Article. Morphological diversity of Sargassum species of Iran. Zahra Noormohammadi1*, Somaye Ghasemzadeh Barki1, Masoud Sheidai2,

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1 Article Morphological diversity of Sargassum species of Iran Zahra Noormohammadi1*, Somaye Ghasemzadeh Barki1, Masoud Sheidai2, Farnaz Rafiee3, Bayram Mohammad Gharanjik4 1- Biology Department, School of Basic sciences, Science and Research Branch, Islamic Azad University (SRBIAU), Poonak, Tehran, Iran 2- Shahid Beheshti University, GC, Faculty of Biological Sciences, Tehran, Iran 3- Faculty of Marine Sciences and Technology, North Tehran Branch, Islamic Azad University, Tehran, Iran 4- Offshore Fisheries Research Center, Chabahar, Iran Geneconserve 10(39): 1-22 Received November 20, 2010 Accepted January 3, 2011 Abstract Sargassum species are one of economically important brown algae in south of Iran. In this study, morphological variation was assessed in three Sargassum species widely distributed species in the southwest of Iran (Oman sea): Sargassum tenerrimum J. Agardh, Sargassum glaucescens J. Agardh and Sargassum ilicifolium C.Agardh. At each site, ten different nonproductive individuals were selected with a minimum distance of 2-3 meters at low tide or snorkeling. We investigated ten measurable and ten categorical morphological parameters from three localities (Chabahar, Quatr and Tang) using ANOVA test as well as factorial analysis. The ANOVA test performed among populations of each species as well as among species showed significant difference (p<0.01) for all quantitative characters used. Different clustering like UPGMA and Neighbor Joining separated populations of each species studied based on measurable characters while both measurable and categorical characters have main rules in differentiation of three species PCA analysis confirmed cluster analysis. In total, both measurable and categorical characters enable to discriminate species while categorical parameters studied did not vary among populations of each species studied. This is the first study on evaluation of inter-population variation of Sargassum species in Iran. Key words: Iran, Morphological characters, Sargassum species Introduction

2 The genus Sargassum C. Agardh (1820) with more than 400 species is the richest genus with more species within the Sargassaceae family recorded by Guiry and Guiry (2007). Different morphological studies has been carried out on Sargassum in several seashores of the world including taxonomic revision in French Polynesia (Mattio et al., 2008) and the northwest pacific (Cheang et al., 2008). In Iran, Børgesen (1939) identified 26 species of brown algae in the Persian Golf seashore. Recently Sohrabipour and Rabii (1996 and 1999) have identified 6 Sargassum species in this area, while Gharanjik (2005) reported different Sargassum species including, S. assimile Harvey, S. swatzii Turner, S. glaucesence J. Agardh, S. cristaefolium (C. Ag.) J. Ag., S. liciforium (Turner) C.Agardh, S. virgatum C. Ag. and S. tenerrimum J. Agardh in Sistan and Baloochestan seashore (Oman sea) located in southeast of Iran. Abdel- Kareem (2009) also reported sixteen Sargassum species in Persian Golf in Arabic (Saudi Arabia) coasts, four of which are also reported by Sohrabipour and Rabii (1999). In the Sistan and Baloochestan seashore in the south of Iran, Sargassum species with more than 40 tons annual biomass production have been considered as economically important brown algae (Hosseini and Gharanjik 2003). Although recently identification of brown algae, especially Sargassum species has attracted attention, in general there is limited information about Sargassum populations and the studies performed mainly concern the identification of species by using morphological characters and no numerical analysis has been performed to identify the intra and inter populations variation. This study attempt to evaluate genetic variation by morphological study of among and within three Sargassum species present in the Oman sea seashores. Materials and Methods Three sargassum species including Sargassum tenerrimum J. Agardh, Sargassum glaucescens J. Agardh and Sargassum ilicifolium C.Agardh (Figure 1) were collected from November to December 2009 (the period of highest density in this region before senescence period) at three coastal sites of the Oman sea namely Chabahar, Tang and Quartz (Table 1, Figure 2). To minimize the effect of temporal and seasonal variations and to retain as much morphological information as possible, the specimens were collected at the same stage, before leaf loss and development of reproductive structures (Ang, 1985). At each site, ten different nonreproductive individuals were selected with a minimum distance of 2-3 meters at low tide or snorkeling. The specimens collected were placed in plastic bags and transported to the laboratory and fixed with 4% formalin seawater solution and mounted on herbarium sheets. The voucher specimens are deposited in Herbarium of Science and Research Branch Islamic Azad University (HSRBIAU). Twenty morphological characters were used for numerical analysis, including ten measurable and ten categorical morphological parameters taken from published materials on Sargassum (Cheang et al., 2008), and species description following Stearn (1983) and Tseng (1983) (Table 2). In measurable characters, as far as possible, ten measure leaves (taken from higher part of specimens) and vesicles in length and width as well as vesicle stalk length per specimen were taken in mm with a caliper to the nearest 0.05 mm. Mean size of each character per specimen used for numerical analyses. In order to determine significant difference for quantitative morphological characters among populations and species studied, analysis of variance (ANOVA) followed by Least Significant Test (LSD) was performed. Species or populations considered as fixed factors without transformation (if data is not percentage) and Bartlet test performed to show data normality.

