geophysical constraints for organic carbon sequestration capacity of zostera marina seagrass meadows and surrounding habitats

LIMNOLOGY and Limnol Oceanogr 00, 2017, 00–00 OCEANOGRAPHY C 2017 The Authors Limnology and Oceanography published by Wiley Periodicals, Inc V on behalf of Association for the Sciences of Limnology and Oceanography doi: 10.1002/lno.10478 Geophysical constraints for organic carbon sequestration capacity of Zostera marina seagrass meadows and surrounding habitats Toshihiro Miyajima,1* Masakazu Hori,2 Masami Hamaguchi,2 Hiromori Shimabukuro,2 Goro Yoshida2 Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Chiba, Japan National Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research and Education Agency, Hatsukaichi, Hiroshima, Japan Abstract To elucidate the factors determining the organic carbon (OC) sequestration capacity of seagrass meadows, the distribution of OC and the fraction of seagrass-derived OC in sediments of the temperate cosmopolitan seagrass Zostera marina meadows and surrounding habitats were investigated in relation to physical properties of sedimentary materials On average, seagrass meadow sediments showed OC levels twofold higher than other shallow nearshore habitats However, offshore sediments often showed greater OC concentrations than average seagrass meadow sediments According to estimations of OC sources based on carbon isotope ratios, 8–55% and 14–24% of OC in nonestuarine seagrass meadow sediments and < 30 m deep offshore sediments, respectively, were assigned to seagrass origin The OC concentration in seagrass meadow and offshore sediments closely correlated to the specific surface area (SSA) of sediment (r2 0.816 and 0.755, respectively; p < 0.0001), with an average OC loading per sediment surface area of approximately 60 lmol m22 In seagrass meadow sediments, the fraction of seagrass-derived OC was also greater in samples with a larger SSA, and the seagrassderived OC occurred preferentially in sediment grains that had a specific gravity exceeding 2.0, namely, in a form closely associated with sediment minerals The OC concentration, the fraction of seagrass-derived OC, and the SSA were positively correlated to the logarithm of areal extent of individual seagrass meadows (p < 0.01) These findings suggest that the OC sequestration capacity of nearshore vegetated habitats is under the primary control of geophysical constraints such as sediment supply rate and depositional conditions Vegetated shallow coastal ecosystems, including intertidal salt marshes, mangroves, and seagrass meadows have been ranked among the most efficient biotic systems for accumulating organic carbon (OC) on an areal basis (McLeod et al 2011; Fourqurean et al 2012) It is estimated that these ecosystems may contribute almost half of OC burial in the global ocean even though they cover < 2% of the ocean surface (Duarte et al 2005) Recent interest has focused on the potential to incorporate these ecosystems, called “blue forests,” into policies for reducing carbon dioxide (CO2) emissions At the same time, there is increased concern about the possibility of CO2 emissions caused by the decline of blue forest ecosystems, including the seagrass meadows (Pendleton et al 2012; Grimsditch et al 2013) High rates of OC accumulation in seagrass meadows are likely the result of specific ecosystem functions such as (1) extremely high primary productivity of seagrasses and associated microalgae, (2) efficient trapping of organic particles within the meadow sediment via its flow-regulation and bottom-stabilization effects, and (3) slowness of remineralization of OC within the meadow sediment due to the anoxic conditions that prevail (Duarte et al 2013) Most of the OC stored in seagrass meadows exists as detrital OC derived from seagrasses and attached algae, seston, and terrestrial organic matter in the underlying sediment (Duarte et al 2013) On average, approximately half of OC stored in seagrass meadow sediment is derived from the primary production of seagrasses and seagrass epiphytes, with the rest being derived from allochthonous sources such as phytoplankton and terrestrial organic matter (Kennedy et al 2010; Miyajima et al 2015) Both the concentration of OC and the fraction of seagrass-derived OC can vary widely depending on geographical and oceanographic settings and seagrass species composition (Kennedy et al 2004, 2010; Serrano et al 2014; Miyajima et al 2015) However, the mechanisms through which these external conditions control OC sequestration in *Correspondence: miyajima@aori.u-tokyo.ac.jp This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made Miyajima et al Carbon sequestration in seagrass meadows In this study, we compared sedimentary OC stocks in temperate cosmopolitan seagrass Zostera marina (eelgrass) meadows and the surrounding coastal habitats, such as unvegetated tidal flats, macroalgal beds, and shallow offshore sediments The relationships of sediment OC to physical properties, such as SSA and density of sediment materials, were used to characterize the properties of OC stored in different habitats Using the stable isotope technique, we also examined how the fraction of seagrass-derived OC in the total sediment OC depended on sedimentological factors Based on the obtained results, we examined the following questions and hypotheses: Is the strong correlation between OC content and the mineral surface also present in sediments of vegetated ecosystems such as seagrass meadows? Is the average OC loading per surface area in these habitats within the range typical of continental margin sediments? If so, can we hypothesize that the capacity of OC sequestration in these habitats is largely determined by external geophysical factors, such as the delivery rate and the granulometric properties of mineral sediment, and that the role of ecosystem functions would be of secondary importance? What are the roles of specific ecosystem functions in OC sequestration? To address the latter question, we compared several subtidal sediments collected from seagrass meadows of contrasting areal extent to demonstrate the role of the ecosystem functions of seagrass meadows on the granulometry and OC of the sediment Finally, using the results from this and other related studies, we discuss the potential of offshore sediment as a remote sink of OC exported from nearshore vegetated habitats seagrass meadow sediments remain poorly understood The lack of this mechanistic understanding has hampered more precise estimation of the geographical distribution and the reliable prediction of future trends of OC stocks in seagrass meadows and other coastal habitats Seagrass meadows and macroalgal beds also export a large fraction of their net primary production to the outer oceans n 1996; Heck et al 2008) OC is exported (Duarte and Cebria from seagrass meadows mainly through washout of detached aged leaves under normal growing conditions, seasonal biomass loss (particularly in annual seagrass populations), and removal of OC stored in the surface sediment by storm surges and tsunamis The exported OC may be transported and stored over the long term in offshore sediments under favorable depositional conditions (Sugimatsu et al 2015) Once appropriately quantified, this process could contribute to carbon sequestration, in terms of an ecosystem service performed by seagrass meadows However, reliable information on the fate of OC exported from seagrass meadows is still lacking The concentration of OC in coastal marine sediments has traditionally been considered to be under the control of several environmental factors, such as productivity in the overlying water column, quality (accessibility and availability to bacteria) of OC, sediment accumulation rates, capacity of the sediment mineral matrix to stabilize organic matter, and the availability of oxygen to benthic heterotrophs (reviewed by Hedges and Keil 1995) It has been considered that the strong correlation that is often observed between the OC concentration and the specific surface area (SSA) of coastal marine sediments suggests an essential role of physical sorption of OC in sediment mineral matrices for stabilization and sequestration of OC (reviewed by Keil and Mayer 2014) A typical OC loading per surface area (or OC/SSA ratio) of between 0.5 mg C m22 and 1.0 mg C m22 (40–80 lmol C m22) has been reported for unvegetated coastal and continental margin sediments (Keil and Hedges 1993; Mayer 1994a) The OC/SSA ratio tends be lower than the typical range when the supply rate of mineral particles is relatively high (Mayer 1994b) or the oxygen exposure period of sediment after deposition is very long (Aller 1998; Hartnett et al 1998) The close association between OC and mineral surface has been confirmed by electron microscopy (Ransom et al 1997; Bennett et al 1999) and density-fractionation techniques (Bock and Mayer 2000) The key role of sorption in the stability of OC has also been demonstrated experimentally (Keil et al 1994b; Zimmerman et al 2004) The empirical global relationship between OC content and the SSA indicates that the capacity of coastal ecosystems to sequester OC in the underlying sediment is globally constrained by the supply rate of the mineral surface available for sorption of OC Other factors, such as the supply rate of OC and oxygen exposure period, would be of local or secondary importance in enhancing and attenuating the OC/SSA ratio Materials and methods Study sites, sample collection, and initial sample processing The Seto Inland Sea located in central Japan (Fig 1a) is a shallow water body (average depth, 37 m; surface area, ca 22,000 km2) with abundant seagrass meadows, of mainly Z marina L (Komatsu 1997) It is located in a warm temperate region of the western North Pacific, enclosed by three large islands of Japan, and connected via narrow straits to both the Pacific Ocean and the Sea of Japan The average sea surface temperature is between 188C and 208C, with seasonal variations being much larger in