups such as Phaeodactylum tricornutm, Skeletonema sp., Crypthecodinium cohnii, Schizochytrium sp., Rhodomonas sp., Pavlova sp. and Isochrysis sp., are commercially explored for
their EPA and DHA contents in aquaculture and pharmaceuticals
(Patil et al., 2007; Mendes et al., 2008; Lang et al., 2011). Similarly,
Chlororachinophytic algae such as Bigelowiella sp., Gymnochlora sp.
and Lotharella sp. that diverged from the secondary endosymbiosis
of green algae and the eukaryotic host also produce C22 PUFAs,
C22:5 n3 and C22:6 n3 (Leblond et al., 2005). Such endosymbiotic

relationship with the FA proles of algae is well supported by the
fact that green and red microalgae and macroalgae inherited their
type II fatty acid synthase (FASII) genes from their cyanobacterial
endosymbiont and brown algae from red algal endosymbiont
(Chan et al., 2012; Ryall et al., 2003). Similarly, two distinct types
of ACCase that catalyze the key step of FA biosynthesis has been
found in algae, heteromeric form in green and red algae of primary
endosymbiotic origin and homomeric ACCase in brown and other
stramenopile algae of secondary endosymbiotic origin, (Huerlimann
and Heimann, 2012). Chan et al. (2012) proposed the co-regulation
of FA biosynthesis and intracellular lipid trafcking in red algae on
the basis of their analysis of 482 EST contigs encoding putative
membrane transporters in Pyropia spp. They also deciphered the
evolution of these transporters and showed that among the three
proteins of transporter complex of chloroplast inner membrane
which is involved in lipid trafcking (TGD1, TGD2 and TGD3),
TGD1 and TGD2 are located in the plastid genome in red algae.
Moreover TGD2 is located in the same operon as accA that encodes
the a-subunit of ACCase while TGD3 is nuclear encoded and
showed strong phylogenetic association with the TGD3 of
51
Fig. 3. Dendrogram obtained from hierarchical cluster analysis of 100 macroalgal samples indexed according to Table 1.
Fig. 4. Principal component and hierarchic analysis of macroalgal samples, indexed from 1 to 100 according to Table 1.
cyanobacteria and higher plants. No homolog for TGD4 involved in

FA import in chloroplast in Arabidopsis was found in EST sequences
or plastid genomes of red alga Pyropia sp. Further, Chan et al.
(2012) also did not nd any candidate gene for any of these
TGD-genes in secondary plastids of stramenopiles. In contrast, all
the three TGD orthologs are located in the nucleus in green algae
(Xu et al., 2008).
Furthermore, the low amounts of C20 PUFAs in Chlorophyta

members as compared to Rhodophyta could be due to the low rate
of conversion of C18 to C20 PUFAs. However, the question arises, if
C22 PUFA production in algae is attributed to secondary endosymbiosis then, how DHA (C22 PUFA) is synthesized in Chlorophyta
species. Whether the enzymes that elongate and desaturate C18
PUFAs also exhibit specicity for C20 PUFAs as substrates or they
52
Fig. 5. Bi-plot of macroalgal species, indexed from 1 to 100 according to Table 1, obtained by PCA analysis of PUFA and nutritional indices data matrix (LA, ALA, GLA, STA, AA,
EPA, DHA, PUFA/SFA, U.I., AI and TI).
inherited some type I polyketide synthase (PKS) genes through

