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Mussel periostracum as a high-resolution archive of soft tissue δ13C records in coastal ecosystems
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ScienceDirect Geochimica et Cosmochimica Acta 260 (2019) 232–243 www.elsevier.com/locate/gca
Mussel periostracum as a high-resolution archive of soft tissue d13C records in coastal ecosystems Liqiang Zhao a,b,⇑, Kotaro Shirai a, Naoko Murakami-Sugihara a, Tomihiko Higuchi a, Kiyoshi Tanaka a a
Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba 277-8564, Japan b Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China Received 2 July 2018; accepted in revised form 23 June 2019; available online 2 July 2019
Abstract Stable carbon isotope ratios (d13C) of mussel soft tissues have been widely used to characterize baseline d13C isoscapes and identify carbon sources at the base of coastal food webs. Extending soft tissue d13C records back in time, however, is extremely challenging due to very limited sample availability. Here, we test if the stable carbon isotopic composition of periostracum (the outermost organic layer of the shell) in the Mediterranean mussel (Mytilus galloprovincialis) can be used as an environmental archive, similar to soft tissue records. In general, spatial and seasonal variations of periostracum d13C values are comparable to those of soft tissues, but apparently the latter are more time-averaged and smoothed. Irrespective of such offset, there is a significant linear correlation between mussel periostracum and soft tissue d13C values (R2 = 0.608, p < 0.001). Moreover, high-resolution d13C analysis of mussel periostracum indicates that it integrates much less time than soft tissue due to metabolically inert nature, consequently being able to record predictable events such as tidal changes and also unpredictable ephemeral events in coastal ecosystems. The present study demonstrates the potential of the periostracum as a viable alternative to the most widely used soft tissues in isotopic studies. Most promisingly, given ample collections over time scales up to hundreds of years and usually stored dry, this technique could be used to extend coastal organic carbon d13C records back in time. Ó 2019 Elsevier Ltd. All rights reserved. Keywords: Stable carbon isotope; Bivalve mollusks; Periostracum; Coastal food web; Otsuchi Bay
1. INTRODUCTION Coastal zones represent one of the most highly diverse, biologically productive and ecologically valuable areas of the world’s oceans (Costanza et al., 1997). However, global coastal ecosystems have degraded at unprecedent rates during recent decades, due to increasing anthropogenic threats such as overharvesting, habitat destruction, eutrophication, hypoxia, pollution, ocean warming and acidification ⇑ Corresponding author at: Atmosphere and Ocean Research
Institute, The University of Tokyo, Chiba 277-8564, Japan. E-mail address: [email protected] (L. Zhao). https://doi.org/10.1016/j.gca.2019.06.038 0016-7037/Ó 2019 Elsevier Ltd. All rights reserved.
(Dutkiewicz et al., 2015; Kirwan and Megonigal, 2013; Temmerman et al., 2013; Wu et al., 2017). Coastal deterioration stems primarily from changes at the base of food webs (Bauer et al., 2013; Hoegh-Guldberg and Bruno, 2010; Middelburg, 2014), fueled by diverse sources and high concentrations of organic carbon (OC) originating from local pelagic and benthic primary production and/or marine and terrestrial import (Christianen et al, 2017; Vlahos and Whitney, 2017; Yokoyama et al., 2005). Consequently, real-time and retrospective monitoring of sources and spatiotemporal dynamics of OC represents a critical step forward in predicting how coastal ecosystems may respond to anthropogenic perturbation.