the inner sections (10–288C) than at the mouth of the strait (17–258C; Yanagi 1984) The tidal range of the spring tide is 3.5–4.0 m at most of the study sites We collected surface sediment cores from intertidal and subtidal seagrass meadows (11 sites), bare areas adjacent to seagrass meadows (2 sites), subtidal macroalgal beds (8 sites), and unvegetated estuarine intertidal flats (5 sites) using a hand-operated knocking corer or a piston corer (Adachi et al 2010) during 2009–2012 Collected cores were immediately cut into 10 cm sections, and in this study only the top 30 cm sections were used for further analyses We processed Miyajima et al Carbon sequestration in seagrass meadows Fig Location of the sampling sites of the surface sediment in the Seto Inland Sea Detailed information can be found in Table [Color figure can be viewed at wileyonlinelibrary.com] by grinding in an automatic mortar for 25 (ALM-200, Nitto Kagaku Ltd., Nagoya, Japan) The influence of grinding on the granulometric properties of samples such as specific surface area and mesopore distribution was insignificant because the area of new surfaces created by grinding was negligible compared to the original surface area of the sediments The final samples were stored in tightly capped glass vials under < 40% relative humidity only one core from each site, except at ZmE2, where three cores were collected from different microhabitats (seagrass coverage) For some of the samples, the full-length OC profiles were described in Miyajima et al (2015) (Table 1) In addition, surface sediments were collected from 23 offshore stations (depth, 9.7–82 m) using a gravity corer during two cruises of the R/V Shirafuji-maru (Japan Fisheries Research and Education Agency) in 2009 and 2011 The top 5-cm sections of all of the offshore cores and subsurface sections (25– 30 cm) of the 2009 cores were used for further analyses All samplings were performed in the western part of the Seto Inland Sea (Fig 1b) Detailed information about the sampling stations is provided in Table Although triplicate cores were collected and analyzed for the 2009 offshore stations (Os15–20, Os22, and Os23), data from only one core from the triplicate are shown here because the difference in the OC concentration within the site was small compared to that among sites All of the sectioned samples were packed in screw-capped polypropylene containers and frozen at 2208C for temporal storage and transportation At the laboratory, the samples were freeze-dried, and the water content was evaluated from the weight lost on drying The dried samples were gently crushed by hand using a mortar and pestle, and passed through a mm mesh stainless sieve to remove large gravels and seagrass rhizomes that were occasionally found in the samples (< 20% and < 1% of bulk weight, respectively) The fraction that passed through the sieve was further homogenized Elemental and isotopic compositions The dried and homogenized sediment samples were subjected to acid treatment to remove inorganic carbon Approximately g of dried sample was placed into screwcapped glass tubes (10 mL), and 2.0 N hydrochloric acid (HCl) solution was added to the sediment dropwise until all the carbonate was converted into CO2 Then, the tubes (without caps) were placed in a vacuum desiccator with a 50-mL beaker containing about 10 g of NaOH pellets as an acid absorber and another beaker containing about 20 mL of concentrated H2SO4 as a desiccant, and kept in vacuo until the sediments were completely dehydrated The NaOH pellets were replaced regularly when they became liquefied due to absorption of HCl and water The drying process normally took 7–10 d The dried sediment was weighed again to check for weight changes due to the acid treatment The concentrations and isotope ratios of OC and total nitrogen (TN) in the treated samples were determined simultaneously by EA-IRMS (FLASH 2000/Conflo IV/DELTA V Miyajima et al Carbon sequestration in seagrass meadows Table Location of sampling sites and sampling dates of the sediment cores used in this study Habitat/Station Latitude (˚N) Seagrass meadows (ZmC) ZmC1 34.23766 Longitude (˚E) Depth* (m) Sampling date Station name in Miyajima et al (2015) Local site name 132.99151 0.9 26 Oct 2011 — Omishima Island ZmC2 34.23110 132.98546 3.7 26 Oct 2011 Z5 Omishima Island ZmC3 ZmC4 34.25776 34.24288 132.97359 132.95203 1.5 1.9 26 Oct 2011 26 Oct 2011 Z4 — Omishima Island Omishima Island ZmC5 34.29136 132.91734 3.0 25 Oct 2011 — Ikunoshima Island ZmC6 ZmC7 34.29671 34.06781 132.91541 132.89949 3.0 0.7 06 Oct 2010 27 Oct 2011 Z3 — Ikunoshima Island Imabari coast ZmC8 34.04272 132.84310 0.6 27 Oct 2011 — Imabari coast Estuarine seagrass meadows (ZmE) ZmE1 34.32424 132.89444 Intertidal 05 Oct 2010 Z1 Hachi tidal flat, Takehara ZmE2 34.32377 132.89385 Intertidal 19 Jun 2012 Z2 Hachi tidal flat, Takehara ZmE3 33.60731 131.23482 Intertidal 17 Oct 2009 Zj Nakatsu tidal flat (Z japonica bed) Bare areas adjacent to meadow (Ba) Ba1 34.29662 132.91504 3.0 06 Oct 2010 B3 Ikunoshima Island Ba2 34.06781 Unvegetated estuarine tidal flat (Tf) 132.89949 0.7 27 Oct 2011 — Imabari coast Tf1 34.32536 132.89594 Intertidal 05 Oct 2010 B1 Kamo river mouth, Tf2 34.32459 132.89485 Intertidal 05 Oct 2010 B2 Takehara Kamo river mouth, Tf3 33.60619 131.23761 Intertidal 16 Oct 2009 Bj Yamaguni river mouth, Nakatsu Tf4 33.62406 131.19647 Intertidal 15 Oct 2009 E1 Yamaguni river mouth, Tf5 33.62583 131.17417 Intertidal 18 Oct 2009 E2 Nakatsu Yamaguni river mouth, Takehara Nakatsu Macroalgal beds (Mb) Mb1 33.52926 132.38878 3.0 20 Jun 2012 — Sadamisaki Peninsula Mb2 33.52926 132.38878 3.0 20 Jun 2012 — Sadamisaki Peninsula Mb3 Mb4 33.45235 33.45235 132.25332 132.25332 2.0 2.0 21 Jun 2012 21 Jun 2012 — — Sadamisaki Peninsula Sadamisaki Peninsula Mb5 33.44535 132.22660 1.5 21 Jun 2012 Ma Sadamisaki Peninsula Mb6 Mb7 33.44535 33.41037 132.22660 132.15018 2.0 2.0 21 Jun 2012 20 Jun 2012 — — Sadamisaki Peninsula Sadamisaki Peninsula Mb8 33.41016 132.14990 1.5 20 Jun 2012 — Sadamisaki Peninsula Offshore stations (Os) Os1 34.41667 133.86500 30.5 10 Sep 2011 — Bisan-Seto Strait Os2 34.27167 133.51833 27.0 10 Sep 2011 — Bingo-Nada Sound Os3 Os4 34.20000 34.08833 133.38167 133.14000 20.8 33.8 10 Sep 2011 10 Sep 2011 — — Bingo-Nada Sound Hiuchi-Nada Sound Os5 34.18167 132.99833 28.7 11 Sep 2011 — Itsuki-Nada Sound Os6 Os7 34.06667 33.78000 132.78333 132.56667 48.5 47.5 10 Sep 2011 09 Sep 2011 — — Itsuki-Nada Sound Iyo-Nada Sound Miyajima et al Carbon sequestration in seagrass meadows TABLE Continued Latitude (˚N) Longitude (˚E) Depth* (m) Sampling date Station name in Miyajima et al (2015) Os8 34.07500 132.31667 24.1 07 Sep 2011 — Aki-Nada Sound Os9 Os10 33.56167 33.42333 132.25500 132.05667 63.5 82.4 09 Sep 2011 09 Sep 2011 — — Iyo-Nada Sound Iyo-Nada Sound Os11 33.36833 132.00833 59.6 09 Sep 2011 — Iyo-Nada Sound Os12 Os13 33.63167 33.83333 131.88500 131.79167 54.9 46.0 09 Sep 2011 07 Sep 2011 — — Iyo-Nada Sound Suo-Nada Sound Os14 33.82667 131.49333 34.9 08 Sep 2011 — Suo-Nada Sound Os15 Os16 33.78333 33.72000 131.44333 131.36500 28.1 20.1 18 Oct 2009 18 Oct 2009 — — Suo-Nada Sound Suo-Nada Sound Os17 33.61667 131.33333 11.3 18 Oct 2009 — Suo-Nada Sound Os18 Os19 33.81667 33.75333 131.25000 131.24833 29.6 18.6 18 Oct 2009 18 Oct 2009 — — Suo-Nada Sound Suo-Nada Sound Os20 33.65333 131.24833 9.8 18 Oct 2009 — Suo-Nada Sound Os21 Os22 33.80000 33.65833 131.17500 131.15000 16.0 9.7 08 Sep 2011 18 Oct 2009 — — Suo-Nada Sound Suo-Nada Sound Os23 33.71667 131.11667 11.3 18 Oct 2009 — Suo-Nada Sound Habitat/Station Local site name * Directly measured depth for offshore stations; depth below datum level for the other sites Advantage, ThermoFisher Scientific, Bremen, Germany) Three to five standard materials of different d13C (233.8& to 210.2&) and d15N (27.8& to 113.8&) values (SI Science Ltd., Saitama, Japan, and Iso-Analytical Ltd., Crewe, UK) were used for daily calibration Samples and standards were € weighed into tin capsules (SANTIS Analytical AG, Teufen, Switzerland) before analysis The measured isotope ratios were represented using conventional d-notation (d13C and d15N, in &) with Vienna Pee-Dee Belemnite and atmospheric N2 as the reference materials The instrumental analytical precision was normally within 61% for the OC and TN concentrations and 0.1& for d13C and d15N However, the subsampling errors for both concentrations and d-values were sometimes twofold to threefold as large as the instrumental errors BELSORP mini II (MicrotracBEL, Osaka, Japan) surface area analyzer The slope of the BET plot at the inflection point that normally appeared between p/p0 0.10 and 0.15 was used for estimating the SSA The particle size distribution analysis of sediment samples was conducted for several seagrass meadow- and tidal flat sediments by GeoAct, Ltd (Kitami, Japan) using core samples that were collected separately The size distribution was determined by a combination of the standard methods, such as sedimentation analysis (for  0.075 mm particles) and dry sieving methods (for > 0.075 mm particles) Density fractionation Selected dried and homogenized sediment samples were subjected to density fractionation by the polytungstate heavy solution method (Sollins et al 2009) A series of heavy solutions (specific gravity of: 1.50, 1.75, 2.0, 2.2, 2.4) were prepared with sodium polytungstate SPT0 (TC-Tungsten Compounds, Grub am Forst, Germany) and ultrapure water The density of the prepared solutions was adjusted using a DMA 35 densitometer (Anton Paar, Graz, Austria) Approximately 2.0 g of the sample was weighed into a 50 mL screwcapped polypropylene centrifuge tube and 25 mL of the lightest heavy solution was added The tube was shaken by a reciprocal shaker at 90 rpm for 30 to disperse and homogenize the sample, and then centrifuged at 2000 g and 208C for 30 using a swing-bucket rotor The supernatant was filtered through pre-weighed 25 mm glassfiber filters GF/F (GE Healthcare Life Science, Pittsburgh, Pennsylvania) under reduced