lateral gene transfer from bacteria like lower unicellular
chlorophytes, Ostrerococcus tauri, Ostrerococcus lucimarinus and
Chlamydomonas reinhardtii (John et al., 2008). Such type I PKS
genes have not been found in any other Plantae lineages neither
in higher plants nor in red algae. However, type III PKS gene has recently been reported in brown alga Sargassum binderi similar to the
higher plants but its role in lipid biosynthesis has not yet been
established (Baharum et al., 2011). Also, Dittami et al., 2012 found
three desaturases similar to D4-desaturases of haptophytes along
with three putative polyketide synthases in brown microalgae
Pseudochattonella farcimen, some of these genes have been
acquired via horizontal gene transfer from the common ancestor
of brown algae and dictyochophyceae. However, the presence
of C22 PUFAs in green macroalgae is quite debatable as no D4desaturase responsible for DHA production in microalgae has
been reported in green macroalgae and thus requires further
investigation.
2.4. Nutritional assessment
The present study revealed that the investigated macroalgae
were rich in nutritionally important PUFAs with their unsaturation
index (U.I.) varying from 104 2.8 (U. lactuca) to 226 2.6 (C. veravalensis) among green; 95.2 2.7 (S. marginatum) to 241.8 5.6 (D.
bartayresiana) among brown and 50.6 1.3 (G. salicornia) to
272 5.2 (P. tenera) among red. Similar indices were documented
for these three phyla in our previous study (Kumari et al., 2010)
and also by Colombo et al. (2006). PCA analysis of PUFA and nutritional indices data matrix (LA, ALA, GLA, STA, AA, EPA, DHA, PUFA/
SFA, UI, AI and TI) to evaluate the distribution of essential FAs in
macroalgae, explained 49% of the variations (PC1 31% and PC2
18%), (Fig. 5). The discriminant variables along axis I were AA,
EPA and UI while along axis II were LNA, DHA, LA, GLA, STA,
PUFA/SFA, AI and TI. The analysis revealed higher amounts of LA
and ALA in all the Chlorophyta members while STA was higher in
Ulva spp. along with the species of Padina, Dictyota, Cystoseira
and S. liforme. DHA which is essential for visual and neurological
development in infants was higher in the species of Halimeda, Udotea, Ulva, Cladophoropsis, Valoniopsis, Acrosiphonia and Chaetomorpha. The species of Gracilaria, Cryptonemia, Rhodymenia and
Coelarthrum had higher loadings of AA while S. cinereum, Lobophora
variegata, D. bartayresiana, Grateloupia licina, as well as species of

