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Some researchers suggest that Zostera japonica has important roles as primary producers in tidal flat ecosystem. Recently, it is known that the methods involving tracers in stable isotope ratio analysis and fatty acid structures stand up to clarify the origin of organic matter. If these methods can be used, it would be possible to clarify the role of organic matter derived from Z. japonica. However, these methods cannot be used yet because the characteristics of stable isotope ratios and fatty acid structures in Z. japonica are not clear. The objectives of this research are the clarification of the characteristics of stable isotope ratio and fatty acid structure in Z. japonica. Moreover, the availability as tracer of organic matter origin was explored; and the characteristics of stable isotope and fatty acid structure of Z. japonica were compared with other primary producers in tidal flat. Results showed that Z. japonica contained large amounts of 18:2n6 and 18:3n3 and they were used as biomarkers of other seagrass-derived organic matter. In addition, Z. japonica also contained LCFAs, which is used as biomarker of terrestrial plants. These results show that LCFAs are not the only biomarkers that can be used to trace the organic matter in terrestrial plant
Journal of Water and Environment Technology, Vol. 9, No.2, 2011 Address correspondence to Yumi Nagahama, Department of Civil and Environmental l Engineering, Tohoku University, Email: nagahama@costep.hucc.hokudai.ac.jp Received May 10, 2010, Accepted February 1, 2011. - 101 - Characterization of the Carbon Stable Isotope Ratio and Fatty Acid Structure of Zostera japonica in Coastal Areas Yumi NAGAHAMA, Munehiro NOMURA, Megumu FUJIBAYASHI, Woo-seok SHIN, Osamu NISHIMURA Department of C ivil and E nvironmental E ngineering, Tohoku University, Se ndai 9 80-8579, Japan ABSTRACT Some researchers suggest that Zostera japonica has important roles as primary producers in tidal flat ecosystem. Recently, it is known that the methods involving tracers in stable isotope ratio analysis and fatty acid structures stand up to clarify the origin of organic matter. If these methods can be used, it would be possible to clarify the role of organic matter derived from Z. japonica. However, these methods cannot be used yet because the characteristics of stable isotope ratios and fatty acid structures in Z. j aponica are not clear. The objectives of this research are the clarification of the characteristics of stable isotope ratio and fatty acid structure in Z. japonica. Moreover, the availability as tracer of organic matter origin was explored; and the characteristics of stable isotope and fatty acid structure of Z. j aponica were compared with other primary producers in tidal flat. Results showed that Z. japonica contained large amounts of 18:2n6 and 18:3n3 and they were used as biomarkers of other seagrass-derived organic matter. In addition, Z. japonica also contained LCFAs, which is used as biomarker of terrestrial plants. These results show that LCFAs are not the only biomarkers that can be used to trace the organic matter in terrestrial plants. Keywords: LCFAs, primary producers, seagrass, tidal flat, Zostera marina, δ 13 C INTRODUCTION The coastal ecosystems contribute to human welfare, both directly and indirectly, because they have high ability for biological production. Seagrasses in particular, serve an important role as organic matter by being a food source in coastal ecosystems. Our previous research suggested that Zostera japonica, a kind of seagrass that grows in the intertidal zone, plays an important role for benthic ecosystems by supplying organic matter for the benthos (Nagahama et a l., 2005). Some researches show that benthic fauna in Z. japonica meadows differ from areas without seagrasses (Posey, 1998; Lee et al., 2001). They consider that the difference in organic matter, both in quality and quantity, cause the difference in benthos. However, the role of the supply of organic matter derived from Z. japonica to benthic invertebrates was not clarified. Recently, some researchers have studied the role of the supply seagrass-derived organic matter by stable isotope ratio and fatty acids as tracers of organic matter (Kharlamenko et al., 2001; Jaschinski et al., 2008). The stable