pressure The filters were washed three times with 10 mL of ultrapure water and Specific surface area and particle size distribution Measurement of the SSA of the sediment was performed by the multipoint Brunauer–Emmett–Teller (BET) method based on N2 gas adsorption under reduced pressure Using the same data, the mean diameter of mesopores (MMD) on sediment grains could also be estimated The dried and homogenized sediment samples were treated at 3508C for 12 h under normal atmosphere to remove most of the organic coating (Keil et al 1997) Weight loss on heating was determined for each individual sample Between 0.5 g and 2.0 g of the treated samples were weighed into glass flasks specific to the gas adsorption measurement, and desiccated further in vacuo (< 1022 kPa) at 3508C for h Immediately after cooling, a multipoint BET measurement was performed with N2 (purity, > 99.9995%) as the adsorbate, using a Carbon sequestration in seagrass meadows 10 2] 2] Miyajima et al ZmC ZmC Os 15 400 800 1200 1600 Salt-corrected OC [µmol C g ] a 2000 Non-estuarine seagrass meadow (ZmC) Estuarine seagrass meadow (ZmE) b Tf NTN 400 800 1200 1600 Salt-corrected OC [µmol C g ] 15 NTN COC 13 Tf ZmC: r = 0.7188, p = 0.0014 Os 10 2000 Bare areas adjacent to meadow (Ba) c Os: r = 0.4384, p = 0.0252 0.06 0.08 0.10 0.12 TN/OC ratio [µmol µmol 0.14 ] Macroalgal bed (Mb) Offshore sediment (Os) Fig The concentrations and the isotope ratios of organic carbon (OC) and total nitrogen (TN) in sediments collected from various habitats of the Seto Inland Sea a, b: plots of d13COC (a) and d15NTN (b) against the concentration of OC c: plot of d15NTN against the atomic ratio of TN/OC Isotope ratios described beside the curves in a and b are the convergence values (C) of the exponential functions, d13COC A exp ([OC] / B) C, for the data plots of non-estuarine seagrass meadow (ZmC, solid circle), offshore (Os, 1), and unvegetated estuarine tidal flat samples (Tf, open triangle) Lines in c are the linear regression lines for the data plots of ZmC and Os samples with the correlation coefficients (r) and the probabilities for the null hypotheses (p) being denoted beside the lines [Color figure can be viewed at wileyonlinelibrary.com] was estimated from the water content of the original wet sediment assuming a pore-water density of 1.025 (i.e., a salinity of 35) To calculate the OC stock per unit area of habitat, the dry bulk density estimated following the method of Miyajima et al (2015) was multiplied by the weightbased concentration determined as above to obtain the concentration per unit volume of original sediment Analysis of the sources of organic matter based on the carbon isotopic composition was performed by the stochastic approach using the IsoSource model developed by Phillips and Gregg (2003) A detailed protocol can be found in Miyajima et al (2015) In the present study, the following endmember d13C values were assumed; for seagrass-derived carbon, the average d13C (210.11& 0.23&, n 5) of Z marina leaves collected in seagrass meadows near Sta Mb6, where terrestrial influence was the lowest of all our sites, was used As the endmember d13C of terrestrial OC, the average value (226.76& 1.71&) of soil samples (n 3) collected at the Yamaguni River beds (near Sta Tf5) and wood debris (n 9) found in the sediment cores collected at Sta Tf1 and Ba1 was used For phytoplankton-derived OC, the asymptotic convergence point (221.58& 0.11&) of the exponential fitting line for the OC–d13COC plot of offshore sediments (Os1–23; Fig 2a) was assumed to represent the endmember value For the calculation, the source increment and mass balance tolerance parameters were set to be 1% and 0.1&, respectively Statistical tests (ANOVA, ANCOVA, and multiple regression) and curve fitting (linear, logarithmic, and exponential models) were conducted using the commercial software packages Aabel NG1 (ver 4; Gigawiz, Tulsa, Oklahoma) and pro Fit (ver 7; QuantumSoft, Uetikon am See, Switzerland), respectively The confidence interval of the exponential curve fitting was evaluated by the Monte Carlo method built into pro Fit with an iteration of 1000, assuming appropriate stored frozen at 2258C until analysis When the volume of particles separated in the supernatant was so large that more than two filters were required to collect all the suspended particles, the pellet was resuspended in 25 mL of the same heavy solution and the separation process was repeated once more to ensure recovery of the low-density particles The pellet was then resuspended in the second-lightest heavy solution and homogenized in the shaker This cycle was repeated using successively heavier heavy solutions, although a centrifugation time of 60 was used for solutions  2.0 g cm23 The wet weight of the pellet confirmed that the amount of heavy solution carried over to the next step was usually < 5% of the added amount The pellet resulting from the centrifugation using the heaviest heavy solution was washed by suspending it twice in 30 mL of ultrapure water and centrifugation, and was then stored frozen at 2258C The filters and the pellet were freeze-dried and weighed to determine the weights of the respective density fractions The samples (including the glassfiber filters) were crushed and homogenized by the agate mortar and quantitatively transferred to 10 mL screw-capped glass tubes The contents were acidified by mL of 1.0 M HCl to remove inorganic carbon as CO2 The tubes were then centrifuged at 1000 g and at 158C for 30 The pellets were washed by suspending them three times in 10 mL of 0.1% NaCl solution in ultrapure water and centrifugation, and then freeze-dried and re-weighed The final samples were analyzed for the concentrations and the isotopic ratios of OC and TN, as described above Data processing The results of the OC and TN concentrations and the SSA were expressed as lmol and m2 per unit weight of salt-free bulk dry sediment The salt content in the sediment sample Miyajima et al Carbon sequestration in seagrass meadows river mouths (group ZmC, 532 75, n 27; p 0.0161) Similar trends and statistically significant differences were also detected for the concentration of TN, except that the difference between ZmE and ZmC was not significant for TN The OC/TN atomic ratio was significantly lower in the Os samples (9.3 0.2) than in any other habitats (p < 0.05) The ZmE samples showed a significantly higher OC/TN ratio (13.0 0.3) than the ZmC (10.9 0.3) and Mb (10.5 0.4) samples The OC/TN ratio of the bare area samples (Ba, 11.9 1.3; Tf, 12.7 0.5) ranged in between these values The stable isotope ratio of sediment OC (d13COC) ranged from 225& to 216& (Fig 2a) For the samples with an OC < 300 lmol g21, the d13COC varied widely and was not apparently habitat-specific However, the d13COC converged in habitat-specific ranges with increasing OC concentration in the ZmC, Os, and Tf groups The convergence values for the Os and Tf groups were 221.6& 0.11& and 225.0& 0.46&, which are typical for marine phytoplankton and terrestrial organic matter, respectively The convergence value for the ZmC group (218.5& 0.33&) was significantly higher than those for Os and Tf, but was still much lower than the d13C typical for Z marina (210.11& 0.23&) The d13COC of the Ba and Mb groups showed a decreasing trend with increasing OC, although no clear convergence values could be determined The d13COC of the ZmE group varied widely, even when the OC concentration was > 1000 lmol g21 The fraction of seagrass-derived OC estimated by the IsoSource model was 8–55% (mean, 31%) and 0–52% (mean, 15%) for ZmC and ZmE habitats, respectively A small contribution of seagrass-derived OC was also determined for Os (mean, 19%), Ba (12%), and Tf (9%) habitats The fraction of terrestrial OC was higher for Tf (59%) and Ba (46%) habitats The IsoSource model was not applied to Mb samples because the endmember d13COC of macroalgal OC was not sufficiently constrained from existing data The stable isotope ratio of TN (d15NTN) varied from 12& to 110& for the samples with an OC < 300 lmol g21 (Fig 2b) It converged within a relatively narrow range between 15& and 17& as the OC concentration increased In contrast to d13COC, difference in the convergence value between habitats was not clear (15.5 0.48& for Os, 14.6 0.79& for Tf, 15.6 0.37& for ZmC) In the samples of the ZmC and Os groups, there was a weak but significant positive correlation between the d15NTN and the TN/OC ratio (Fig 2c) For reference, the d15N of Z marina leaves collected around Sadamisaki Peninsula (Sta Mbx) and Hiroshima Bay (near Sta Os8) ranged between 13.9& to 15.2& and 17.2& to 18.5&, respectively, and that of macroalgae (Sargassum spp.) collected around Sadamisaki Peninsula was 15.0& 1.60& (n 13) (unpublished data) The TN/OC ratio of these macrophytes was mostly lower than in the sediment samples (0.033–0.070 for Z marina leaves, 0.046 0.017 for Sargassum spp.; magnitudes of errors for both independent and dependent variables (explained in the figure caption) Satellite image analysis Of the seagrass meadows studied, we selected eight subtidal sites (ZmC in Table 1) where the influence of river-borne terrestrial inputs was relatively small, and the areal extent of each seagrass meadow was estimated by satellite image analysis (Sagawa et al 2008) We used multispectral satellite images AVNIR2 (10 m resolution) taken from the Advanced Land Observing Satellite (ALOS1) during the growing season of Z marina (winter to early summer) of 2010 and 2011 Image processing and analysis were performed using the geographic information system (GIS) software ArcGIS 10 (ESRI, Redlands, California) Water bodies deeper than 10 m, where no seagrasses could grow, were masked based on bathymetric maps available from the Japan Hydrographic Association Unmasked shallow coastal areas were classified further into seven classes: unvegetated sandy or muddy areas; patchy seagrass distribution (< 50% coverage); dense seagrass distribution ( 50% coverage); rocky macroalgal