Pyropia, Acanthophora, Gelidiella, Gelidiopsis and Polysiphonia had
higher loadings of EPA. Both AA and EPA are precursors of prostaglandins, thomboxanes and other eicosanoids, which inuence
inammation processes and immune reactions (Gebauer et al.,
2006; Gillies et al., 2011). Further, all the species had nutritionally
benecial n6/n3 ratio ranging from 0.1 0.1:1 (Ulva beytensis) to
1.7 0.7:1 (C. javanica) in green, from 0.4 0.01:1 (D. hauckiana)
to 3.9 1.8 (D. pinnatida) in brown (except H. cuneiformis) and
from 0.5 0.1:1 (Polysiphonia ferulacea) to 3.6 0.7:1 (Botryocladia
botryoides) in red with the exception of other Gracilaria spp.,
Hypnea spinella, R. sonderii and Cryptonemia undulata (p 6 0.001,
Table 2), that reported higher n6/n3 ratios exceeding the ratio of
n6/n3 < 5:1 recommended by World Health Organization (WHO)
(WHO and FAO joint consultation, 1995; Simopoulos, 2002).
According to the recent FAO report, 2010, if a diet contains the recommended amount of essential FAs then, whether the n6/n3 ratio
of 4/1 or1/1 or <1 is meaningless. It should be ensured that n3 PUFAs are in substantial amount (equal/greater than n6 PUFAs) in the
food product. Thus, macroalgae exhibiting such low n6/n3 FAs
could help in decreasing low density lipoproteins, cholesterol and
prevent inammatory, cardiovascular and nervous system disorders. In addition, the PUFA/SFA ratio, which is one of the important
parameter to assess the nutritional quality of the lipid fraction of
food, was in accordance to the nutritional guidelines, P0.4 in all
the seaweeds except seven red algae, Jania rubens, S. monoliformis,
Laurencia sp., Solieria robusta, H. musciformis, Sarconema scinaioides
and G. salicornia (Department of Health, 1994). The artherogenic
(AI) and thrombogenic (TI) indices were < 3 in all the macroalgal
species studied except Laurencia sp., Gracilaria textorii, G. salicornia
and G. corticata v. cylindrica. Similar AI and TI values were observed
for different macroalgae in our previous work (Kumar et al., 2011)
and TI values were comparable to those of meat (1.081.58) and
milk-based products (2.1). Recently, Lpez-Lpez et al. (2009)
demonstrated that the addition of macroalgae such as Undaria
sp., Himanthalia sp. and Pyropia sp. to meat products improved
their thrombogenic and atherogenic indices thereby illustrating
their potential in development of healthier lipid formulations.
Despite the health-benetting n6/n3 ratio displayed by most of
the investigated macroalgae, microalgae have been considered better promising sustainable sources of essential FAs (especially, EPA
and DHA) for commercial use till date as they are capable of
producing much more lipids than any conventional crops. However, many of the investigated macroalgal species contained comparable ALA, AA and EPA contents. Most of the green macroalgal
species such as Ulva, Caulerpa, Monostroma, Bryopsis, Udotea and
Acrosiphonia had ALA contents in the range of 1448% of TFAs comparable to those of Dunaliela and Chlorella commercially utilized for
ALA (Zhukova and Aizdacher, 1995; Lang et al., 2011). Further, C.
auriculata, Caulerpa verticillata, all the brown algae except P. tetrastomatica, D. ciliolata and S. cinereum and red algae had AA contents
1025% of TFAs. C. undulata and R. sonderi had AA content >30% of
TFAs while G. corticata showed higher content >50% of TFAs that
is higher than commercial sources of microlagal AA such as
Porphyridium sp. (34.7% of TFA) and Parietochloris sp. (46% of TFA)
(Guil-Guerrero et al., 2001; Lang et al., 2011). Similarly, EPA contents of the species Pyropia, Halymenia, Polysiphonia, Acanthophora,
Gelidium and Gelidiella (2035% of TFAs) were comparable to those
of Phaeodactylum tricornutum (1214% of TFAs), Pavlova lutheri
(1028% of TFAs), Nannochloropsis spp., Thalassiosira sp., Chaetoceros
spp. (1218% of TFAs), Nitzschia sp., Skeletonema sp., Chattonella
spp. and Navicula sp. (2026% of TFAs) (Marshall et al., 2002;
Pratoomyot et al., 2005; Lang et al., 2011) But, the DHA content
in macroalgae was very low (<7% of TFAs found in Halimeda sp.)
as compared to the species of Schizochytrium, Crypthecodinium,
Scripsiella and Peridinium (3050% of TFAs) (Leblond et al., 2006;
Fan et al., 2007; Mendes et al., 2008). The only bottleneck in the utilization of macroalgae for pure oil-based products such as PUFA-oils
is the low lipid content of macroalgae as compared to microalgae.
Conversely, this low lipid content rich in PUFAs is a boon for their
utilization as whole macroalgae in both fresh and dried form in
human nutrition and aquaculture as it helps in improving cardiac,
mental health and combating inammatory diseases.
3. Conclusion
The macroalgae investigated in this study revealed low lipid
contents but exhibited nutritionally high amounts of essential n6 and n-3 PUFAs (LA, ALA, STA, AA, EPA and DHA). The health beneting n6/n3 ratio in macroalgae offers their utilization in the formulation of functional foods and nutraceuticals. The species of
green macroalgae investigated could be utilized as potential
sources of LA and ALA while the species of Ulva, Padina, Dictyota,
Cystoseira and S. liforme for STA. The red algal species of Gracilaria,
Cryptonemia, Rhodymenia and Coelarthrum could be utilized for AA
while Pyropia, Polysiphonia, Acanthophora, Gelidiella, Gelidiopsis, G.
licina, S. cinereum, L. variegata and D. bartayresiana for EPA. The
contents of ALA in green, AA and EPA in most of the red macroalgae
were comparable with those of commercially utilized microalgae.
Thus, an appropriate choice of macroalgae can be utilized as ingredients in the formulation of low fat foods and PUFA rich nutraceuticals that would improve the quality of human diet and also
reduce the dependency on traditional terrestrial sources. Further,
the statistical analysis demonstrated that FA based chemotaxonomic relationships were conserved at different taxonomic ranks
within the phyla and corroborated well with their molecular phylogenies. This signies the use of FA signatures as an additional
chemotaxonomic tool for closer understanding the relationships
among different species at higher ordinal levels such as families,
orders and phyla. Moreover, the green, red and brown macroalgae
acquired their ability of producing long-chain PUFAs from the coupling of chloroplastic desaturase enzymatic systems of their
respective endosymbionts and FA desaturase and elongase system
of the eukaryotic host. Although the inheritance of DHA production
by chlorophyta members is not yet understood, further phylogenetic investigations coupled with increased availability of EST sequences may decode this enigma.
53
4. Materials and methods