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Stable carbon isotope analysis serves as a useful tool for inferring sources and flows of OC in aquatic and terrestrial ecosystems (Middelburg, 2014). Stable carbon isotope ratios (d13C) of primary producers in marine, freshwater and terrestrial food webs differ distinctly due to differences in d13C values of carbon substrate assimilated, i.e., dissolved inorganic carbon (DIC) or atmosphere carbon dioxide, and isotopic fractionation during photosynthetic carbon fixation (Fry, 2006). Marine benthic primary producers have d13C values between 18 and 13‰ (Currin et al., 1995; Riera et al., 1999), while marine pelagic primary producers often display a narrow range of 22 to 20‰ (Creach et al., 1997; Currin et al., 1995). The average d13C value of carbon transferred from terrestrial sources to the oceans is rather similar to that found in C3 plants varying slightly from 27 to 26‰ (Peterson and Fry, 1987). Riverine phytoplankton and their detritus are typically characterized by d13C values (40 to 30‰) much lower than those of marine and terrestrial derived carbon (Hamilton and Lewis, 1992; Hellings et al., 1999). Values of OC d13C falling between these extremes are often interpreted as indicating mixing of differential carbon components, and the relative contribution of each source can be estimated through end-member mixing analysis (Fry, 2006). However, documenting the isotopic composition of OC sources at the base of the coastal food web (i.e., isotopic baseline) is still fraught with difficulty, due largely to its heterogeneous nature (Compton et al., 2013). Hence, exploring proxies of this isotopic baseline is becoming imperative. d13C records of bivalve soft tissues have been used to decipher sources and dynamics of OC in coastal food webs with great success (e.g., Briant et al., 2018; Dubois et al., 2014; Gillikin et al., 2006a, 2006b; Oczkowski et al., 2014; Yokoyama et al., 2005; Zhao et al., 2013a, 2013b), because d13C values typically exhibit little (<1‰) or no trophic enrichment (Fry, 2006). Through d13C analyses of multiple bivalve species at the landscape scale, for example, Christianen et al. (2017) found that the Dutch Wadden Sea intertidal food webs were sustained by OC originating from microphytobenthos (i.e., benthic primary producers). Therefore, a retrospective analysis of archived soft tissues may offer the potential to track OC baselines and food web dynamics, and document long-term coastal ecosystem changes increasingly modified by human activities (e.g., Chasar et al., 2005; Syva¨ranta et al., 2006; Vander Zanden et al., 2003). Despite such promise, however, this approach has not been routinely applied, due largely to concerns that preservation of soft tissues in formalin (and/or ethanol) can potentially induce shifts in d13C values through the incorporation of isotopically light carbon from preservatives (e.g., Carabel et al., 2009; Delong and Thorp, 2009; Kaehler and Pakhomov, 2001; Liu et al., 2013). Another constraint comes from the limited availability of long-term preserved soft tissues, with most only dating back over few decades (Delong and Thorp, 2009). On the contrary, skeletal hard parts of primary consumers, especially bivalve shells, are likely collected over time scales up to hundreds of years and usually stored dry, such as
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in museums, institutes, and personal collections, thereby sparking interest in exploring their potential as archives of d13C in coastal ecosystems. Bivalve shells precipitate in the extrapallial fluid (EPF), a thin liquid film located between the outer mantle epithelium (OME) and inner shell surface (Wheeler, 1992). In the EPF, the crystallization of inorganic calcium carbonate is mediated by organic matrix secreted from the OME (Wheeler, 1992), the latter referred to as carbonate-bound organic matter (CBOM). Individual crystals are subsequently deposited on the inner surface of periostracum, a protective organic layer covering the shell. It is, therefore, reasonable to assume that during shell building, isotopic characteristics of the OME (and hence whole soft tissue) can be potentially archived and retained by the CBOM and periostracum. Unlike soft tissues which are continuously degrading and accruing new material consequently resulting in temporal