beds; land; clouds; and other water surfaces, by comparison with available information, such as direct observation, low-altitude aerial photos, and acoustic mapping Approximately 30 polygons in the ALOS images that were known to belong to one of the seven classes were chosen, and RGB brightness bands in the polygons were extracted to determine the spectra typical to each class Next, supervised classification with a maximal likelihood method was employed to identify areas with patchy and dense seagrass distribution on the satellite images The habitat classification obtained by this method was generally consistent with direct underwater observation The areal extent of seagrass meadows was determined by summing the pixel counts (1 pixel 100 m2) Results Organic carbon and total nitrogen in sediments The concentration of OC in the sediments tested varied widely from 35 lmol C g21 to 1890 lmol C g21 It was, on average, more than twofold higher in Z marina meadows (697 lmol C g21 67 lmol C g21, mean SE, n 39) and offshore sediment samples (group Os; 922 104, n 31) than in the bare areas adjacent to meadows (Ba; 256 70, n 8), the unvegetated intertidal flats (Tf; 193 32, n 13), macroalgal beds (Mb; 156 23, n 23), and a Zostera japonica meadow (ZmE3; 344 64, n 3) The OC in either of the former two habitats was significantly higher than either of the latter four habitats (p < 0.05; ANOVA with Scheffe’s post hoc test) There was no significant difference between Z marina meadows and Os samples, or between the bare area (Ba, Tf) and Mb samples The OC concentration in sediments of estuarine seagrass meadows (group ZmE including the Z japonica meadow, 922 85, n 15) was significantly higher than the other Z marina meadows that were distant from Miyajima et al Carbon sequestration in seagrass meadows Table Dependence of OC, the OC/TN ratio, d13COC, and d15NTN of Os samples on water depth and longitude of sampling sites (n 23) evaluated by multiple regression analysis Predictors* Depth [m] Criteria OC [lmol g Slope 21 ] Longitude [˚E] p Slope Multiple regression p R2 p 222.2 0.0004 2193 0.1204 0.559 0.0003 OC/TN [mol/mol] d13COC [&] 20.037 0.023 0.18) for all variables Specific surface area and grain size distribution The SSA evaluated by the BET method for Os samples (20.9 1.86 m2 g21, n 31) was, on average, more than twofold higher than any habitats in the shallower nearshore areas (p < 0.0001) A significant negative correlation was found between the SSA and water depth of the offshore sites (r2 0.4551, p 0.0011), and sediments with a relatively large SSA (> 25 m2 g21) occurred only in depth ranges of 9– 30 m (Fig 3a) Of the shallower nearshore habitats, seagrass meadow sediments (8.24 1.12 m2 g21, n 27 for ZmC; 9.19 0.77, n 15 for ZmE) had a larger SSA than nonmeadow sediments (3.02 0.49, n for Ba; 4.73 0.19, n 23 for Mb; 5.21 0.36, n 13 for Tf), although only the difference between ZmE and Ba was marginally significant according to an ANOVA (p 0.0441) The difference in the SSA between seagrass meadow and nonmeadow sediments was apparently related to the grain size distribution of the sediment (Fig 4) In particular, the fraction of size range between 0.002 and 0.1 mm, i.e., silt size class, increased from < 15% in an unvegetated tidal flat sediment (SSA, 3.97 m2 g21) to > 80% in a Z marina meadow sediment Miyajima et al Carbon sequestration in seagrass meadows Fig Relationship of the specific surface area (SSA) of offshore sediment samples and the water depth from which the samples were collected (a), mean mesopore diameter (MMD) with standard deviation of sediment mineral particles from each habitat (b), and plots of the OC concentration (c) and the OC/TN atomic ratio (d) against SSA Lines in a and c are linear regression lines for offshore (Os, 1) and non-estuarine seagrass meadow (ZmC, solid circle) samples, with the correlation coefficients (r) and the probabilities for the null hypotheses (p) being denoted beside the lines In b, the MMD of Os samples was averaged separately for shallow (< 30 m) and deep (> 30 m) sediments [Color figure can be viewed at wileyonlinelibrary.com] Fig Particle size distribution of surface sediment samples collected from four different habitats The specific surface area (SSA) of each sample is described in the legend Inset: plot of SSA against weight-average grain size Data were fitted to a 2/3-power-law curve (dashed line): SSA 1.87 (average grain size)22/3 [Color figure can be viewed at wileyonlinelibrary.com] Fig OC concentration (bar), d13COC (open circle), d15NTN (solid circle), and OC/TN atomic ratio (triangle) of density-fractionated sediment samples collected from non-estuarine seagrass meadows (a – f), a bare area adjacent to the seagrass meadow (g, h), an estuarine seagrass meadow (i), a macroalgal bed (j), unvegetated tidal flat (k, l), and offshore sites (m – p) The density fraction is denoted below the abscissa (g cm23) OC is represented as a concentration per unit weight of bulk sediment (i.e., before density separation) [Color figure can be viewed at wileyonlinelibrary.com] Miyajima et al Carbon sequestration in seagrass meadows Fig Dependence of OC concentration (a), fraction of seagrass-derived OC (b), SSA (c), and OC/SSA ratio (d) on the areal extent of individual seagrass meadows for the sediment samples collected from seven non-estuarine seagrass meadows Dotted line and shaded area are the best-fit exponential regression curve (y A exp (–x / B) 1C) and its 68.3% (1r) confidence interval Curve fitting was conducted for A, B, and C using the software pro Fit and assuming the following estimation errors for x and y: error for areal extent – 0.1 km2; OC and SSA – 10% of estimates; fraction of seagrassderived OC – standard deviation given in the outputs from the IsoSource model; OC/SSA ratio – 15% of estimates Only the fitted B value is shown in each panel The confidence interval was estimated by the Monte-Carlo approach using the same software and 1,000 iterations The assumed estimation errors are represented by horizontal and vertical lines associated with each data point [Color figure can be viewed at wileyonlinelibrary.com] tested In contrast, the OC in the  2.0 g cm23 density fractions was < 51% for the samples from the Ba (Fig 5g,h), Mb (Fig 5j), and Tf (Fig 5k,l) habitats The OC content per weight of individual density fraction was highest in the < 1.50 or 1.50–1.75 g cm23 fractions, often exceeding 10 mmol C g21, and decreased in the heavier fractions The OC content in the 2.0–2.2 and 2.2–2.4 g cm23 fractions of the ZmC and Os samples was between 1.2 and 2.6 mmol C g21, and was not significantly higher than that in the other habitats However, the OC content in the heaviest > 2.4 g cm23 fraction was higher in the ZmC, ZmE, and Os samples (0.17–0.50 mmol C g21) than in the Ba, Mb, and Tf samples (0.07–0.17 mmol C g21) The OC/TN ratio was always higher in the lighter density fractions than the heavier fractions (Fig 5) The ratio in the lightest fraction varied from 16 to 48 depending on the site, The OC concentration was significantly positively correlated to MMD for Mb (r 0.8711, p 0.0007), Tf (r 0.6830, p 0.0447), and Os (r 0.6485, p 0.0016) but not for ZmC, ZmE, or Ba In the Os and Mb samples, the OC/TN ratio and the d13COC significantly correlated positively and negatively, respectively, to the MMD (p < 0.02) Density fractionation The OC in the sediment samples collected from the nonestuarine seagrass meadows and offshore sites was associated mainly with relatively heavier sediment particles The fraction of OC found in the density fraction of  2.0 g cm23 was 80–92% for ZmC and Os sediments (Fig 5a–c,e,m–p), except ZmC4 (59%; Fig 5d) In these sediments, and also in a ZmE sediment (Fig 5i), the fraction between 2.2 and 2.4 g cm23 contained the most abundant OC among the fractions 11 Miyajima et al Carbon sequestration in seagrass meadows decreased from 0.65 in the smallest meadow to 0.18–0.27 in meadows larger than 0.4 km2 The SSA of the sediment consistently increased with the areal extent of the meadows (Fig 6c), while the OC/SSA ratio did not clearly depend on the meadow size (Fig 6d) The correlation with the logarithm of the meadow area was significant for the SSA (r 0.9238, p < 0.0001) but not for the OC/SSA (r 0.2338, p 0.25) The B and C values of the exponential model for SSA were estimated as 0.48 0.14 km2 and 21.1 2.3 m2 g21, respectively but converged to 7–10 in the fractions heavier than 2.2 g cm23 The d13COC also varied widely depending on site in the lighter density fractions, from 228.3& at Tf1 to 217.3& at ZmC2 (Fig 5); it was higher in the ZmC sites than in the Ba, Mb, and Tf sites, with intermediate values observed in the ZmE and Os sites The d13COC in the fractions heavier than 2.2 g cm23 was less variable, being typically higher in the ZmC samples (220.4& to 218.0&) than in the other samples (225.7& to 219.7&) The d15NTN generally increased from the lighter to the heavier density fractions In all but the Os samples, the d15NTN was the highest at a fraction of 2.2–2.4 g cm23 The d15NTN of the fractions heavier than 2.2 g cm23 always ranged between 15.1& and 16.9&, except in the sample collected from Os3 ( 7&) which represents the eastmost sampling station Discussion Characteristics of OC stored in seagrass meadow sediments The density fractionation of seagrass meadow sediments showed a noteworthy accumulation of OC in heavier density fractions (> 2.2 g cm23; Fig 5a–f) As naturally occurring organic matter, such as cellulose and lignin, commonly have densities of 1.2–1.6 g cm23 (Gibson 2012), the occurrence of OC in density fractions > g cm23 indicates that the majority of OC stored in seagrass meadow sediments was in close association with mineral particles (e.g., Arnarson and Keil 2001) Since aluminosilicate minerals that constitute most of the sediment minerals around the study sites typically have densities ranging from 2.3 to 2.9 g cm23 (Deer et al 1992), the presence of OC in a density range slightly lighter than