isotope ratio and fatty acid analysis as food tracers demonstrate the role of Z. japonica as food in benthic ecosystems. However, organic matter derived from Z. japonica could not be traced because the characteristics of the carbon stable isotope ratio and fatty acid structure of Z. japonica have not been known. In this study, we aim to clarify the characteristics of carbon stable isotope ratio and fatty acid structure of Z. japonica. Moreover, we also aim to consider the difference of stable Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 102 - HK MG NG KT KG HK MG NG KT KG Table 1 Sampling conditions and cooperating institutions during sampling. Site Code Sampling Month-Year Water depth (m) Water temp. (℃) n Cooperating Institutions Z.jap Poronuma, Hokkaido Pref. HK Jun-09 0.5 14 3 Wakkanai Fisheries Research Institute Sarufutsu-mura gyogyou kyodo kumiai Katsurashima, Miyagi Pref. MG Jun-06 0.5 14 9 Our team Toyota, Niigata Pref. NG Jun-09 3.5 21 3 Sado suisan gijyutsu center Tai coast, Kyoto Pref. KT Jun-09 2.6 24 3 Fisheries Technology Department; Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Center Oshima, Kagawa Pref. KG Jun-09 1.5 26 3 KAGAWA-SUISHI・AKASIOKEN Z.mar Katsurashima, Miyagi P ref. MG Sep-08 1.5 14 3 Our team Toyota, Niigata Pref. NG Jun-06 3.5 22 3 Sado suisan gijyutsu center Monjyu coast, Kyoto Pref. KT Jun-09 1 24 3 Fisheries Technology Department; Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Center Oshima, Kagawa Pref. KG Jun-09 2 26 3 KAGAWA-SUISHI・AKASIOKEN Z. japonica Z. marina isotope ratio and fatty acid structure of Z. japonica with other primary producers in coastal areas. Our research promotes the use of stable isotope ratio and fatty acid structure of Z. japonica for the study of tidal flat ecosystems. MATERIALS AND METHODS Sample collection In June 2006, samples of Z. japonica were collected in Katsurashima at Matsushima Bay, Miyagi Prefecture (38° 20’ 7.4N, 141° 5’ 14.7W). Using a shovel, Z. japonica samples were collected at the center of the meadows, and then the samples were frozen immediately upon reaching the laboratory. In September 2008, Z. marina were collected from the sublittoral zone in the same area. The methods of collection and preparation were the same as Z. japonica. In addition, in June 2009, Z. japonica and Z. marina were collected in Hokkaido Prefecture, Niigata Prefecture, Kyoto Prefecture, and Kagawa Prefecture. The seagrasses were frozen after collection. The research institutes of each prefecture cooperated for these sample collections. Information regarding the sampling site and sample collection are shown in Table 1 and Fig.1. The water temperatures were Fig. 1 - Sampling sites for seagrasses Table 1 - Sampling conditions and cooperating institutions during sampling Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 103 - based on the data of Japan Meteorological Agency and only the water temperature in Niigata Prefecture was measured during sampling. In the laboratory, the samples were defrosted by running water. Next, the leaves and stems were immediately separated from the roots and rhizomes, and these samples were granulated after freeze-drying. The granulated samples were stored in a freezer (-20ºC). Analysis of carbon stable isotope The granulated samples were used for analysis. Carbon stable isotope ratio was determined using a mass spectrometer (DELTA plus; Finnigan Mat, USA). Isotope ratios are represented in the delta notation as shown below: Pee Dee Belemnite (PDB) was used as the reference to δ 13 C and analytical error was within ± 0.2‰ for δ 13 C. Analysis of fatty acids The methods of Meziane and Tsuchiya (2000) were used for the extraction of lipids. A gas chromatograph (GC-17A; Shimadzu, Japan) was used for the analysis. Fatty acid methyl esters (FAME) were separated with a capillary column (CP-Select CB for FAME, 100m × 0.25mm i.d.). The standards used were PUFA No.3 (47085-U, SUPELCO, USA); Supelco TM 37 Component FAME Mix (47885-U, SUPELCO, USA); Bacterial Acid Methyl Esters Mix (47080-U, SUPELCO, USA); and 6 kinds of long-chain fatty acids (LCFAs). These standards were used to obtain 56 kinds of fatty acids. Therefore, the contents of each fatty acid were calculated based on the quantity of lipid, the content ratios of all the fatty acids, and the ratio of each fatty acid. RESULTS