integration of stable isotope records (Gillikin et al., 2017), shell organics are metabolically inert (Saleuddin and Petit, 1983), i.e., once deposited the isotopic information is locked in place. An increasing number of studies have recently tested the use of bivalve shell CBOM as an archive of stable nitrogen isotopes (d15N) in coastal and estuarine ecosystems with promising results (e.g., Carmichael et al., 2008; Gillikin et al., 2017; Graniero et al., 2016; Kovacs et al., 2010; O’Donnell et al., 2003; Versteegh et al., 2011; Whitney et al., 2019), and d15N records have been extended back over long-time scales (Black et al., 2017; Darrow et al., 2017). In particular, Gillikin et al. (2017) demonstrated the feasibility of direct combustion with minimal processing for CBOM d15N measurement, paving the way for high resolution d15N reconstructions. However, determining d13C values in shell CBOM by means of direct combustion is clearly not feasible given an extremely large CaCO3 percent (typically 95–99% in the bulk shell; Marin et al., 2008). Hence, the study of d13C records in shell periostracum has recently attracted renewed attention. For example, Delong and Thorp (2009) reported that d13C values derived from the periostracum of freshwater mussel (Amblema plicata) were comparable to those of soft tissues, and longterm (>110 years) d13C records from A. plicata periostracum have been recently developed, demonstrating the promise of this technique in tracking OC dynamics and developing baseline isoscapes in freshwater ecosystems. In a pilot study to date to explore the periostracum as a potential environmental proxy in marine bivalves (Arctica islandica), Wanamaker and Luzier (2014) demonstrated that d13C and d15N values varied with shell growth, indicating that the periostracum may record ambient environmental conditions during the lifetime of A. islandica. Recently, Whitney et al. (2019) reported that the significant correlation of periostracum d15N values with contemporaneous CBOM d15N values of the same A. islandica shell, which suggests that shell periostracum preserves a similar d15N record to that encoded into the CBOM. More promisingly, source amino acid d15N analysis of A. islandica periostracum reveals that bulk periostracum d15N values can reflect the d15N values of the clam’s food source (Whitney
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et al., 2019). However, whether shell periostracum and soft tissue d13C values are strongly correlated on spatial and temporal scales remains to be tested. The present study aims to explore the potential of marine bivalve shell periostracum as an archive for d13C studies in coastal ecosystems. Firstly, to investigate if shell periostracum can provide the same or parallel information as soft tissues, a comparison between their d13C values will be conducted over a wide range of spatial and temporal scales. Then, d13C time-series extracted from one single periostracum will be analyzed to investigate if shell periostracum can be used to reconstruct d13C changes at a highresolution. These results may promote the routine use of d13C records in marine shell periostracum as a tool for studying important ecological questions, especially regarding OC sources and dynamics at the base of food webs in coastal ecosystems. 2. MATERIALS AND METHODS 2.1. Study area Otsuchi Bay (39°210 N, 141°580 E) is a semi-enclosed bay located on the Sanriku Ria coast of Northern Japan (Fig. 1). The bay opens northeast toward the North Pacific Ocean, with a length of approximately 8 km and width of 3 km. Three rivers, the Otsuchi River, the Kozuchi River and the Unosumai River, flow into the bay, with the highest discharge (about 30 m3 s1) occurring in summer and lowest (6 m3 s1) in winter (Anbo et al., 2005). Freshwater inflow is also one of the primary driving forces resulting in highly stratified water column in the bay from spring through to early autumn. During the stratification period, low saline surface water mass with an average depth of 10 m flows out of the bay and a dense bottom water mass moves in an opposite direction (Otobe et al., 2009). The pelagic food webs in the Bay are supported not only by local primary production but also high riverine inputs of
Fig. 1. Bathymetry map showing sampling sites in Otsuchi Bay and one site designated as the control in Funakoshi Bay.