aluminosilicate minerals (i.e., 2.2 g cm23) has been considered to indicate that the OC is present as an organo-mineral complex (Bock and Mayer 2000) The linear correlation between OC concentration and SSA (Fig 3c) further suggests that organic matter adsorbed to the surface of sediment minerals was the predominant form of OC stored in the seagrass meadow sediments The accumulation of OC in the heavier density fractions, as well as the linear relationship between OC and SSA, was common to nonestuarine seagrass meadow (sample group ZmC), macroalgal bed (Mb), and offshore sediments (Os), although the OC concentration was much lower in Mb samples than in ZmC and Os samples The average OC loading per surface area was 60 lmol C m22, which is consistent with previously reported OC loading for continental margin sediments (Keil et al 1994a; Mayer 1994a) These facts suggest that among shallow coastal habitats, only seagrass meadows can accumulate OC in a similar manner to offshore sediment with respect to the relationship to the mineral surface The observation that the density distribution pattern of OC was almost identical between the surface (i.e., recent; Fig 5a,e) and deep (old; Fig 5b,f) sediments within the same cores indicates the long-term stability of OC adsorbed to the mineral surface The origins, chemical structure, and exact mechanisms responsible for the stability of such mineral-associated OC are still under debate (Keil and Mayer 2014) While the chemical composition of such organic matter could be Influence of areal extent of seagrass meadows Among the sediment cores collected from nonestuarine seagrass meadows, ZmC5 and ZmC6 were sampled from the same meadow, while the other six cores were collected at different meadows (Table 1) The areal extent (patch size) of these seven different seagrass meadows ranged from 0.091 to 1.222 km2 The OC concentration of sediments collected from these sites consistently increased with the increasing areal extent of seagrass meadows (Fig 6a), showing a statistically significant correlation with the logarithm of the meadow area (r 0.8686, p 0.0002) For the seagrass meadows with an area of < 0.25 km2, the average OC concentration in the sediment and the OC stock in the top 30 cm were < 200 lmol C g21 and < 10 mmol cm22, respectively The OC concentration and the stock increased to > 400 lmol C g21 and around 20 mmol C cm22 for meadows of > 0.3 km2 The overall trend of dependence of OC on the areal extent could be approximated by an exponential saturation model formulated as y A exp(2x/B) C, where y is the OC concentration and x is the areal extent of the meadows The coefficient B and its standard deviation were estimated at 0.36 0.09 km2 after fitting to the curve, and the saturation value C was 1090 90 lmol C g21 The d13COC also tended to increase with the increasing size of the meadows The fraction of seagrass-derived OC in the sediment OC, which could be estimated stochastically from the d13COC using the IsoSource model, rapidly increased with the increasing areal extent of the meadows when the area was < 0.4 km2, was constant or slightly decreased with increasing area for meadows larger than 0.4 km2 (Fig 6b), and was significantly correlated with the logarithm of the meadow area (r 0.5584, p 0.0099) The B and C values of the exponential approximation were estimated as 0.091 0.057 km2 and 0.415 0.024, respectively The fraction of plankton-derived OC was relatively constant (0.27–0.47 with a mean of 0.38) and did not depend on the seagrass meadow area The fraction of terrestrial OC 12 Miyajima et al Carbon sequestration in seagrass meadows samples, except for ZmC2, and the d13COC of the lighter fractions was particularly low in the nonmeadow Ba and Tf samples (Fig 5g–i,k) This trend suggests that terrestrial OC with low d13C (typically < 225&) was supplied and stored in these sediments, except for ZmC2 In the case of the Ba and Tf samples, the terrestrial OC with a low d13C is often the dominant fraction of sedimentary OC (Fig 2a) Compared to marine OC, terrestrial OC appears to be stored preferentially as independent organic particles not tightly associated with sediment minerals In other words, marine OC appears to be converted more readily into mineral-associated OC during early diagenesis than terrestrial OC The d13COC of ZmC sediments tended to converge to a value (218.5&) higher than the Os samples as the OC concentration increased (Fig 2a) This is ascribed to the contribution of seagrass detritus to the sedimentary OC pool, because seagrasses have much higher d13C levels than pelagic plankton Epiphytic microalgae also have relatively higher d13C levels (215.4& to 212.6&; Takai et al 2004) As seagrass meadows provide favorable substrates for the attachment of epiphytic microalgae, detritus derived from these microalgae could be an additional source of high-d13C OC to the seagrass meadow sediments OC with a relatively high d13C (220.5& to 218.0&) was accumulated in the density fractions of > 2.0 g cm23 of ZmC sediments (Fig 5a–f) This suggests that the OC derived from seagrasses and epiphytic microalgae could be converted to mineral-associated organic matter during early diagenesis High d13C (219.8&) in the heaviest (> 2.4 g cm23) fraction of the macroalgal bed Mb8 sediment (Fig 5j) suggests that high-d13C OC derived from macroalgae was also preferentially preserved in sediment as mineral-associated organic matter The heaviest density fraction of the surface sediment of Ba1 (Fig 5g) also exhibited slightly higher d13C (219.7&) than Os, which implies that seagrass-derived detritus exported from adjacent seagrass meadows was accumulated in sediments of the surrounding bare areas, as previously suggested by Kennedy et al (2010) In contrast, the d13COC of ZmE samples was often lower than that of ZmC samples with similar OC concentrations (Fig 2a) The d13C of the density fractions heavier than 2.0 g cm23 of a ZmE sample (Fig 5i) was also lower (222.4& to 220.9&) than the same fractions of ZmC samples This difference could primarily be attributed to the accumulation of river-borne soil particles that had originally been coated by terrestrial (low-d13C) OC to the sediments of estuarine habitats such as ZmE and Tf These mineral particles coated by terrestrial OC may be partially transported across coastal areas and even to offshore sediment, and effectively lower the baseline d13COC of the bulk sediment as well as that of the heavier density fractions of these habitats (e.g., Weijers et al 2009) In addition, primary producers, including seagrasses and epiphytic algae in estuarine environments, exhibit lower d13C than their nonestuarine counterparts because the d13C of the dissolved inorganic carbon (DIC) in highly complex and diverse, the organic fraction detached from sediment particles by mechanical and chemical treatments is often predominated by high-molecular-weight (HMW; > 10 kDa) substances, such as polysaccharides and peptides (Miyajima et al 2001; Nunn and Keil 2005) A plausible precursor for such HMW substances would be extracellular polymeric substances produced by bacteria upon decomposition of plankton- or plant-derived particulate organic matter (POM) that are originally not associated with minerals (Chenu 1993; Puget et al 1999; Decho 2000) The abundance of bacterial exopolymer networks on sediment mineral particles has also been confirmed by electronmicroscopic studies (Ransom et al 1997, 1999; Bennett et al 1999) Therefore, the mineral-associated OC is likely to be secondary products generated during early diagenesis While the d13C signature of organic matter is not significantly modified by decomposition compared to the natural variability of d13C among different OC sources (Macko et al 1994; Meyers 1994), the d15N usually increases with the processes of bacterial reworking during early diagenesis (Freudenthal et al 2001; Lehmann et al 2002; Kohzu et al 2011) Therefore, d13COC will be useful for tracing source organic matter even when it has been transformed into mineral-associated OC through early diagenesis, while d15N may be used as an indicator for the degree of bacterial reworking In the case of seagrass meadow sediments, the d15NTN of the heavier density fractions was generally higher than the lighter fractions (Fig 5a–f,i), which suggests that OC adsorbed to mineral surfaces had undergone bacterial reworking more extensively than OC not tightly associated with minerals This conclusion is consistent with the trend previously suggested for continental margin sediments by the amino acid degradation index (Arnarson and Keil 2001, 2007) The OC/TN ratio of sediment organic matter converged to around 10 as the SSA increased for both the offshore and seagrass meadow sediments (Fig 3d) This implies either that the organic matter accumulated as organomineral complex in these two types of sediments was derived from common precursors, mostly bacterial macromolecules (Harrison and Mann 1975), or that nitrogen played some essential role in the stabilization of mineral-associated OC (Knicker et al 1996; Burdige 2007) The asymptotic relationship between the concentration and the d13C of OC in Os (Fig 2a) indicated that the d13C representative of OC accumulating in the offshore habitat was around 221.6&, which is within the range of d13C previously reported for pelagic POM and zooplankton in the Seto Inland Sea (Takai et al 2002, 2004; Miller et al 2010) The d13COC of the density fractions of > 2.0 g cm23, in which most of the OC in the offshore samples had accumulated, was also in a range between 221.7& and 220.9& (Fig 5m– p) In contrast, the d13COC for fractions < 2.0 g cm23 was consistently lower than this range A similar trend of d13COC across density fractions was observed for all the tested 13 Miyajima et al Carbon sequestration in seagrass meadows consist of labile organic compounds, such as carbohydrates and amino acids and have been suggested to be the predominant carbon source for indigenous microbial communities (Wood and