AND DISCUSSION Characteristics of carbon stable isotope in Z. japonica The mean δ 13 C of Z. japonica was -12.4 ± 1.6‰ in the leaves and stems, and -11.6 ± 1.5‰ in the roots and rhizomes. Meanwhile, Z. marina in the leaves and stems has -9.7 ± 1.0‰, and in the roots and rhizomes has -9.6 ± 1.0‰. Fig. 2 shows that the leaves and stems as well as the roots and rhizomes of Z. j aponica have significantly lower δ 13 C than those of Z. marina (n = 12, p < 0.01). Kharlamenko et al. (2000) showed that δ 13 C of Z. marina was -6.7 ± 0.1‰ in the leaves and -8.9 ± 0.1‰ in the rhizomes. Svensson et al. (2007) showed that δ 13 C of Z. tasmanica was -9.9 ± 0.1‰. Also, Hemminga et al. (1996) showed that the distribution of δ 13 C in seagrasses had a single-peaked pattern, and the mode value of pattern was about -10‰. These results suggest that δ 13 C of Z. japonica tend to be lower than the δ 13 C of some kinds of seagrass such as Z. marina. We consider that the δ 13 C of Z. japonica became lower than Z. marina because Z. japonica might have used enough carbon for photosynthesis. Generally, Z. japonica grows up in a shallower zone than Z. marina . Seawater in the intertidal zone should contain more CO 2 than in the sublittoral zone because the seawater in the intertidal zone CCR RRC standardsample 1213 13 10001 … (1) … (2) Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 104 - is disturbed by breaking waves. Typically, δ 13 C of plants decrease when there is enough carbon to be used. In fact Z. noltii, which often grows in the intertidal zone, has δ 13 C of -14.4 ± 1.6‰ (Carlier et al., 2007). Next, we compared the δ 13 C in the leaves and stems with the δ 13 C in the roots and rhizomes of Z. japonica. We thought that it was important to divide them into several parts. We often find seagrass leaves piling up in the upper side of coastal areas. In addition, benthic ecosystems might have influenced the roots and rhizomes rather than the leaves and stems. If we can separate the leaves and stems from the roots and rhizomes, we might be able to study the deciduous effect of the respective parts of Z. japonica. The δ 13 C in the roots and rhizomes seems to be larger than the δ 13 C in the leaves and stems. For the same species of seagrass, when the δ 13 C of the parts were compared, the difference is lesser than when the δ 13 C between species of Z. marina and Z. japonica were compared as seen in Fig. 2. Actually, δ 13 C in the leaves and stems significantly differ from δ 13 C in the roots and rhizomes of Z. japonica collected in Hokkaido Prefecture (n = 3, p < 0.05). There may be different mechanisms of carbon transport to depend on for stable isotope in each part of Z. japonica . Furthermore, carbon isotope fractionation may differ from each part of Z. japonica. In addition, the origin of dissolved inorganic carbon used by the leaves and stems may differ from that of the roots and rhizomes. For example, the δ 13 C of HCO 3 - is higher than δ 13 C of CO 2 in sea water. Characteristics of fatty acid structure in Z. japonica We found about 30 kinds of fatty acids in the leaves and stems and about 36 kinds of fatty acids in the roots and rhizomes of Z. japonica . We focused on the fatty acid content of Z. japonica , because we needed to exclude the influence of a few other organisms, such as bacteria, periphyton and sessile animals. We define the fatty acids with > 5% content as dominant fatty acids and Fig. 3 shows the dominant fatty acids in the leaves and stems of Z. japonica in each sampling site. The term “other” in the x-axis designates the sum of all the fatty acids with < 5% content. The three fatty acids 16:0, 18:2n6c, 18:3n3 were found > 15% in the leaves and stems of all the samples of Z. -14 -13 -12 -11 -10 -9 -8 LS RR δ 13 C（‰） Z.japonica Z.marina Fig.2 Comparison of δ 13 C in Z. japonica and Z. marina. LS: leaves and stems RR: roots and rhizomes -14 -13 -12 -11 -10 -9 -8 LS RR δ 13 C（‰） Z.japonica Z.marina Fig.2 Comparison of δ 13 C in Z. japonica and Z. marina. LS: leaves and stems RR: roots and rhizomes Fig. 2 - Comparison of δ 13 C in Z. japonica and Z. marina. LS: leaves and stems RR: roots and rhizomes Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 105 - japonica. The average fatty acid content in the leaves and stems were 31.2 ± 7.2% for 16:0, 22.0 ± 5.4% for 18:2n6c and 