ACTA BOT. CROAT. 76 (2), 2017 177 Acta Bot. Croat. 76 (2), 177–182, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2017-0008 ISSN 0365-0588 eISSN 1847-8476 Genetic diversity of Potamogeton pectinatus L. in Iran as revealed by ISSR markers Shabnam Abbasi1, Saeed Afsharzadeh2*, Hojjatollah Saeidi3 Department of Biology, University of Isfahan, 81746-73441, Isfahan, Iran Abstract – Potamogeton pectinatus L. is a widespread aquatic species distributed widely in aquatic ecosys- tems of Iran. In this study, inter simple sequence repeat (ISSR) markers were used to assess the genetic diver- sity of 35 accessions and 175 individuals of P. pectinatus collected from different regions of Iran. In total, 123 polymorphic DNA fragments were amplified from five combinations of ISSR primers. The ISSR based prin- ciple coordinate analyses (PCoA) demonstrated four different groups mostly corresponding with their geo- graphic origins (North, Kerman/Fars, Centre and Southwest). The most variable populations were found in the central region of Iran possibly as a consequence of the larger number of samples from that region. The result of molecular variance (AMOVA) attributed 11% of the total genetic variation among and 89% within population variation. The results showed high levels of intra-regional and low inter-regional gene flow be- tween clones, although the Northern accessions were clearly differentiated from the others. There was a low correlation between genetic distance and geographic distance of accessions. The results of STRUCTURE analysis suggested the presence of three genetic groups of this species in Iran, mostly adapted to different ecological conditions. Our results cover one of the gaps of different studies worldwide. In addition, our re- sults confirm high levels of genetic diversity of P. pectinatus in Iran. Key words: genetic diversity, Iran, ISSR, Potamogeton pectinatus, Potamogetonaceae * Corresponding author, e-mail: s.afshar@sci.ui.ac.ir Introduction Potamogeton L. (Potamogetonaceae) comprises about 100 species and 50 interspecific hybrids worldwide (Wieg- leb 1988, Sculthorpe 1967), 14 species of which occur in Iran (Dinarvand 2009, 2011, Abbasi et al. 2015). Among them, P. pectinatus L. is one of the most diverse submerged aquatic species (Sandsten et al. 2005, Wiegleb and Kaplan 1998). This species harbors many useful physiological traits such as tolerance to a wide range of nutrients, ability to grow in oligotrophic to eutrophic waters (Triest et al. 2010), capacity to improve water quality by absorbing nu- trients (Lone et al. 2013) and immunomodulatory activity (Kumar et al. 2012). These traits have made the species a potentially important organic tool for cleaning up polluted waters by absorption of heavy metals (Demirezen and Ak- soy 2004, Ren et al. 2006). Potamogeton pectinatus reproduces both sexually and vegetatively through propagules emerging from the rhi- zomes (van Wijk 1989) and waterfowl probably have an important role in the species dispersal (Green et al. 2002). Accessions of P. pectinatus have been analysed using isozyme (Hettiarachchi and Triest 1991), RAPD (Mader et al. 1998, Hangelbroek et al. 2002), ISSR (King et al. 2002) and AFLP (Han et al. 2014) markers. In most cases there was a geographic pattern of diversity and also a correlation between genetic diversity and migration pathways of water birds (Mader et al. 1998, Hangelbroek et al. 2002). Triest and Fenart (2013) indicated a correlation between genetic structure of clones and habitat type in this species. It has been shown that morphological variation within this species is mostly correlated with ecological conditions (Kaplan 2002), and therefore morphological characters are not precise indicators for evaluation of a population’s