3 Different clustering methods including UPGMA (Unweighted Paired Group using Arithmetic Average), Neighbor Joining (NJ), maximum parsimony as well as ordination plot based on Principal Components Analysis (PCA) were performed by using both quantitative and qualitative characters. Co-phenetic correlation and bootstrapping (100 replicates) were performed to check the fit of dendrograms obtained (Podani 2000) and choosing the best method. PCA also may show better spatial grouping based on most variable characters and avoiding involve all data input. For numerical analyses, the mean of quantitative characters were used while qualitative characters were coded as binary or multistate characters (Table 2). Factor analysis was used to identify the most variable morphological characters among species studied. Morphological data were standardized (Mean = 0, variance = 1) for factor analysis. The same is true for cluster analysis used to determine taxonomic and Euclidean distances. The fit of dendrograms were checked by estimating co-phenetic correlation and bootstrap values (Podani, 2000). Results and Discussion Ten quantitative characters of the three Sargassum species were analyzed and their means and standard deviation were determined (Table 3). ANOVA test (Table 3) performed among populations of each species as well as among species showed significant difference (p<0.01) for all quantitative characters used except maximum vesicle length and width which were not shown significant differences (p>0.01) among species studied (Table 3). LSD test showed that almost all quantitative characters differed significantly among three species studied (data not shown). However, no significant difference was observed between S. ilicifolium and S. tenerrimum for maximum leaf length and also between S. ilicifolium and S. glaucescens for maximum of leaf length to leaf petiole length ratio although these characters were significant in ANOVA test when all 3 species were analyzed together. Principle component analysis identified 3 factors which explain 94.5 % of the total variance. The first factor comprised about 70.3% of total variance, while second and third factors comprise about 16.3% and 7.8% of total variances respectively (Table 4). In the first component, high positive correlation was observed for all categorical characters (> 0.8) as well as maximum leaf length to maximum leaf width ratio and leaf petiole length characters (> 0.7). However, the rest of measurable characters showed high negative variation (> - 0.7) with this component. The Second principle component showed high positive variation in vesicle characters (> 0.6) with no variation in categorical characters, while the third component revealed positive correlation only with the leaf length (> 0.7). No variation was observed among populations in Sargassum species for categorical characters. These results show that both measurable and categorical characters may be used to identify and discriminate Sargassum species, while categorical parameters are not useful for this task because of being invariable in populations of each species while enable to discriminate Sargassum species. Cheang et al. (2008) as well as Wong et al., 2004 also reported that measurable characters specially leaf and vesicle sizes are the most important morphological parameters to be used for discriminating Sargassum species. In respect to sea surface temperature (SST) and effects of salinity levels on morphological characters, no differences observed in these physicochemical factors (Table 1) among three localities studied. Cheang et al. (2008) suggested possible influence of changes in SSTs on leaf and vesicle morphologies of Sargassum species