25.7 ± 8.4% for 18:3n3 (n = 15). The sum of these fatty acids was about 80%. Meanwhile, Fig. 4 shows the dominant fatty acids in the roots and rhizomes in each sampling site. The average fatty acid content in the roots and rhizomes was 28.1 ± 4.8% for 16:0 and 33.6 ± 8.1% for 18:2n6c. In addition, the roots and rhizomes from all sampling sites contain 9.8 ± 2.5% of 18:3n3. From the results obtained, it is possible to conclude that 16:0, 18:2n6c and 18:3n3 are the typical fatty acids of Z. japonica. However, we think that the characteristics of the fatty acid structure in the leaves and stems differ from those of the roots and rhizomes because the content of the fatty acid 18:3n3 in the leaves and stems differ from that of the roots and rhizomes. Therefore, we use principal components analysis (PCA) for the 29 kinds of fatty acids with > 1% content in Z. japonica collected in each sampling site (Fig. 5). The PCA ordination plot shows that the leaves and stems were clearly separated from the roots and rhizomes at around PC1 = 0. These results show that the difference in parts as leaves and roots is the main factor to consider for the difference in fatty acid structures of Z. japonica . Furthermore, these results especially the characteristics of PC1-axio, suggest that 18:2n6c and LCFAs were contained in greater amounts in the roots and rhizomes than in the leaves and stems, and 18:3n3 was contained in lesser amounts in the roots and rhizomes than in the leaves and stems. The content of 18:2n6c, 18:3n3 and LCFAs in the leaves and stems was compared with that in the roots and rhizomes. The roots and rhizomes contain more 18:2n6 and LCFAs than the leaves and stems (n = 15, p < 0.01). Meanwhile the leaves and stems contain more 18:3n3 than the roots and rhizomes (n = 15, p < 0.01). Further studies are needed to clarify why there was such a difference between each part of Z. japonica. 0 5 10 15 20 25 30 35 40 45 16:0 16:1n7 18:0 18:2n6c 18:3n3 other Fatty acids Fatty acid content (%) HK MG NG KT KG Fig.3 Dominant fatty acid content in the leaves and stems at each sampling site． HK：Hokkaido Pref. MG：Miyagi Pref. NG：Niigata Pref. KT：Kyoto Pref. KG：Kagawa Pref. 0 5 10 15 20 25 30 35 40 45 16:0 18:1n9c 18:2n6c 18:3n3 24:0 LCFAs other Fatty acids Fatty acid content (%) HK MG NG KT KG Fig.4 Dominant fatty acid content in the roots and rhizomes at each sampling site． HK：Hokkaido Pref. MG：Miyagi Pref. NG：Niigata Pref. KT：Kyoto Pref. KG：Kagawa pref. 0 5 10 15 20 25 30 35 40 45 16:0 16:1n7 18:0 18:2n6c 18:3n3 other Fatty acids Fatty acid content (%) HK MG NG KT KG Fig.3 Dominant fatty acid content in the leaves and stems at each sampling site． HK：Hokkaido Pref. MG：Miyagi Pref. NG：Niigata Pref. KT：Kyoto Pref. KG：Kagawa Pref. 0 5 10 15 20 25 30 35 40 45 16:0 18:1n9c 18:2n6c 18:3n3 24:0 LCFAs other Fatty acids Fatty acid content (%) HK MG NG KT KG Fig.4 Dominant fatty acid content in the roots and rhizomes at each sampling site． HK：Hokkaido Pref. MG：Miyagi Pref. NG：Niigata Pref. KT：Kyoto Pref. KG：Kagawa pref. Fig. 3 - Dominant fatty acid content in the leaves and stems at each sampling site HK: Hokkaido pref, MG: Miyagi Pref, NG: Niigata Pref. KT: Kyoto Pref, KG: Kagawa Pref Fig. 4 - Dominant fatty acid content in the roots and rhizomes at each sampling site HK: Hokkaido Pref, MG: Miyagi Pref, NG: Niigata Pref, KT: Kyoto Pref, KG: Kagawa Pref Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 106 - Difference of carbon stable isotope ratio and fatty acid characteristics in Z. japonica and in primary producers If we use stable isotope ratios or fatty acids as biomarkers to elucidate the origin of organic matter in coastal areas, we must clarify the difference from the characteristics of stable isotope ratios and fatty acid structures in Z. japonica to those in primary producers. Coastal areas have some kinds of primary producers such as terrestrial plants, benthic microalgae, periphyton of seagrass leaves and phytoplankton. The δ 13 C of Z. japonica (-11.8 ± 1.5‰) significantly differs from the δ 13 C (-27.3 ± 1.7‰, n = 27, p < 0.05) of terrestrial plants (Carlier et al., 2007; Svensson et al., 2007; Yokoyama, 2008; Abrantes et al., 2009); benthic microalgae (-17.6 ± 4.4‰, n = 10, p < 0.05) (Svensson et al., 2007; Yokoyama, 2008; Abrantes et al., 2009); periphyton (-16.8 ± 4.4‰, n = 5, p < 