ge- netic structure. Of the several molecular markers developed so far, inter simple sequence repeat (ISSR) markers developed by Ziet- kiewicz et al. (1994) have been widely used to detect ge- netic similarities in plants (Zietkiewicz et al. 1994, Mousav- ifard et al. 2015, Akhavan et al. 2015). Due to high polymorphism, only a few ISSR loci, (as few as five to sev- en primer pairs) are sufficient to obtain reliable information about genetic diversity (Matesanz et al. 2011). In the present study, we used ISSR markers to evaluate genetic diversity within Iranian germplasm of P. pectinatus ABBASI S., AFSHARZADEH S., SAEIDI H. 178 ACTA BOT. CROAT. 76 (2), 2017 and elucidate the patterns of diversity in contrast to geo- graphic distribution and dispersal mechanisms. Regarding the progressive drought in Iran which can result in loss of the genetic diversity of this species, we hope that the results of this study will provide useful information for design of more efficient conservation strategies. Materials and methods Plant material A total of 35 accessions of P. pectinatus (each composed of five individuals) were collected from different regions of Iran during July – October 2014. The herbarium voucher specimens are deposited in the herbarium of the University of Isfahan. Accessions were morphologically identified ac- cording to Flora Iranica (Dandy, 1971); Flora Europaea (Dandy 1980), Flora of Turkey (Uotila 1984), Flora of Iraq (Dandy 1985), Flora Palaestina (Feinbrun-Dothan 1986), monograph of Wiegleb and Kaplan (1998). Geological fea- tures and ecological conditions of collecting sites acces- sions were divided into four geographic groups (Center, North, Southwest, Kerman/Fars). Accessions code, locality and other details regarding the plant materials used in this study are provided in Table 1. Tab. 1. Geographical information of the studied Potamogeton pectinatus accessions in Iran; N – North, C – Center, KF – Kerman-Fars, SW- Southwest. Accession code Population Locality Location Coordinates Elevation (m) Pot-p1 N Mazandaran, Tonekobon toward Ramsar River N:36, 47 E:50, 55 –20 Pot-p8 N Mazandaran, Zaghmarz River N:36, 29 E:52, 53 –3 Pot-p7 N Mazandaran, Valasht Lake N:36, 27 E:51, 32 983 Pot-p11 N Gilan, Siahrood River N:36, 18 E:52, 53 62 Pot-p25 N Delijan, Delijan River N:30, 06 E:52, 55 1674 Pot-p18 N Gilan, Langrood River N:44, 53 E:47, 45 76 Pot-p28 N Gilan, Sefidrood River N:37, 22 E:50, 10 –2 Pot-p16 N Gilan, Amirkelaie River N:37, 16 E:50, 13 76 Pot-p32 N Gilan, Siahrood River N:36, 18 E:52, 53 62 Pot-p2 C Isfahan, Hojatabad River N:32, 30 E:50, 50 1902 Pot-p3 C Isfahan, Hamzeali River N:31, 50 E:51, 04 2303 Pot-p4 C Isfahan, Chamaseman Wetland N:32, 24 E:21, 22 1750 Pot-p5 C Chaharmahal va Bakhtiari, Boroujen River N:31, 57 E:51, 19 2128 Pot-p6 C Isfahan, Chamheidar River N:32, 27 E:50, 59 1780 Pot-p9 C Chaharmahal va Bakhtiari, Gandoman Wetland N:31, 48 E:51, 05 2254 Pot-p12 C Chaharmahal va Bakhtiari, Chaghakhor Wetland N:31, 55 E:50, 55 2320 Pot-p15 C Isfahan, Jarghoie River N:32, 09 E:52, 37 1413 Pot-p19 C Isfahan, Cheshmedimeh Spring N:32, 30 E:50, 12 2133 Pot-p21 C Isfahan, Polechoom River N:32, 35 E:51, 46 1578 Pot-p33 C Chaharmahal va Bakhtiari,, Lordegan, Barm Spring N:31, 34 E:51, 12 1635 Pot-p10 KF Kerman, Gogher River N:29, 29 E:56, 38 2740 Pot-p14 KF Kerman, Yaschaman River N:29, 27 E:56, 37 2824 Pot-p20 KF Fars, Hasanabad River N:29, 39 E:53, 20 1621 Pot-p30 KF Fars, Komjan Wetland N:29, 40 E:53, 08 1625 Pot-p31 KF Fars, Komjan Wetland N:29, 40 E:53, 08 1625 Pot-p34 KF Fars, Sivand River N:30, 06 E:52, 55 1717 Pot-p35 KF Kerman, Yaschaman River N:29, 27 E:56, 37 2824 Pot-p13 SW Shushtar, Pole Bande Mizan River N:32, 03 E:48, 51 127 Pot-p17 SW Khuzestan, Izeh toward Lordegan River N:32, 03 E:48, 51 1307 Pot-p22 SW Shushtar, Shushtar River N:32, 03 E:48, 51 127 Pot-p23 SW Khuzestan, Abadan, Minoo Island N:30, 20 E:48, 13 128 Pot-p24 SW Khuzestan, Shadegan Wetland Wetland N:30, 15 E:48, 19 137 Pot-p26 SW Khuzestan, Miangaran Wetland N:31, 52 E:49, 52 925 Pot-p27 SW Khuzestan, Abadan toward Ahwaz Lake N:30, 26 E:48, 11 110 Pot-p29 SW Khuzestan, Susangerd River N:31, 47 E:47, 55 113 GENETIC DIVERSITY OF POTAMOGETON PECTINATUS L. ACTA BOT. CROAT. 