Hayasaka 1981; Vichkovitten and Holmer 2005; Bouillon and Boschker 2006) Such microbial processes are thought to promote the generation and accumulation of mineral-associated organic matter in the seagrass meadow sediments, raising the OC load on the mineral surface up to “carrying-capacity” level (Mayer 1994b; Keil and Mayer 2014) within a relatively short time span The present study also revealed that the OC content of seagrass meadow sediments could vary by two orders of magnitude from site to site (25–1310 lmol C g21; Fig 2a), and that such variability was primarily explained by the difference in the spatial extent of individual seagrass meadows (Fig 6a) This finding indicates that, although seagrass meadows have a potential ecosystem function of carbon sequestration as mentioned above, the degree to which this potential is developed depends on the size of the habitat Breaking down the OC concentration into products of the SSA and the OC loading per unit surface area (OC/SSA), the SSA depended on the areal extent of the seagrass meadows (Fig 6c) but the OC/SSA ratio seemed to be much less sensitive to the meadow size (Fig 6d) The dependence patterns of the OC concentration and the SSA on the areal extent were similar to each other This suggests that the OC concentration in sediments is principally constrained by the capacity of the meadow to accumulate sediment materials with high SSA, such as silt and clay, which is dependent on the size of the meadow The net accumulation rate of sediment is determined by the balance between the gross supply rate and the resuspension and removal rate of sediment particles, and seagrass meadows of Z marina can influence these rates by modifying the hydrodynamics of the overlying seawater by canopy friction (Fonseca et al 1982; Fonseca and Fisher 1986) As the attenuating effect of a meadow on water current and turbulence is expected to increase with increasing flow path length across the meadow, the average flow rate and turbulent intensity would be reduced as the dimension of the meadow increases Concomitantly, finer suspended particles will be deposited to sediment, and sediment resuspension will be suppressed, resulting in accumulation of high-SSA mineral particles in the sediment This is likely the mechanism that leads to the relationship mentioned above between the SSA and the meadow size Conversely, the lack of dependence of the OC/SSA ratio on the meadow size implies that, irrespective of spatial extent, seagrass meadows could provide sufficient OC to fill up available sites of adsorption on mineral surface The fraction of seagrass-derived OC in sediment OC, as estimated by the d13C-based model, also apparently increased with increasing areal extent of seagrass meadows, and was saturated at around 0.3 km2 (Fig 6b) However, the data also showed that the variation of the estimated fraction was relatively large for smaller seagrass meadows This estuaries is usually significantly lower than marine DIC due to mixing with freshwater DIC that typically has low d13C (Fry 2002) Such low d13C values characteristic of estuarine primary producers may also be responsible for the lower d13C in the ZmE samples OC originally derived from marine primary producers, including seagrasses, macroalgae, phytoplankton, and epiphytic microalgae, seems to be stored in the sediment mainly as organic matter adsorbed to the surface of sediment minerals, while OC derived from terrestrial plants tends to remain as organic particles not tightly associated with mineral particles This difference may be related to differences in the accessibility of organic matter for indigenous bacteria, i.e., compared to terrestrial organic matter transported by rivers that is usually characterized by low degradability and high C/N ratios, marine organic matter is considered to be more readily utilized by sedimentary bacteria to produce cellular and extracellular materials that can persist over the long term in sediments by the sorptive preservation mechanism (Hedges et al 1997; Zonneveld et al 2010) Therefore, unless the terrestrial loading of OC is overwhelming, the capacity of marine habitats to sequester OC in the underlying sediment would be primarily constrained by two factors: the supply rate of OC produced by marine primary producers, and the accumulation rate of the mineral surface available for OC to adsorb Accumulation of sediment OC as an ecosystem function of seagrass meadows The present study showed that meadows of Z marina stored, on average, more than twofold higher concentration of OC in the underlying sediments than other nearshore unvegetated habitats and macroalgal beds in the temperate coastal region of the Seto Inland Sea, Japan This conclusion is consistent with recent suggestions that seagrass meadows represent a hot spot of natural carbon sequestration on Earth (Duarte et al 2005; McLeod et al 2011; Duarte et al 2013) Furthermore, seagrass meadows can accumulate OC predominantly as mineral-associated organic matter, which represents the mature, stable form of OC sequestered in the continental margin sediments This feature was not evident for the other habitats except for the shallow (< 30 m) offshore sediments A significant fraction of the adsorbed OC in seagrass meadow sediments is likely derived from seagrasses and epiphytic microalgae, i.e., autochthonous organic matter These features altogether can be regarded as one of the major ecosystem functions specific to seagrass meadows The capacity of seagrass meadows to generate OC-rich sediment is considered to rely partially on the role of the below-ground seagrass tissues The roots and rhizomes of seagrasses continuously release photosynthetic products (dissolved organic matter and oxygen) that stimulate microbial activity in the surrounding sediments (Wetzel and Penhale 1979; Moriarty et al 1986) Organic exudates from the roots 14 Miyajima et al Carbon sequestration in seagrass meadows Table Comparison of the sedimentation and the OC accumulation rates among different habitats Sedimentation rate* Water depth [m] OC [lmol C g21] [g cm22 y21] Station # Reference† OC accumulation rate [mmol C m22 y21] ZmC2 ZmC3 3.7 1.5 1090 780 0.077 0.076 Z5 Z4 A A 844 591 ZmC6 3.0 936 0.028 Z3 A 261 3.0 428 0.036 B3 A 152 Os2 Os8 27.0 24.1 1150 1270 0.19 0.20 50 12 B C 2180 2550 Os13 46.0 510 0.11 55 D 561 Os14 Os20 34.9 9.8 672 1340 0.165 0.19 62/69‡ 75 D D 1110 2540 Os21 16.0 Su-121 E 2850 Habitat/Sample Seagrass meadows Bare area adjacent to meadow Ba1 Offshore sites 1430 0.20 14 210 137 Pb/ Cs method in Refs B–E *The sedimentation rate was estimated by the C method in Ref A and the †References: A, Miyajima et al (2015); B, Hoshika and Shiozawa (1984b); C, Hoshika and Shiozawa (1984a); D, Hoshika and Shiozawa (1985); E, Komai et al (2015) ‡Because site Os14 (depth, 34.9 m) was located near the midpoint between Sta 62 (38.7 m) and 69 (31.3 m) of Ref D, the average sedimentation rate of the latter two stations was used in this case transported by rivers Therefore, to model the carbon sequestration rate of seagrass meadows, information on the factors controlling the delivery of soil particles to the meadows, such as the geology, land use, and climate conditions of the watersheds, and hydrodynamics of coastal water from the river mouths to the meadows, will be indispensable phenomenon may be interpreted in terms of the edge effect (Ricart et al 2015) At the edge of a seagrass meadow, sediment may receive a relatively large amount of organic and inorganic matter transported from outside of the meadow, compared to the inner part of the meadow, resulting in a relatively low fraction of autochthonous OC in the sediment OC pool even when the production rate of autochthonous OC is similar at the edge and the inner part The proportion of area influenced by such an edge effect would be greater for the smaller seagrass meadows This is probably the reason for the relatively smaller fraction of seagrass-derived OC estimated with a greater variance in smaller seagrass meadows Therefore, it seems reasonable, at present, to consider that the autochthonous OC production per unit area does not depend on the size of the seagrass meadow, albeit the average fraction of autochthonous OC remaining in the sediment increases with the meadow size The capacity of seagrass meadows to sequester OC in the sediment depends on both high autochthonous OC production and efficient accumulation of high-SSA mineral particles However, at our study sites, the OC sequestration seemed to be limited, not by the OC production rate, but by the supply rate of mineral surface area available for the sorptive preservation of OC An important implication of this finding is that the carbon sequestration as an ecosystem function of seagrass meadows cannot be achieved by the performance of seagrass meadows alone, but requires a continuous supply of available mineral surfaces, such as silt and clay from outside of the meadows Quantitatively, the most important source of silt and clay are terrestrial soils Shallow offshore sediment as another hot spot of OC sequestration Offshore sediments at our study site showed remarkable negative correlations of OC (Table 2) and SSA (Fig 3a) with water depth, which indicates that fine sediment particles such as silt and clay, as well as OC adsorbed to them, preferentially accumulate in a relatively shallow sea floor This trend is presumably attributable to the hydrodynamic structure of the Seto Inland Sea (Inouchi 1982), where strong tidal currents often occur in relatively deep channel areas and preclude fine sediment from accumulating there, while silt and clay particles loaded by rivers tend to deposit primarily on the shallow zones along the coastline We found that shallow offshore sediments (water depth, 9–30 m) often contained a higher concentration of OC (up to 1890 lmol C g21) than most OC-rich seagrass meadow sediments (Fig 2a) This suggests that shallow offshore environments may represent another hotspot of OC sequestration We