organic matter, the relative contribution of the latter decreasing gradually with distance from the mouth of the river (Wada et al., 1987). Evidently, the ecosystem biogeochemistry in Otsuchi Bay is dynamic and multiple carbon sources showing distinct isotopic values vary temporally and spatially. Accordingly, a large d13C gradient of particulate organic carbon, which is an essential prerequisite to fulfill the task in the present study, can be expected along the coastal of Otsuchi Bay. As shown in Fig. 1, seven sites in Otsuchi bay which are affected by riverine discharge to different degrees were deliberately selected. One control site in Funakoshi Bay which is not affected by freshwater runoff was designated. 2.2. Study species The Mediterranean mussel, Mytilus galloprovincialis, is an invasive bivalve species along the Pacific coast of Japan (Wilkins et al., 1983). In Otsuchi Bay, M. galloprovincialis is a dominant inhabitant in rocky intertidal and shallow subtidal zones. Its shell is subdivided into three major layers: protective organic layer (periostracum), calcitic prismatic layer and aragonitic nacreous layer (Fig. 2). Generally, the thickness of the latter two layers varies significantly over the whole lifespan, while the thickness of the periostracum remains relatively constant (Fig. 2). Shells of M. galloprovincialis grow by periodic deposition of calcium carbonate on the inner surface of the periostracum, accordingly forming periodic growth increments and lines in the prismatic layers (Richardson et al., 1990). 2.3. Mussel collection and preparation At each sampling site, more than 10 adult mussels of similar shell length (5–6 cm) were randomly collected from the quay walls in Otsuchi Bay during low tide on 23 April, 06 June and 29 September in 2016 and 6 January 2017, corresponding to spring, summer, autumn and winter, respectively. Water samples were collected simultaneously for the measurements of oxygen isotopes, which can be used to indicate the intensity of riverine discharge (Zhao et al., 2019). Upon arrival in the laboratory, mussels were immediately stored at 20 °C for further analysis. After thawing at room temperature, the adductor muscle and most recently formed shell periostracum (as illustrated in Fig. 2) were carefully dissected using a scalpel, freeze-dried and homogenized. One mussel shell collected on 6 January 2017 at the site of Southern Nebama (in the vicinity of the mouth of the Unosumai River) was used for high-resolution carbon isotopic analysis. More specifically, prior to taking samples, the periostracum was cleaned thoroughly by lightly brushing to remove any surface contamination. Afterward, periostracum (>200 lg) was scraped off equidistantly (ca. 3 mm) along the axis of maximum shell growth. To constrain the timing and duration of shell formation, shell carbonate materials (>100 lg) were sequentially taken at approximately 0.4–0.5 mm resolution along the axis of maximum shell growth (Fig. 2) for oxygen isotopic analysis. Finally, periostracum materials were placed into open silver capsules and acid-cleaned (1 ml of
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Fig. 2. Shells of the Mediterranean mussel Mytilus galloprovincialis. (a) High-resolution periostracum sampling following the axis of maximum shell growth. (b) Schematic of the shell cross section showing major shell structures. (c) Magnification of the shell ventral margin indicating the most newly formed shell periostracum.