76 (2), 2017 179 DNA extraction and PCR From each sample, total DNA was extracted following the method developed by Gawel and Jarret (1991) from the leaves of each accession. In order to perform ISSR analysis, forty combinations of ISSR primers (Blair et al. 1999) were tested, from which five primer pairs (Tab. 2) amplifying de- tectable and polymorphic DNA fragments were selected for further analysis from genomic DNA of P. pectinatus acces- sions. The PCRs were carried out in a 15 µL volume with 250 nM of each primer (Tab. 2), 0.2 mM of each dNTP, 1.5 mM MgCl2, 1 U Taq polymerase, and 50–100 ng of genomic DNA. After 4 min at 95 °C, PCR was followed by 45 cycles of 1 min at 95 °C, 1 min at annealing temperature (50–56 °C; Tab. 3), 2 min at 72 °C, followed by a final extension step of 10 min at 72 °C. PCR products were detected by 2% agarose and ethidium bromide staining under UV light. Data analysis The presence (1) or absence (0) of each band (DNA fragment amplified in PCR) was scored and genetic similar- ity was calculated based on Jaccard (1908) similarity coef- ficients. Correlation between genetic distances and geo- graphic distances (r) was measured using Mantel test statistics (Mantel, 1967) implemented in Genalex software (ver 6.5; Peakall and Smouse 2006). To assess genetic di- versity, basic parameters including Nei`s gene diversity in- dex (h), Shannon index (I), percentage of polymorphic loci (PPL) and mean expected heterozigosity (He) were calcu- lated from the data using POPGENE software ver. 1.32 (Yeh et al. 2000). Analysis of molecular variance (AMO- VA) was performed to calculate the proportion of intra-ac- cession and inter-accession genetic diversity using Genalex 6.5 software. The principal coordinates analysis (PCoA) was performed using Genalex 6.5. To infer the genetic structure of sampled populations, STRUCTURE software was used. Results In the present study, genetic diversity was examined in Potamogeton pectinatus based on ISSR markers. In total, 5 ISSR primer combinations (Tab. 2) amplified 123 DNA fragments from genomic DNA of 35 accessions of P. pecti- natus. The number of bands per primer ranged from 18 to 30 with an average of 24.6 bands per primers pair. The highest and the lowest numbers of bands were produced from the primer combinations ISSR 823 (30 bands) and ISSR 811 – ISSR 812 combination (18 bands), respectively. In PCoA 2D plot (Fig. 1), grouping mainly followed geographic origin. In the PCoA 2D plot, accessions collect- ed from the North were grouped and the southern acces- sions were also loosely grouped. Accessions collected from Isfahan, Kerman and Fars however showed no grouping re- lated to their geographic origin. In the Structure analysis, accessions were divided into three clusters (K=3, red, green and blue in Fig 2, which can be regarded as genetic populations). Accessions collected from the North of Iran and accessions collected from the South (Kerman-Fars) were more uniform than those of the center and southwest (Fig. 2). Mantel test showed a low correlation between genetic distance and geographic distance (r = 0.240, p = 0.02). In analysis of molecular variance (AMOVA), 11% of total genetic variation was attributed to the between and 89% to the within population differentiations and the amount of PhiPT