tentatively calculated the accumulation rates of OC in the sediments of several seagrass meadow and offshore sites using the OC concentrations in sediments reported in this study, and existing data of sediment accumulation rates measured at the same or proximate locations (Table 3) The calculated OC accumulation rate at six offshore sites was 15 Miyajima et al Carbon sequestration in seagrass meadows changes, such as sea level rise and intensified water stratification, affect the OC stocks in shallow offshore sediment is also a high priority for future research approximately onefold to fivefold higher than the average OC accumulation rate for seagrass meadows Although this difference may be partially due to differences in methodologies among studies, this comparison suggests that OC may be sequestered in offshore sediment at rates at least comparable to those of seagrass meadows The range of d13COC in Os samples (Figs 2a, 5m–p) was similar to the range typical of planktonic materials In fact, the IsoSource model indicated that 45–50% of OC stored in surface sediment of Os was plankton-derived OC, with seagrasses contributing 14–24% as an OC source Because seagrasses rarely occur below a depth of m in the Seto Inland Sea, the putative seagrassderived OC in sediments deeper than m is thought to be transported from nearshore seagrass meadows by sediment erosion and redeposition This is consistent with several similarities we found between the shallow offshore and seagrass meadow sediments, such as the range of the MMD (Fig 3b), the strong correlation between OC and SSA (Fig 3c), and the accumulation of OC in the denser fractions (Fig 5a–f,m–p) As the carbon sequestration capacity of vegetated coastal habitats is widely believed to be a global potential sink of atmospheric CO2, increasing concern has arisen about the potential emission of CO2 that might occur should these habitats decline due to coastal development and climate change (Pendleton et al 2012) In fact, historical OC deposits in the top layer of seagrass meadow sediments could be lost by erosion within a  short period after vegetation loss (Macreadie et al 2015; Marba et al 2015) In the case of the Seto Inland Sea, it has been estimated that > 70% (ca., 170 km2) of the seagrass meadows that were present in the 1950s have been lost during the third quarter of the 20th century due to reclamation and shore protection works, eutrophication (reduced light penetration), and dragnet fisheries (Komatsu 1997) Meanwhile the standing stock of OC in the top meter of six seagrass meadow sediments from Seto Inland Sea (Z1–5, Zj in Table 1) has been estimated to range from 450 to 750 mol C m22 (Miyajima et al 2015) Assuming that half (i.e., the top 50 cm) of the stock was eroded and subsequently oxidized into CO2 within several decades of the loss of vegetation, an emission of 1.7–2.8 Mt of CO2 is expected to have been caused to date by the loss of seagrass meadows mentioned above However, the actual fate of the OC contained in the eroded seagrass meadow sediments is largely unknown If the seagrass meadows are followed by shallow flat-bottomed areas, the eroded OC-rich sediment may run off into them, which may act as a secondary OC sink It is thus possible that the OC-rich sediments found at shallow offshore sites in this study were partly derived from former seagrass meadows that had disappeared during the above period or earlier Thus, future research should concentrate on quantitative evaluation of the transport processes of sediment and OC from nearshore vegetated habitats to the offshore sea floor, with the explicit inclusion of time scales At the same time, how global warming and the associated environmental References Adachi, H., H Yamano, T Miyajima, and M Nakaoka 2010 A simple and robust procedure for coring unconsolidated sediment in shallow water J Oceanogr 66: 865–872 doi: 10.1007/s10872-010-0070-4 Aller, R C 1998 Mobile deltaic and continental shelf muds as suboxic, fluidized bed reactors Mar Chem 61: 143– 155 doi:10.1016/S0304-4203(98)00024-3 Arnarson, T S., and R G Keil 2001 Organic–mineral interactions in marine sediments studied using density fractionation and X-ray photoelectron spectroscopy Org Geochem 32: 1401–1415 doi:10.1016/S0146-6380(01) 00114-0 Arnarson, T S., and R G Keil 2007 Changes in organic matter–mineral interactions for marine sediments with varying oxygen exposure times Geochim Cosmochim Acta 71: 3545–3556 doi:10.1016/j.gca.2007.04.027 Bennett, R H., B Ransom, M Kastner, R J Baerwald, M H Hulbert, W B Sawyer, H Olsen, and M W Lambert 1999 Early diagenesis: Impact of organic matter on mass physical properties and processes, California continental margin Mar Geol 159: 7–34 doi:10.1016/S00253227(99)00003-1 Bock, M J., and L M Mayer 2000 Mesodensity organo-clay associations in a near-shore sediment Mar Geol 163: 65–75 doi:10.1016/S0025-3227(99)00105-X Bouillon, S., and H T S Boschker 2006 Bacterial carbon sources in coastal sediments: A cross-system analysis based on stable isotope data of biomarkers Biogeosciences 3: 175–185 doi:10.5194/bg-3-175-2006 Burdige, D J 2007 Preservation of organic matter in marine sediments: Controls, mechanisms, and an imbalance in sediment organic carbon budgets? Chem Rev 107: 467– 485 doi:10.1021/cr050347q Chenu, C 1993 Clay-or sand-polysaccharide associations as models for the interface between micro-organisms and soil: Water related properties and microstructure Geoderma 56: 143–156 doi:10.1016/0016-7061(93)90106-U Decho, A W 2000 Microbial biofilms in intertidal systems: An overview Cont Shelf Res 20: 1257–1273 doi: 10.1016/S0278-4343(00)00022-4 Deer, W A., R A Howie, and J Zussman 1992 An introduction to the rock-forming minerals, 2nd ed Pearson Education Ltd n 1996 The fate of marine autoDuarte, C M., and J Cebria trophic production Limnol Oceanogr 41: 1758–1766 doi:10.4319/lo.1996.41.8.1758 Duarte, C M., J J Middelburg, and N Caraco 2005 Major role of marine vegetation on the oceanic carbon cycle Biogeosciences 2: 1–8 doi:10.5194/bg-2-1-2005 16 Miyajima et al Carbon sequestration in seagrass meadows , and I Hendriks 2013 Duarte, C M., H Kennedy, N Marba Assessing the capacity of seagrass meadows for carbon burial: Current limitations and future strategies Ocean Coast Manag 83: 32–38 doi:10.1016/j.ocecoaman 2011.09.001 Fonseca, M S., J S Fisher, J C Zieman, and G W Thayer 1982 Influence of the seagrass, Zostera marina L., on current flow Estuarine Coast Shelf Sci 15: 351–364 doi: 10.1016/0272-7714(82)90046-4 Fonseca, M S., and J S Fisher 1986 A comparison of canopy friction and sediment movement between four species of seagrasses with reference to their ecology and restoration Mar Ecol Prog Ser 29: 15–22 doi:10.3354/ meps029015 Fourqurean, J W., and others 2012 Seagrass ecosystems as a globally significant carbon stock Nat Geosci 5: 505–509 doi:10.1038/ngeo1477 € fer, M Zabel, and G Freudenthal, T., T Wagner, F Wenzho Wefer 2001 Early diagenesis of organic matter from sediments of the eastern subtropical Atlantic: Evidence from stable nitrogen and carbon isotopes Geochim Cosmochim Acta 65: 1795–1808 doi:10.1016/S0016-7037(01)00554-3 Fry, B 2002 Conservative mixing of stable isotopes across estuarine salinity gradients: A conceptual framework for monitoring watershed influences on downstream fisheries production Estuaries 25: 264–271 doi:10.1007/ BF02691313 Gibson, L J 2012 The hierarchical structure and mechanics of plant materials J R Soc Interface 9: 2749–2766 doi: 10.1098/rsif.2012.0341 Grimsditch, G., J Alder, T Nakamura, R Kenchington, and J Tamelander 2013 The blue carbon special edition— introduction and overview Ocean Coast Manag 83: 1–4 doi:10.1016/j.ocecoaman.2012.04.020 Harrison, P G., and K H Mann 1975 Detritus formation from eelgrass (Zostera marina L.): The relative effects of fragmentation, leaching, and decay Limnol Oceanogr 20: 924–934 doi:10.4319/lo.1975.20.6.0924 Hartnett, H E., R G Keil, J I Hedges, and A H Devol 1998 Influence of oxygen exposure time on organic carbon preservation in continental margin sediments Nature 391: 572–574 doi:10.1038/35351 Heck, K L., T J B Carruthers, Jr., C M Duarte, A R Hughes, G Kendrick, R J Orth, and S W Williams 2008 Trophic transfers from seagrass meadows subsidize diverse marine and terrestrial consumers Ecosystems 11: 1198–1210 doi:10.1007/s10021-008-9155-y Hedges, J I., and R G Keil 1995 Sedimentary organic matter preservation: An assessment and speculative synthesis Mar Chem 49: 81–115 doi:10.1016/0304-4203(95) 00008-F Hedges, J I., R G Keil, and R Benner 1997 What happens to terrestrial organic matter in the ocean? Org Geochem 27: 195–212 doi:10.1016/S0146-6380(97)00066-1 Hoshika, A., and T Shiozawa 1984a Sedimentation rates and heavy metal pollution of sediments in the Seto Inland Sea: Part Hiroshima Bay J Oceanogr Soc Jpn 40: 115–123 doi:10.1007/BF02302492 (in Japanese with English summary) Hoshika, A., and T Shiozawa 1984b Sedimentation rates and heavy metal pollution of sediments in the Seto Inland Sea Part Hiuchi-Nada J Oceanogr Soc Jpn 40: 334–342 doi:10.1007/BF02303337 Hoshika, A., and T Shiozawa 1985 Sedimentation rates and heavy metal pollution of sediments in the Seto Inland Sea: Part Suo-Nada J Oceanogr Soc Jpn 41: 283–290 doi:10.1007/BF02109235 Inouchi, Y 1982 Distribution of bottom sediments in the Seto Inland Sea––the influence of tidal currents on the distribution of bottom sediments J Geol Soc Jpn 88: 665–681 (in Japanese with English summary) doi: 10.5575/geosoc.88.665 Keil, R G., and J I Hedges 1993 Sorption of organic matter to mineral surfaces and the preservation of organic matter in coastal marine sediments Chem Geol 107: 385–388 doi:10.1016/0009-2541(93)90215-5 Keil, R G., E Tsamakis, C B Fuh, J C Giddings, and J I Hedges 1994a Mineralogical and textural controls on the organic composition of coastal marine sediments: Hydrodynamic separation using SPLITT-fractionation Geochim Cosmochim Acta 58: 879–893 doi:10.1016/0016-7037 (94)90512-6 Keil, R G., D B Montluc¸on, F G Prahl, and J I