4 studied northwestern Pacific areas. Different clustering methods performed using both quantitative and qualitative morphological characters produced similar results (Figures 3 and 4). Both NJ and UPGMA clustering grouped 3 species populations separately. However, dendrogram obtained through UPGMA method (Figure 4) showed the best fit of tree obtained to the original morphological data with the highest cophenetic coefficient (r = 0.98). Three major groups/clusters were identified. The first cluster is mainly comprised of three S. tenerrimum populations. Each population forms a distinct group by significant variation (p < 0.05) in measurable characters except maximum vesicle length to maximum vesicle width ratio and vesicle stalk length (p > 0.05). No significant difference was observed in categorical characters among 3 populations of this species. One specimen of Chabahar population (T-Ch5) stands far from the other in this population because of differences in vesicle characters (Figure 3). The second group consists of S. ilicifolium populations while S. glaucescens populations make the third group. Populations of these two species clustered separately and differed significant in all measurable characters except leaf width for S. ilicifolium populations and leaf width as well as leaf petiole length for S. glucescens populations. In PCA ordination S. ilicifolium and S. tenerrimum populations stand close while S. glaucescens populations stand far from others. The position of these species in the PCA1 axis is due to all categorical characters and all measurable characters except leaf length (Figure 5). In the PCA2 axis, populations of S. ilicifolium and S. tenerrimum populations are separated which is due to vesicle characters (PC2, Table 4; Figure 5). In general both measurable and categorical characters studied can differentiate Sargassum species from each other and may be used in taxonomy of Sargassum. In inter and intra population studies measurable characters are suitable characters and could discriminate population of each species while categorical characters with lack of variations among populations are not useful in this level. Acknowledgments The authors gratefully acknowledge Science and Research Branch, Islamic Azad University (SRBIAU). Thanks are kindly given to Mr. B. M. Gharanjik for the assistance in sample collection. References Abdel-Kareem MSM (2009). Phenetic Studies and New Records of Sargassum Species (Fucales, Phaeophyceae) from the Arabian Gulf Coast of Saudi Arabia Acad. J. Plant Sci. 2 (3): Agardh CA (1820). Species Algarum Rite Cognitae, cum Synonymis, Differentiis Specificis et Descriptionibus Succinctis. Vol. 1(1). Berling, Lund, Sweden, pp Ang PO (1985). Regeneration studies of sargassum siliquosum J. Ag. And S. paniculatum J. Ag. (Sargassaceae, phaeophyta). Bot. Mar. 28: Børgesen F (1939). Marine algae from the Iranian Gulf. In: Danish Scientific Investigations in Iran (K. Jessen and R. Sparck, eds.). Einar, Munksgaard, Copenhagen 1:

5 Cheang CC, Chu KH and Ang PO (2008). Morphological and genetic variation in the populations of Sargassum hemiphyllum (Phaeophyceae) in the Northwestern Pacific. J. Phycol. 44: Guiry MD and Guiry GM (2007). AlgaeBase Version 4.2. World-wide electronic publication. National University of Ireland, Galway. Available at: (accessed on 5 June 2007). Gharanjik BM (2005). Determination of biomass and expansion of Algae and preparation of Persian Golf and Oman Sea Algae Atlas. Project No: 84/538, Offshore Fisheries Research Center, Published in: Research Institute of Fisheries of Iran. Hosseini MR, Gharanjik BM (2003). Determination of biomass and expansion of Sargassum glaucesens in Iran. Iran. J. Fisheries 11(3): Mattio L, Payri CE and Stiger-Pouvreau V (2008). Taxonomic revision of Sargassum (Fucales, Phaeophyceae) from French Polynesia based on morphological and molecular analysis. J. Phycol. 44: Podani J (2000). Introduction to the Exploration of Multivariate Data. English translation. Backhuyes Publishers, Leide, pp Sohrabipour J and Rabii R (1996). New records of Algae for Persian Gulf and flora of Iran. Iran. J. Bot. 7 (1): Sohrabipour J and Rabii R (1999). A list of marine Alexandria University for his kind help and reviewing the algae of seashores of Persian Gulf and Oman manuscript. Sea in the Hormozgan Province. Iran. J. Bot. 8: Stearn WT (1983). Botanical Latin. New ed. Fitzhenry and Whiteside, Toronto, Canada, pp Tseng CK (1983). Order Fucales, Family Sargassaceae. In Tseng CK [Ed.] Common Seaweeds of China. Science Press, Beijing, China: Wong CL, Gan SY and Phang CM (2004). Morphological and molecular characterization and differentiation of Sargassum baccularia and S. polycystum (Phaeophyta). J. Appl. Phycol. 16: Table 1. Sample details of Sargassum populations detected in the study. Abbreviation: Ch = Chabahar; Qu = Quatr; Ta = Tang; SST = Sea Surface Temperature population species Sample site SST/Salinity Ch Sargassum tenerrimum Chabahar, Sistan & Balochestan /35pptprovince; N 61 39, E ppt Sargassum glaucescens Sargassum ilicifolium Qu Sargassum tenerrimum Sargassum glaucescens Quatrz, Hormozgan province, N 61 30, E / 35ppt- 40ppt Sargassum ilicifolium Ta Sargassum tenerrimum Tang, Hormozgan province, N 59 54, E /35ppt- 39ppt