was 0.11. The amount of gene flow be- tween populations (geographic regions) was indirectly cal- culated from PhiPT as Nm = 0.25[(1/PhiPT)–1] = 2. In calculation of Shannon index, which readily trans- lates into heterozygosity, the highest observed heterozygos- Tab. 2. Sequences and annealing temperatures of primers (Blair et al. 1999). Primer ID Sequence (5’→ 3’) Tm ISSR 807 AGAGAGAGAGAGAGAGT 50 UBC 872 GATAGATAGATAGATA 38 ISSR 823 TCTCTCTCTCTCTCTCC 52 ISSR 826 ACACACACACACACACC 52 ISSR 811 GAGAGAGAGAGAGAGAC 52 ISSR 812 GAGAGAGAGAGAGAGAA 50 UBC 873 GACAGACAGACAGACA 48 Tab. 3. Primer combinations, annealing temperature (Ta), percentage of polymorphism, total (T.b.) and average (A.b.) number of bands produced by primers used for inter simple sequence repeat (ISSR). P.b. – number of polymorphic bands; P.p. – percentage of polymor- phism; B.s. – band size range (bp); PIC -polymorphism information content. Primer combination Ta (°C) T.b. P.b. P.p. B.s. A.b. PIC ISSR807+ISSR872 52.1 28 28 100 250–2000 7 0.33 ISSR823 45.7 30 30 100 200–3000 7.5 0.19 ISSR826 44 22 22 100 310–1500 5.5 0.22 ISSR811+ISSR812 48.7 18 18 100 150–700 4.5 0.21 ISSR811+ISSR873 53.9 25 25 100 100–2000 6.25 0.14 Total 123 123 1.09 Average 24.6 24.6 0.218 ABBASI S., AFSHARZADEH S., SAEIDI H. 180 ACTA BOT. CROAT. 76 (2), 2017 ity (0.230) was calculated within the Isfahan (C) population and the lowest one (0.193) was calculated within the Ker- man-Fars (KF) population. The highest polymorphism ratio was calculated between germplasm collected from the Cen- ter of Iran (66.67%) and the lowest one (49.59%) from the South (KF) (Tab. 4). Genetic structure of accessions is shown in Fig. 2. Fig. 1. Principle coordinate analysis (PCoA) 2D plot based on in- ter simple sequence repeat (ISSR) data, the numbers are accession numbers (1–35) of Potamogeton pectinatus from Iran, divided into 4 geographic regions: C – Central, N – North, KF – Kerman/ Fars, S – South West. Fig. 2. Map of the collection site and population structure of the 35 accessions of Potamogeton pectinatus from Iran, grouped in 4 geo- graphic regions (1 – North, 2 – Center, 3 – South, 4 – Southwest) analyzed using inter simple sequence repeat (ISSR) markers. Structure analysis results revealed three clusters (green = cluster 1, red = cluster 2, blue = cluster 3) for 4 regions for the wild Iranian P. pectinatus gene pool. Each vertical column represents one accession. Tab. 4. Genetic diversity within populations of Potamogeton pec- tinatus in Iran revealed by inter simple sequence repeat (ISSR) data. N.a. – number of accessions; P – percentage of polymor- phism at population level; Ae – mean effective number of alleles; Ho – mean observed heterozygosity (Shannon index); He – mean expected heterozygosity (unbiased); h – Nei’s gene diversity. Population N.a. P (%) Ae Ho He h Center 10 66.67 1.201 0.230 0.137 0.13 North 9 56.91 1.201 0.218 0.134 0.13 Kerman/Fars 7 49.59 1.172 0.193 0.117 0.11 Southwest 9 58.54 1.193 0.221 0.134 0.13 Average 57.93 0.215 0.130 GENETIC DIVERSITY OF POTAMOGETON PECTINATUS L. ACTA BOT. CROAT. 76 (2), 2017 181 Discussion Potamogeton pectinatus is distributed along Zagros and Alborz mountains in Iran, in rivers, wetlands, springs, lakes and swamplands characterized by a vast range of environ- mental conditions. Our sampling covered ecologically dif- ferent habitats of the species in Iran. Regarding the species geographic distribution in different ecological conditions, some kind of adaptation-based variation was expected within the Iranian gene pool of this species that may not be well reflected in terms of morphological characters. As Wang et al. (2012) have noted, the mixed mode of repro- duction has an important effect on the genetic structure of P. pectinatus. Asexual