0.5 mol HCl) overnight to prevent potential contamination from shell carbonate (Delong and Thorp, 2009). It should be emphasized here that to reduce the loss of soluble organic components, all materials were not rinsed after acid treatment (Schlacher and Connolly, 2014). These silver capsules were subsequently dried on a hot plate at 50 °C for 48 h, sealed and placed in a desiccator. In addition, 30 specimens were randomly selected from different sampling sites to assess the differences in d13C values between acid-cleaned and uncleaned periostracum materials. 2.4. Carbon and oxygen isotopic analysis via EA-IRMS and CF-IRMS Approximately 1–2 mg homogenized soft tissue and periostracum materials were weighed into tin capsules, which were then folded and subsequently measured with an elemental analyzer–isotope ratio mass spectrometer (EA-IRMS; FLASH 2000/Conflo IV/DELTA V Advantage, Thermo Fisher Scientific) at Atmosphere and Ocean Research Institute, The University of Tokyo. The daily calibration was conducted based on standard material Ala (L-Alanine powder, SI Science, Ltd., Saitama, Japan), of which the d13C value is traceable against the international reference materials (e.g., NBS19 and Sucrose ANU). The instrumental analytical error was estimated based on a daily reproducibility of the standard analyses (n > 7) and on average, the value was better than ±0.2‰ (1 standard deviation). Measured carbon isotopic ratios were reported relative to the Vienna Pee Dee Belemnite (VPDB) scale and expressed in the conventional delta (d) notation in per mil (‰). Regarding oxygen and carbon isotopic analysis of the shell, 100–200 lg carbonate materials were weighed into glass vials, flushed with pure helium and dissolved with 100% phosphoric acid at 72 °C for one hour. Liberated carbon dioxide (CO2) gas was analyzed using a continuous
flow-isotope ratio mass spectrometer (CF-IRMS; DELTA V Plus, Thermo Fisher Scientific) coupled to an automated carbonate reaction device (GasBench II, Thermo Fisher Scientific) housed at the same institute. Measured isotopic data were then calibrated against NBS-19 (d18O = 2.20‰, d13C = +1.95‰). The external reproducibility was better than ±0.23‰ and ±0.05‰, respectively for d18O and d13C (1 standard deviation) based on repeated analysis of an in-house standard. Shell carbonate d18O and d13C values were calculated against the VPDB and expressed in the same manner as d13C value of organic materials. Detailed analytical conditions were previously reported (Shirai et al., 2018). Seawater samples were analyzed using a L2120-I wavelength-scanned cavity ring-down spectroscopy isotopic water analyzer Isotopic (Picarro, Sunnyvale, CA, USA). Before introduction into the analyzer, samples were filtered using a membrane filter (pore size: 0.45 lm, Toyo Roshi Kaisha) to reduce suspended particles and prevent blocking of the sampling line. The analytical uncertainty was better than ±0.10‰ (1 standard deviation) based on repeated analyses of Milli-Q water. d18Owater values were calibrated against the Vienna Standard Mean Ocean Water (V-SMOW). 2.5. Statistical analysis All data were statistically analyzed using SPSS 19.0 software. The Shapiro-Wilk’s test and Levene’s F-test were used to examine whether measured data are normally distributed and vary equally across groups. Then, two-way analysis of variance (Two-way ANOVA) followed by the LSD post-hoc was performed to test the combined effects of sampling site and season on d13C values of mussel soft tissue and periostracum, respectively. The correlation between the d13C values of mussel soft tissue and correspondingly most recently formed portion of the perios-
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tracum was computed by means of Pearson correlation analysis. Statistically significant differences were set at p values < 0.05. 3. RESULTS 3.1. Comparison of d13C data between acid-cleaned and uncleaned mussels Without exception, acid-cleaned mussels showed lower d13Cperio values in compared to those in uncleaned specimens, exhibiting large offsets ranging from 0.35 to 5.13‰ (Fig. 3). A paired Student’s t-test confirmed that there is a statistically significant difference in d13C values between acid-cleaned and uncleaned periostracum materials.