6 Sargassum glaucescens Sargassum ilicifolium Table 2. List of morphological parameters and their coding used in this study Measurable parameters: LL LW LL/W LP LL/P VL VW VL/W VS VL/S Description Maximum leaf length Max. leaf width Max. leaf length to Max. leaf width ratio Leaf petiole length Max. leaf length to leaf petiole length ratio Max. vesicle length Max. vesicle width Max. vesicle length to Max. vesicle width ratio Vesicle stalk length Max. vesicle length to vesicle stalk length ratio Categorical parameters: LT LS LM Leaf tip (1= Apices blunt, 2=Obtuse, 3= Narrow rounded Leaf shape (1= Elongated ellipsoid, 2= Elongated ellipsoid or obovated, 3= Elongated-lanceolate) Leaf margin (1 = Sinuate dentate, 2= Dentate VM VSt VA LC M Axis outward margin more dentate than inward, 3= Dentate) Vesicle margin (1= Sharp mucronate, 2= Ear like wing, 3= Mucronate) Vesicle stalk (1= Spherical, 2= Ovate, 3= Subspherical, ovate Vesicle arrangement (Single - on branch beside lamela ) Leaf color (1= Greenish brown, 2= Yellowish brown) Midrib (1= Central, 2= Central-apix, 3= Central, vanishing below apices ) 1=Round, 2=Cylindrical Thallus 1= Slightly twisted, 2=Tense zigzag, 3= Ssmooth Table 3. Comparison of 10 morphological characters in S. tenerrimum, S. glaucescens and S. ilicifolium (mean ± S.D.) and ANOVA test (F and significance level) Morphological S. tenerrimum S. S. ilicifolium F Sig. character (cm) glaucescens LL 4.10± ± ± LW 0.54± ± ± LL/W 7.54± ± ± LP 0.33± ± ± LL/P 12.63± ± ± VL 0.45± ± ± VW 0.35± ± ± VL/W 1.29± ± ±

7 VS 0.30± ± ± VL/S 1.50± ± ± Table 4. Three first principle components based on PCA analysis in three species studied (Abbreviation as in Table 2). Legend to Figures 1-5. Figure 1. Thallus of the three Iranian Sargassum species studied: Sargassum glaucescens (a), Sargassum tenerrimum (b) and Sargassum ilicifolium (c). Figure 2. Location of the three collection localities in Sistan and Baloochestan seashores. Figure 3. Neighbor Joining dendrogram based on all morphological characters. (The numbers above branches indicate bootstrap values based on 100 replicates). Figure 4. UPGMA dengrogram based on all morphological characters in three species of Sargassum populations studied. Abbreviations: Tch = Chabahar population of S. tenerrimum; TTa = Tang population of S. tenerrimum; TGu = Guatr population of S. tenerrimum; GCh = Chabahar population of S. glaucescens; GTa = Tang population of S. glaucescens; GGu = Guatr population of S. glaucescens; ICh = Chabahar population of S. ilicifolium; ITa = Tang population of S. ilicifolium; IGu = Guatr population of S. ilicifolium Figure 5. PCA ordination based on all morphological characters in three Sargassum populations studied ( S. tenerrimum, + S. ilicifolium, S. glaucescens).

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