reproduction is likely to be responsi- ble for short-distance gene flow, while sexual reproduction is expected to be long-distance dispersal (van Wijk 1989). Therefore, the pattern of diversity in this species could not be influenced by only ecological adaptations. We used ISSR markers to demonstrate the patterns of diversity and test these hypotheses. The results of this study showed that the Iranian gene pool of P. pectinatus harbors a high level of heterozygosity (Ho = 0.25, the highest amount of heterozygosity for domi- nant markers are 0.50). As freshwater and brackish water ecosystems are usually geographically separated, one may expect that aquatic plants in these regions are genetically isolated by distance (see for example Triest et al. 2010). In the case of P. pectinatus, this species grows both in fresh water, brackish water and rivers in a vast range of ecologi- cal conditions. Therefore it has probably experienced dif- ferent adaptations which possibly resulted in higher genetic diversity. Accessions collected from the Center showed higher genetic diversity than those collected from other re- gions. This region is frequently visited by migratory birds coming from northern and southern regions (Sehhatisabet and Khaleghizadeh 2013), which can easily bring seeds that are characterized by different genotypes to this region, re- sulting in higher genetic diversity. The northern accessions of the species were closely clustered and showed low ge- netic diversity (Fig 1). This lower genetic diversity could be the result of lower levels of ecological variation present in this region. Such a situation is also observed in southern (KF) accessions, those that grouped together in dendrogram and PCoA 2D plot. Hangelbroek et al. (2002) indicated that seeds rather than vegetative structures are responsible for gene flow within populations of P. pectinatus. This species is an im- portant food source for many herbivorous waterfowls and water birds (Triest et al. 2010). As seeds can be easily trans- ported by water birds, it can be concluded that seeds are more probably responsible for long distance gene flow. Iran is on the migration way of birds coming from the northern and southern territories. Therefore they have probably an important role in gene flow among isolated populations as in the central region of Iran. As shown in PCoA 2D plot, accessions were clearly grouped in correspondence to their geographical origin. This can be interpreted as an indication for establishment of specific genotypes in different regions with high intra-regional and low inter-regional gene flow between clones. The clones sampled in the north are geneti- cally more similar than the ones from the other regions and such finding can be a result of a relative uniformity of the ecological conditions in the Northern region. The STRUCTURE diagram divided accessions into three clusters (red, blue and green, Fig. 2). The results cor- responded with the grouping in PCoA. The genetic structur- ing suggested that there are three genetic groups of the spe- cies in Iran, which presumably adapted to different ecological conditions. The result of Mantel test, which showed no significant correlation between genetic and geo- graphic distance, indicates that dispersal occurs in different directions and probably with different mechanisms (sexual and asexual) involved in the dispersal of the species (Wang et al. (2012). The results of present study were partly in contrast with the results of Mader et al. (1998) which showed a correlation between genetic distance and geo- graphic distance. In that study, the analyzed accessions were collected from Europe, North Africa, North and South America, whereas our collection originated from a smaller region (Iran), in