lected near the mouths of all three rivers in Otsuchi Bay. It is worth noting here that in comparison to contemporaneous d13Ctissue records, some d13Cperio values are much more scattered displaying larger standard deviations, as illustrated in Fig. 4. Likewise, significant offsets between periostracum and soft tissue d13C values occurred occasionally (Fig. 4). Without exception, these offsets occurred in summer and autumn. On average, nevertheless, d13Cperio and d13Ctissue records compared reasonably to each other, thereby yielding a high degree of similarity. A significant linear relationship between mussel periostracum and soft tissue d13C values in all contemporaneous specimens (R2 = 0.608, p < 0.001) was subsequently computed (Fig. 5), with a slope of 1.335 and intercept of 6.292. 3.3. High-resolution shell periostracum d13C profiles
3.2. Spatial and seasonal variations of d13C values 13
Spatial and seasonal variations in mussel soft tissue d C records are shown in Fig. 4. d13Ctissue values displayed significant differences in the effects of sampling site and season as well as interaction between these two factors (Table 1). Mussels inhabiting the river mouths in Otsuchi Bay exhibited evident seasonality in d13Ctissue records. Specifically, mussels collected at the site of Aka in the vicinity of the Otsuchi River and Kozuchi River mouths showed the lowest d13Ctissue values in summer, while at sites of Northern Nebama and Southern Nebama close to the Unosumai River mouth, maximum and minimum d13Ctissue values were recorded in spring and autumn, respectively. No evident seasonality was shown in mussels collected in the outer part of the bay (e.g., at sites of Shirahama and Keisen). For better comparison of mussel soft tissue and periostracum d13C values, the latter was plotted in the same diagram (Fig. 4). As in the case of soft tissue, d13Cperio values extracted from the ventral margin of the shell (representing the most recently deposited periostracum portion) varied significantly in space and time (Table 1), with striking seasonal fluctuations being most pronounced in specimens col-
Fig. 6 depicts high-resolution d13C analysis of shell periostracum in one specimen randomly collected at the site of Southern Nebama on 6 January 2017. Its ontogenetic age was estimated according to major growth lines visible on the outer periostracum (forming exclusively during winter; Richardson et al., 1990) and then verified by d18Oshell oscillations. As shown in Fig. 6a, the individual chosen herein was two-years old. It has been shown that blue mussels (Mytilus edulis) precipitate their shells in oxygen isotopic equilibrium with the ambient seawater (Wanamaker et al., 2007). Therefore, d18Oshell values reconstructed from d18Owater and temperature recorded on the day of collection can function as a time gauge, by which d18Oshell profiles and corresponding shell portion can be contextualized (Zhao et al., 2019). At the site of Southern Nebama, d18Owater values varied seasonally (0.75‰ on 23 April, 1.75‰ on 06 June, 4.95‰ on 29 September in 2016 and 0.14‰ on 6 January 2017, respectively). As shown in Fig. 6b, the onset and end of shell growth were constrained to mid-March and late-November, according to reconstructed seawater temperatures of 8.3 °C and 13.8 °C using the Eq. (1) calibrated by Wanamaker et al. (2007). To place the shell
Fig. 3. Differences in d13C values between acid-cleaned and uncleaned shell periostracum materials.
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Fig. 4. Spatial and seasonal variations of mussel soft tissue and the most newly formed shell periostracum d13C records. Values were expressed as average ± standard deviation (n = 2–5). At each locality, significant differences between contemporaneous soft tissue and periostracum d13C values were indicated by asterisk (p < 0.05).
Table 1 Summary of two-way ANOVA analyses on the interactive effects of sampling season and site on mussel soft tissue and periostracum d13C signatures. Source of variations
df
MS
F
p
Soft tissue Season Site Season site
3 7 21
0.735 0.394 0.247
11.287 6.052 3.794
<0.001 <0.001 <0.001
portion into a precise temporal context, an interpolated method was applied. Specifically, d18Oshell data with known date of sampling (0.90‰ on 23 April, 0.77‰ on 06 June, 3.48‰ on 29 September in 2016 and 1.44‰ on 6 January 2017, respectively) were measured and inserted into the d18Oshell time-series, by which the shell can be temporally contextualized on sub-seasonal scales. Td18 O ð CÞ¼ 16:33 4:48 ðd18 Oshell d18 Owater Þ 13
Periostracum Season Site Season site
3 7 21
1.465 1.125 0.794
7.105 5.365 3.801
<0.001 <0.001 <0.001
ð1Þ
In comparison to the seasonality in d Cperio records demonstrated in Fig. 4, high-resolution d13Cperio profiles showed that, despite exhibiting an overall decreasing trend in warmer months, 13Cperio values increased gradually over a certain period of time from June through September,
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Given that mussel d13Cshell record can likely be used as an indicator of large d13CDIC (and hence salinity) differences (Gillikin et al., 2006a, 2006b), the degree to which d13Cperio value correlates with that of d13Cshell was evaluated. As shown in Fig. 6c, d13Cperio and d13Cshell profiles were significantly correlated (p < 0.05). However, only up to 19% variations in d13Cperio were explained by d13Cshell changes. 4. DISCUSSION 4.1. Inter-individual variability in periostracum d13C values
Fig. 5. Simple linear regression of mussel periostracum d13C values (obtained from the most newly formed portion) plotted against corresponding soft tissue d13C values. The grey dashed line is the line of identity (i.e., the 1:1 line).