Hedges 1994b Sorptive preservation of labile organic matter in marine sediments Nature 370: 549–552 doi:10.1038/ 370549a0 Keil, R G., L M Mayer, P D Quay, J E Richey, and J I Hedges 1997 Loss of organic matter from riverine particles in deltas Geochim Cosmochim Acta 61: 1507– 1511 doi:10.1016/S0016-7037(97)00044-6 Keil, R G., and L M Mayer 2014 Mineral matrices and organic matter, p 337–359 In H Holland and K Turekian [eds.], Organic Geochemistry, (P G Falkowski and K H Freeman, eds.), 2nd ed V 12 Treatise on Geochemistry, Elsevier doi:10.1016/B978-0-08-095975-7.01024-X Kennedy, H., E Gacia, D P Kennedy, S Papadimitriou, and C M Duarte 2004 Organic carbon sources to SE Asian coastal sediments Estuarine Coast Shelf Sci 60: 59–68 doi:10.1016/j.ecss.2003.11.019 Kennedy, H., J Beggins, C M Duarte, J W Fourqurean, M , and J J Middelburg 2010 Seagrass Holmer, N Marba sediments as a global carbon sink: Isotopic constraints Global Biogeochem Cycles 24: GB4026 doi:10.1029/ 2010GB003848 Knicker, H., A W Scaroni, and P G Hatcher 1996 13C and 15 N NMR spectroscopic investigation on the formation of fossil algal residues Org Geochem 24: 661–669 doi: 10.1016/0146-6380(96)00057-5 17 Miyajima et al Carbon sequestration in seagrass meadows composition Geochim Cosmochim Acta 65: 1455–1466 doi:10.1016/S0016-7037(00)00612-8 Miyajima, T., M Hori, M Hamaguchi, H Shimabukuro, H Adachi, H Yamano, and M Nakaoka 2015 Geographic variability in organic carbon stock and accumulation rate in sediments of East and Southeast Asian seagrass meadows Global Biogeochem Cycles 29: 397–415 doi: 10.1002/2014GB004979 Moriarty, D J W., R L Iverson, and P C Pollard 1986 Exudation of organic carbon by the seagrass Halodule wrightii Aschers And its effect on bacterial growth in the sediment J Exp Mar Biol Ecol 96: 115–126 doi: 10.1016/0022-0981(86)90237-6 Nunn, B L., and R G Keil 2005 Size distribution and amino acid chemistry of base-extractable proteins from Washington coast sediments Biogeochemistry 75: 177–200 doi:10.1007/s10533-004-6546-9 Pendleton, L., and others 2012 Estimating global “blue carbon” emissions from conversion and degradation of vegetated coastal ecosystems PloS One 7: e43542 doi: 10.1371/journal.pone.0043542 Phillips, D L., and J W Gregg 2003 Source partitioning using stable isotopes: Coping with too many sources Oecologia 136: 261–269 doi:10.1007/s00442-003-1218-3 Puget, P., D A Angers, and C Chenu 1999 Nature of carbohydrates associated with water-soluble aggregates of two cultivated soils Soil Biol Biochem 31: 55–63 doi: 10.1016/S0038-0717(98)00103-5 Ransom, B., R H Bennett, R Baerwald, and K Shea 1997 TEM study of in situ organic matter on continental margins: Occurrence and the “monolayer” hypothesis Mar Geol 138: 1–9 doi:10.1016/S0025-3227(97)00012-1 Ransom, B., R H Bennett, R Baerwald, M H Hulbert, and P J Burkett 1999 In situ conditions and interactions between microbes and minerals in fine-grained marine sediments: A TEM microfabric perspective Am Mineral 84: 183–192 doi:10.2138/am-1999-1-220 Ricart, A M., P H York, M A Rasheed, M Perez, J Romero, C V Bryant, and P I Macreadie 2015 Variability of sedimentary organic carbon in patchy seagrass landscapes Mar Pol Bull 100: 476–482 doi:10.1016/j.marpolbul.2015.09.032 Sagawa, T., A Mikami, T Komatsu, N Kosaka, A Kosako, S Miyazaki, and M Takahashi 2008 Technical Note Mapping seagrass beds using IKONOS satellite image and side scan sonar measurements: A Japanese case study Int J Remote Sens 29: 281–291 doi:10.1080/01431160701269028 Serrano, O., P S Lavery, M Rozaimi, and M Mateo 2014 Influence of water depth on the carbon sequestration capacity of seagrasses Global Biogeochem Cycles 28: 950–961 doi:10.1002/2014GB004872 Sollins, P., M G Kramer, C Swanston, K Lajtha, T Filley, A K Aufdenkampe, R Wagai, and R D Bowden 2009 Sequential density fractionation across soils of contrasting mineralogy: Evidence for both microbial- and mineral- Kohzu, A., and others 2011 Direct evidence for nitrogen isotope discrimination during sedimentation and early diagenesis in Lake Kasumigaura, Japan Org Geochem 42: 173–183 doi:10.1016/j.orggeochem.2010.10.010 Komai, Y., K Yonebayashi, N Katsumi, T Irizuki, A Tsujimoto, and M Okazaki 2015 Changes in sediment environment of Seto Inland Sea estimated from carbon and nitrogen stable isotope ratios and meiobenthos communities J Jpn Soc Water Environ 38: 39–47 doi: 10.2965/jswe.38.39 (in Japanese with English summary) Komatsu, T 1997 Long-term changes in the Zostera bed area in the Seto Inland Sea (Japan), especially along the coast of the Okayama Prefecture Oceanol Acta 20: 209–216 Open Access version: http://archimer.ifremer.fr/doc/00093/20391/ Lehmann, M F., S M Bernasconi, A Barbieri, and J A Mckenzie 2002 Preservation of organic matter and alteration of its carbon and nitrogen isotope composition during simulated and in situ early sedimentary diagenesis Geochim Cosmochim Acta 66: 3573–3584 doi:10.1016/ S0016-7037(02)00968-7 Macko, S A., M H Engel, and Y Qian 1994 Early diagenesis and organic matter preservation—a molecular stable carbon isotope perspective Chem Geol 114: 365–379 doi:10.1016/0009-2541(94)90064-7 Macreadie, P I., S M Trevathan-Tackett, C G Skilbeck, J Sanderman, N Curlevski, G Jacobsen, and J R Seymour 2015 Losses and recovery of organic carbon from a seagrass ecosystem following disturbance Proc R Soc B 282: 20151537 doi:10.1098/rspb.2015.1537 , N., and others 2015 Impact of seagrass loss and subMarba sequent revegetation on carbon sequestration and stocks J Ecol 103: 296–302 doi:10.1111/1365-2745.12370 Mayer, L M 1994a Relationships between mineral surfaces and organic carbon concentrations in soils and sediments Chem Geol 114: 347–363 doi:10.1016/0009-2541(94)90063-9 Mayer, L M 1994b Surface area control of organic carbon accumulation in continental shelf sediments Geochim Cosmochim Acta 58: 1271–1284 doi:10.1016/00167037(94)90381-6 McLeod, E., and others 2011 A blueprint for blue carbon: Toward an improved understanding of the role of vegetated coastal habitats in sequestering CO2 Front Ecol Environ 9: 552–560 doi:10.1890/110004 Meyers, P A 1994 Preservation of elemental and isotopic source identification of sedimentary organic matter Chem Geol 114: 289–302 doi:10.1016/0009-2541(94)90059-0 Miller, T W., K Omori, H Hamaoka, J Shibata, and O Hidejiro 2010 Tracing anthropogenic inputs to production in the Seto Inland Sea, Japan—a stable isotope approach Mar Poll Bull 60: 1803–1809 doi:10.1016/ j.marpolbul.2010.06.002 Miyajima, T., H Ogawa, and I Koike 2001 Alkali-extractable polysaccharides in marine sediments: Abundance, molecular size distribution, and monosaccharide 18 Miyajima et al Carbon sequestration in seagrass meadows marina L.) root exudate J Exp Mar Biol Ecol 50: 153– 161 doi:10.1016/0022-0981(81)90047-2 Yanagi, T 1984 Seasonal variation of water temperature in the Seto Inland Sea J Oceanogr Soc Jpn 40: 445–450 doi:10.1007/BF02303072 Zimmerman, A R., J Chorover, K W Goyne, and S L Brantley 2004 Protection of mesopore-adsorbed organic matter from enzymatic degradation Environ Sci Technol 38: 4542–4548 doi:10.1021/es035340 Zonneveld, K A F., and others 2010 Selective preservation of organic matter in marine environments; processes and impact on the sedimentary record Biogeosciences 7: 483– 511 doi:10.5194/bg-7-483-2010 controlled soil organic matter stabilization Biogeochemistry 96: 209–231 doi:10.1007/s10533-009-9359-z Sugimatsu, K., H Yagi, K Abo, K Tarutani, M Hori, G Yoshida, H Shimabukuro, and A Nakayama 2015 A coupled particle tracking-carbon cycle modeling system for sedimentary organic carbon derived from drifting seagrass in Seto Inland Sea J Jpn Soc Civil Eng B2 71: I_1387–I_1392 doi:10.2208/ kaigan.71.I_1387 (in Japanese with English abstract) Takai, N., Y Mishima, A Yorozu, and A Hoshika 2002 Carbon sources for demersal fish in the western Seto Inland Sea, Japan, examined by 13C and 15N analyses Limnol Oceanogr 47: 730–741 doi:10.4319/lo.2002.47.3.0730 Takai, N., A Yorozu, T Tanimoto, A Hoshika, and K Yoshihara 2004 Transport pathways of microphytobenthos-originating organic carbon in the food web of an exposed hard bottom shore in the Seto Inland Sea, Japan Mar Ecol Prog Ser 284: 97–108 doi: 10.3354/meps284097 Vichkovitten, T., and M Holmer 2005 Dissolved and particulate organic matter in contrasting Zostera marina (eelgrass) sediments J Exp Mar Biol Ecol 316: 183–201 doi: 10.1016/j.jembe.2004.11.002 Weijers, J W H., S Schouten, E Schefuß, R R Schneider, and J S Sinninghe Damste 2009 Disentangling marine, soil and plant organic carbon contributions to continental margin sediments: A multi-proxy approach in a 20,000 year sediment record from the Congo deep-sea fan Geochim Cosmochim Acta 73: 119–132 doi:10.1016/j.gca.2008.10.016 Wetzel, R G., and P A Penhale 1979 Transport of carbon and excretion of dissolved organic carbon by leaves and roots/rhizomes in seagrasses and their epiphytes Aquat Bot 6: 149–158 doi:10.1016/0304-3770(79)90058-5 Wood, D C., and S S Hayasaka 1981 Chemotaxis of rhizoplane bacteria to amino acids comprising eelgrass (Zostera Acknowledgments The authors are grateful to Bo Thamdrup, Robert Howarth, and an anonymous reviewer for helpful comments on an earlier version of the manuscript Field survey assistance at the Seto Inland Sea was provided by staff from the Takehara Marine Science Station of Hiroshima University and the R/V Shirafuji-maru of the Fisheries Research Agency GIS analysis assistance was provided by Hideki Hamaoka Isao Koike, Tsuneo Ono, Masahiro Nakaoka, and Tomohiro Kuwae also provided valuable suggestions for interpreting the data This study was financially supported by a Grant in Aid for the Promotion of Global Warming Countermeasures from the Fisheries Agency of the Ministry of Agriculture, Forestry and Fisheries, Japan, and by JSPS Grants in Aid for Scientific Research Grant Numbers 25291098 and 25257305 Conflict of Interest None declared Submitted 25 January 2016 Revised 26 June 2016 Accepted 18 October 2016 Associate editor: Bo Thamdrup 19