which habitats are frequently visited by mi- gratory birds. Therefore, it can be concluded that on a geo- graphically small scale, as in the present case, inter popula- tions gene flow occurs more frequently than on a larger scale, as in the collection of Mader et al. (1998). According to our results, P. pectinatus is genetically highly diverse. Therefore a greater sampling would be valu- able for planning a conservation strategy, but not a broad diversity analysis such as that carried out here. Future re- search should be oriented towards investigation of genetic basis of drought resistance. Acknowledgements This work was a part of the PhD thesis of the first author at the University of Isfahan. The authors are grateful to the University of Isfahan for their support. References Abbasi, S., Afsharzadeh, S., Dinarvand, M., 2015: Potamogeton friesii, a new record for the Flora of Iran. Iranian Journal of Botany 21, 39–42. Akhavan, A., Saeidi, H., Rahiminejad, M. R., Zarre, S., Blattner, F. R., 2015: Interspecific relationships in Allium subgenus Melanocrommyum sections Acanthoprason and Asteroprason (Amaryllidaceae) revealed using ISSR markers. Systematic Botany 40, 706–715. Blair, M. W., Panaud, O., McCouch, S. R., 1999: Inter simple se- quence repeat (ISSR) amplification for analysis of microsatel- lite motif frequency and finger printing in rice (Oriza sativa L.). Theoretical and Applied Genetic 98, 780–792. Dandy, J. E., 1971: Potamogetonaceae. In: Rechinger, K. H. (ed.), Flora Iranica 83. Akademisch Druk- und Veragsanatalt, Graz, Austria. Dandy, J. E., 1980: Potamogetonaceae. In: Tutin, T. G., Heywood, V. H., Burges, N. A., Moore, D. M., Valentine, D. H., Walters, ABBASI S., AFSHARZADEH S., SAEIDI H. 182 ACTA BOT. CROAT. 76 (2), 2017 S. M., Webb, D. A. (eds.), Flora Europaea 5, 7–11. Cambridge University Press, Cambridge. Dandy, J. E., 1985: Potamogetonaceae. In: Townsend, C.C, Guest, E. (eds.), Flora of Iraq 8, 19–26. Department of Agriculture and Agrarian Reform Republic of Iraq, Baghdad. Demirezen, D., Aksoy, A., 2004: Accumulation of heavy metals in Typha angustifolia and Potamogeton pectinatus living in Sul- tan Marsh) Kayseri, Turkey). Chemosphere 56, 685–696. Dinarvand, M., 2009: Two new records of the genus Potamogeton (Potamogetonaceae) for the aquatic flora of Iran. Iranian Jour- nal of Botany15, 164–166. Dinarvand, M., 2011: New record of the genus Potamogeton (Po- ta mogetonaceae) for the aquatic flora of Iran. Iranian Journal of Botany 17, 230–232. Feinbrun-Dothan, N., 1986: Potamogetonaceae. In: Zohary, M. (ed.), Flora Palaestina. Jerusalem. 4, 6–12. Gawel, N. J., Jarret, R. L., 1991: A modified CTAB extraction pro- cedure for Musa and Ipomoea. Plant Molecular Biology Re- porter 9, 262–266. Green, A. J., Figuerola, J., Sanchez, M. L., 2002: Implications of waterbird ecology for the dispersal of aquatic organisms. Acta Oecologica 23, 177–189. Han, Q., Wang, G., Li, W., Liu, F., 2014: Genetic diversity of Potamogeton pectinatus L. in relation to species diversity in a pair of lakes of contrasting trophic levels. Biochemical Sys- tematics and Ecology 57, 60–66. Hangelbroek, H., Ouborg, N. J., Santamaria. L., Schwenk, K., 2002: Clonal diversity and structure within a population of pondweed Potamogeton pectinatus foraged by bewicks swans. Molecular Ecology 11, 2137–2150. Hettiarachchi, P., Triest, L., 1991: Isozyme polymorphism in the genus Potamogeton (Potamogetonaceae). Opera Botanica 4, 87–114. Jaccard, P., 1908: Nouvelles recherches sur la distribution florale. Bulletin de la Société Vaudoise des Sciences Naturelles 44, 223–270. Kaplan, Z., 2002: Phenotypic plasticity in Potamogeton (Potamo- getonaceae). Folia Geobotanica 37, 141–170. King, R. A., Gornall, R. J., Preston, C. D., Croft, J. M., 2002: Pop- ulation differentiation of Potamogeton pectinatus L. in the Baltic Sea with reference to waterfowl dispersal. Molecular Ecology 11, 1947–1956. Kumar, A. A., Meena, V. S., Chattopadhyay, S., Panigrahi, K. C., 2012: Novel immunomodulatory effect of Gracilaria verru- cosa and Potamogeton pectinatus extracts on in vitro activa- tion of T cells. International Journal of Pharmaceutical and Life Sciences 2, 233–239. Lone, P. A., Bhardwaj, A. K, Bahar, F. A., 2013: A study of com- parative purification efficiency of two species of Potamogeton (submerged macrophyte) in wastewater treatment. Interna- tional Journal of Scientific Research 3, 1–5. Mader, E., Viersen, W.V., Schwenk, K., 1998: Clonal diversity in the submerged macrophyte potamogeton pectinatus L. in- ferred from nuclear and cytoplasmic variation. Aquatic Bota- ny 62, 147–160. Mantel, N., 1967: The detection of disease clustering and a gener- alized regression approach. Cancer Research 27, 209–220. Matesanz, S., Gimeno, T. E., de la Cruz M., Escudero A., Val- ladares, F., 2011: Competition may explain the fine-scale spa- tial patterns and genetic structure of two co-occurring plant congeners. Journal of Ecology 99, 838–848. Mousavifard, S. S., Saeidi, H., Rahiminejad, M. R., Shamsadini, M., 2015: Molecular analysis of diversity of diploid Triticum species in Iran using ISSR markers. Genetic Resources and Crop Evolution 62, 387–394. Peakall, R., Smouse, P. E., 2006: GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and re- search. Molecular Ecology Notes 6, 288–295. Ren, D., Peng, G., Huang, H., Wang, H., Zhang, Sh., 2006: Effect of rhodoxanthin from Potamogeton crispus L. on cell apopto- sis in Hela cells. Toxicology in Vitro 20, 1411–1418. Sandsten, H., Beklioglu, M., Ince, O., 2005: Effect of waterfowl, large fish and periphyton on the spring growth of Potamoge- ton pectinatus L. in lake Mogan Turkey. Hydrobiologia 537, 239–5478. Sculthorpe, C. D., 1967: Biology of aquatic vascular plants. Ed- ward Arnold (Publishers) Ltd., London. Sehhatisabet, M. E and Khaleghizadeh, A., 2013: A review of cur- rent knowledge of radio-tracking of waterbirds and raptors in Iran. Podoces 8, 22–30. Triest, L., Thi, V. T., Thi, D. L., Sierens, T., Geert, A. V., 2010. Genetic differentiation of submerged plant populations and taxa between habitats. Hydrobiologia 656, 15–27. Triest, L., Fenart, S., 2013: Clonal diversity and spatial genetic structure of Potamogeton pectinatus in managed pond and river populations. Hydrobiologia 737, 145 – 161. Uotila, P., 1984: Potamogetonaceae. In: Davis, P. H. (ed.), Flora of Turkey and the East Aegean Islands. Edinburgh 8, 17–22 van Wijk, R. J., 1989: Ecological studies on Potamogeton pectina- tus L.: general characteristics, biomass production and life cy- cles under field conditions. Aquatic Botany 31, 211–256. Wang, B., Lin, Y., Guo, Y., Cui, X., 2012: The relative contribu- tion of sexual and asexual reproduction to genetic variation in natural populations of the pondweed Potamogeton pectinatus. Israel Journal of Ecology and Ecology 58, 27–38. Wiegleb, G., 1988: Notes on pondweeds outlines for a mono- graphical treatment of the genus Potamogeton L. Repertorium specierum novarum regni vegetabilis 99, 249–266. Wiegleb, G., Kaplan, Z., 1998: An account of the species of Pota- mogeton L. (Potamogetonaceae). Folia Geobotanica 33, 241– 316. Yeh, F. C., Yang, R., Boyle, T. J., Ye, Z., Xiyan, J. M., 2000: POP- GENE 32: Microsoft windows-based freeware for population genetic analysis. Edmonton: Molecular biology and biotech- nology center, University of Alberta. Zietkiewicz, E., Rafalski, A., Labuda, D., 1994: Genome finger- printing by simple sequence repeat (SSR) anchored poly- merase chain reaction amplification. Genomics 20, 176–183.