0.05) (Kharlamenko et al ., 2001; Carlier et al ., 2007; Yokoyama, 2008); and phytoplankton or particulate organic matter (-21.1 ± 2.3‰, n = 10, p < 0.05) ( Kharlamenko et al., 2001; Schaal, 2008; Yokoyama, 2008; Matsuo et al., 2009). The above-mentioned values reveal that the δ 13 C of seagrass is obviously higher than the δ 13 C of oceanic primary producers. Based on the fatty acid characteristics, some fatty acids are used as biomarkers to primary producers. Table 2 shows the biomarker fatty acid of primary producers in tidal flat by previous researches. According to some researchers as shown in Table 2, the biomarker fatty acids of seagrasses are 18:2n6 and 18:3n3. However, Fig.4 shows that Z. japonica when the origin of organic matter in coastal area is studied. Meanwhile, the PCA ordination plot shows the leaves and stems of Z. japonica were not clearly separated from the leaves and stems of Z. marina (Fig.6). This result shows that there are no differences observed between the fatty acid structures of Z. japonica and Z. marina. Fig. 5 - PCA of both part Z. japonica collected in each sampling site LS: leaves and stems, RR: roots and rhizomes, HK: Hokkaido Pref, MG: Miyagi Pref, NG: Niigata Pref, KT: Kyoto Pref, KG: Kagawa Pref Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 107 - CONCLUSIONS This study focused on the carbon stable isotope and fatty acid structure of Z. japonica because stable isotope and fatty acids are useful methods to clarify the carbon cycle in the ecosystem, and Z. japonica may have an important role in tidal flat ecosystems. Our research shows the characteristics of carbon stable isotope and fatty acid structure of Z. japonica. Furthermore, we compared the characteristics of carbon stable isotope and fatty acid structure of Z. japonica with those of the other primary producers in tidal flat areas. The mean of δ 13 C in Z. japonica significantly differ from the δ 13 C of other primary producers such as terrestrial plants, benthic microalgae, periphyton and phytoplankton because Z. japonica may have difficult system of photosynthesis compared to these plants and algae. In addition, the leaves and stems as well as the roots and rhizomes of Z. japonica have significantly lower δ 13 C than those of Z. marina. This might be because Z. japonica uses sufficient quantities of carbon for photosynthesis. However, it is debatable whether other seagrasses as Z. noltii have the same trend of δ 13 C or not, as Z. japonica and Z. marina. Meanwhile, the results related to fatty acids showed that 16:0, 18:2n6c and 18:3n3 are most probably the typical fatty acids of Z. japonica. In addition, the roots and rhizomes contained more 18:2n6 and LCFAs than the leaves and stems. Meanwhile, the leaves Table 2 Biomarker fatty acids of primaly producers in coastal areas based on previous researches. Biomarker FA producer Ref. a,i-15:0 Bacteria Meziane et al. (2000), Alfaro et al. (2006) 18:1n7 Aerobic bacteria Arts et al. (1998), Jaschinski et al. (2008) 18:1n9 Brown algae Alfaro et al. (2006) 18:2n6 Seagrass or Green macroalgae Meziane et al. (2000), Kharlamenko et al. (2001), Alfaro et al. (2006) 18:3n3 Seagrass Kharlamenko et al. (2001), Alfaro et al. (2006), Jaschinski et al. (2008) Meziane et al. (2000), Kharlamenko et al. (2001) Alfaro et al. (2006), Jaschinski et al. (2008) 22:6n3 Dinoflagellates Arts et al. (1998), Kharlamenko et al. (2001), Alfaro et al. (2006) LCFAs Terrestrial or Vascular plants Meziane et al. (2000), Alfaro et al. (2006) 20:5n3 Diatom Fig. 6 - PCA of the each parts of leaves and stems in Z. japonica and Z. marina. Z. jap: Zostera japoncia, Z. mar: Zostera marina, HK: Hokkaido Pref, MG: Miyagi Pref, NG: Niigata Pref, KT: Kyoto Pref, KG: Kagawa Pref Table 2 - Biomarker fatty acids of primaly producers in coastal areas based on previous researches Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 108 - and stems contained more 18:3n3 than the roots and rhizomes. However, many previous researchers use LCFAs as biomarker of terrestrial plants, but it is necessary to consider the LCFAs derived from Z. japonica when the origin of organic matter in coastal areas is studied. Acknowledgment This research was supported in part by Grants-in-Aid for Scientific Research (KAKENHI 09J06022, 21360249 and 21760413). 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