4.1. Chemicals
For the identication and quantication of FAs, following analytical grade standards were used: 37-component F.A.M.E. Mix
C4-C24 (Supelco, USA), 7-hexedecenoic acid methyl ester (C16:1,
n7) and stearidonic acid methyl ester (C18:4, n3) (Cayman chemicals, USA). The internal standard nonadecanoic acid (C19:0) was
purchased from Sigma. All the solvents used (such as chloroform,
methanol, dichloromethane and hexane) were of HPLC grade and
other reagents of analytical grade.
4.2. Algal samples

Macroalgal samples were collected in triplicates during low tide
periods during MarchOctober 2011 from different sites along the
Gujarat coast, India. The species of Pyropia and Monostroma were
collected during August, 2010 from Maharashtra coast, India. Fully
grown thalli of each alga which were inundated below the small
layer of water (about 0.52.0 cm) were chosen for this investigation. A detailed list of macroalgal samples along with the description of their habitat and the geographical co-ordinates are
presented in Supplementary Table 2. The temperature, salinity
and pH of seawater were also recorded at each collection sites
and shown in Supplementary Table 3. The samples were wrapped
in wet tissue towels and transported to the laboratory in cool conditions in an ice box. They were immediately cleaned thoroughly
with autoclaved seawater to remove the epiphytes and other undesired foreign matter from the surface of algal fronds. The cleaned
fronds were then blotted with tissue paper, weighed to determine
the fresh weight, thereafter, 0.5 g of each sample was immediately
frozen in liquid nitrogen in triplicate and stored at 40 C, until the
analysis commenced.
4.3. Lipid extraction, fatty acid methyl esters preparation and GCMS
analysis
Lipids were extracted by modied Bligh and Dyer method using
chloroformmethanolphosphate buffer (pH 7.5) (1/2/0.9), as
modied by Kumari et al. (2011). The fatty acids were converted
to their fatty acid methyl esters (FAMEs) by transmethylation of lipid samples with 1 ml of 1% NaOH in methanol, followed by heating for 15 min at 55 C, adding 2 ml of 5% methanolic HCl and again
heated for 15 min at 55 C then adding 1 ml milli-Q water (Carreau
and Dubacq, 1978). Nonadecanoic acid was used as internal standard and FAMEs were extracted in hexane.
The GCMS analysis of FAME samples was carried out on a QP2010 gas chromatographymass spectrometer (GC-2010 coupled
with GCMS QP-2010) equipped with an autosampler (AOC5000) from Shimadzu (Japan) using a RTX-5 fused silica capillary
column, 30 m 0.25 mm 0.25 lm (Rastek). Helium (99.9% purity) was used as the carrier gas with the column ow rate of
1 ml/min and the pre-column pressure of 49.7 kPa. The column
temperature regime was 40 C for 3 min, followed by a 5 C/min
ramp up to 230 C followed by 40 min at 230 C. The injection volume and temperature was 0.2 ll and 240 C and the split ratio was
1/30. The mass spectrometer was operated in electron compact
mode with electron energy of 70 eV. Both the ion source temperature and the interface temperature were set at 200 C. FAME peaks
were identied by comparison of their retention times with
authentic standards by GCMS Post run analysis and quantied
by area normalization.
54
4.4. Indices
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Pseudochattonella farcimen (Dictyochophyceae). Protist 163, 143161.
AI C12 : 0 C14 : 0 C16 : 0=n-3 PUFAs n-6 PUFAs MUFAs; and

TI C14 : 0 C16 : 0 C18 : 0=0:5n-6 PUFAs 3n-3 PUFAs n3=n6 PUFAs:
4.5. Statistical and multivariate analysis

All analytical determinations were performed in triplicate and
the mean values were recorded. The percentages of fatty acids
were compared by analysis of variance (ANOVA) with values signicant at p 6 0.001. The multivariate analyses were performed
using software Unscrambler X (Camo, USA) to establish the relationship between and within different orders and families belonging to the three phyla and to evaluate the distribution of essential
FAs in macroalgal samples. Three principal component analyses
(PCA) were performed separately on FA, FA groups and
PUFA + nutritional indices data matrices of 100 macroalgal samples. FA data matrix included all the FAs reported in Table 2 with
the exception of dodecanoic acid (C12:0), pentadecanoic acid
(C15:0), heptadecanoic acid (C17:0), 10-heptadecenoic acid
(C17:0, n-7), eicosanoic acid (C20:0), 11-eicosenoic acid (C20:1,
n-9), docosanoic acid (C22:0), 13-docosenoic acid (C22:1, n-9)
and tetradocosanoic acid (C24:0) due to their insignicant
amounts and lack of correlation with the data matrix which otherwise might led to misclassication of species. Such variables could
also increase the number of outliers owing to their higher contribution to the score/loading values. Further, FA groups data matrix
included seven variables; SFA, MUFA, PUFA, C18 PUFA, C20 PUFA,
n-3 PUFAs and n-6 PUFAs and PUFA + nutritional indices data matrix included LA, ALA, GLA, STA, AA, EPA, DHA, PUFA/SFA, UI, AI and
TI. In addition, a dendrogram that was obtained by Ward hierarchical clustering, using squared Euclidean distance (Ward, 1963) described by the rst 8 principal components with eigen-values >1.
Ward linkage is an agglomerative clustering algorithm which starts
with n singleton clusters (each consisting of one element of the
data set) and merges two clusters on the basis of a similarity
measure.
Acknowledgments
We would like to thank Chief Conservator of Forest and Wildlife, Jamnagar and Ministry of Forest and Environment, Govt. of
Gujarat, for permitting to collect some algal samples investigated
in this study from Kalubhar Island (off-Vadinar Coast), Marine National Park, Gujarat. We also thank Head, Analytical Sciences, CSIR
CSMCRI for permitting us to use the GC facility and Dr. S.K. Mandal
for sharing the data of physico-chemical parameters for some collection spots in this study. The rst author thanks CSIR for the
award of senior research fellowship (SRF).
Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2012.
10.015.

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Phytochemistry 86 (2013) 4456

Contents lists available at SciVerse ScienceDirect

Fatty acid proling of tropical marine macroalgae: An analysis

synthesized by humans and are thus obtained only through dietary

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P. Kumari et al. / Phytochemistry 86 (2013) 4456

But, there is no consensus among the researchers regarding the

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TL (mg/g fr. wt.)

TL (mg/g fr. wt.)

FAs (TFAs). The major FAs detected in Chlorophyta members were

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P. Kumari et al. / Phytochemistry 86 (2013) 4456

2006; Chakraborty and Santra, 2008; Ortiz et al., 2010; Kumari

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P. Kumari et al. / Phytochemistry 86 (2013) 4456

Pyropia acanthophora var. brasilensis

Rhodophyta and Phaeophyta although it explained only 36% of the

2009; Silberfeld et al., 2010; Tronholm et al., 2010; Pareek et al.,

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P. Kumari et al. / Phytochemistry 86 (2013) 4456

de novo similar to the green and red microalgae as explained by

C22:5 n3 and C22:6 n3 (Leblond et al., 2005). Such endosymbiotic

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P. Kumari et al. / Phytochemistry 86 (2013) 4456

cyanobacteria and higher plants. No homolog for TGD4 involved in

Furthermore, the low amounts of C20 PUFAs in Chlorophyta

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P. Kumari et al. / Phytochemistry 86 (2013) 4456

inherited some type I polyketide synthase (PKS) genes through

variegata, D. bartayresiana, Grateloupia licina, as well as species of

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P. Kumari et al. / Phytochemistry 86 (2013) 4456

4. Materials and methods

4.2. Algal samples

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The unsaturation index (U.I.) was calculated by multiplying the

AI C12 : 0 C14 : 0 C16 : 0=n-3 PUFAs n-6 PUFAs MUFAs; and

4.5. Statistical and multivariate analysis

Appendix A. Supplementary data

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