beyond which decreases occurred between October and November (Fig. 6a). A similar decrease in d13Cperio value was also evident from April to June.
As shown in Fig. 4, d13Cperio values observed at the same site and time show variability between individuals, even larger than seen in soft tissues. Such individual variability could be ascribed to serval potential causes. While d13C value typically exhibits little (<1‰) trophic enrichment (Fry, 2006), factors such as food quality and quantity, ontogenetic age and physiological condition can affect carbon isotope fractionation (reviewed by Fry, 2006; Caut et al., 2009). In the present study, effects of food and age can be ruled out, given that mussels inhabited the same area and were practically the same age (two-years old according to major growth lines clearly visible in the periostracum). On the other hand, mussels which were randomly selected for d13C analysis had different shell length. It is, therefore, reasonable to assume that these individuals could have
Fig. 6. High-resolution isotopic analysis of the shell. (a) d18O and d13C values measured from shell periostracum and corresponding carbonate materials. The grey solid line demonstrates the position of the major growth lines visible on the periostracum. d18Oshell and d13Cperio data with known date were used to place the shell portion into a temporal context. (b) Instrumental temperature, salinity and precipitation time-series recorded in the middle of Otsuchi Bay were shown. Seasonal changes of d18Oseawater at the site of Sothern Nebama were plotted together. d18Oshell-reconstructed temperatures (8.3 °C and 13.8 °C) were used to constrain the timing of seasonal shell growth which was indicated by grey arrows. (c) Simple linear regression of mussel periostracum d13C values plotted against corresponding shell carbonate d13C values.
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different metabolic rates. Metabolic fractionation of carbon isotope has been well documented in aquatic animals (Blair et al., 1985; Focken and Becker, 1998), as the lighter 12C is preferred in metabolism (Fry, 2006). Variations in carbon assimilation and excretion caused by metabolic activities can therefore partially explain the inter-individual variability seen in d13Cperio records. The contamination resulting from shell carbonate may also play a role. Shell carbonate is considerably enriched in 13C. In the case of M. galloprovincialis, d13Cshell values varied between 3.5 and 0.8‰ (Fig. 5a). While scrapping off periostracum, the presence of even small amounts of carbonate that flaked off the shell may increase the value of d13Cperio, as exemplified in Fig. 3. A detailed examination of the data also confirmed that some specimens showed unreasonably high d13Cperio data. This finding suggests that the duration of acidification (e.g., 24 h in the present study) may not be sufficient to remove all carbonate. Further evidence in support of such assumption comes from similar observations from Delong and Thorp (2009), according to which several A. plicata individuals still showed anomalously high d13Cperio values following 48-h acidification. Therefore, it is paramount to thoroughly clean the periostracum materials with acid of sufficient duration to eliminate the contamination of intracrystalline organic coating carbonates (Delong and Thorp, 2009). 4.2. Offsets between shell periostracum and soft tissue d13C values In general, d13C records extracted from the most recently deposited shell periostracum exhibited similar variations in space and time compared to those of soft tissues (Fig. 4). Nevertheless, mussels that grew at the same site and time exhibited significant differences in d13C value between these two tissues (up to 0.92‰), occurring exclusively in summer and/or autumn (Fig. 4). The finding is in line with the observation in the freshwater mussel A. plicata (Delong and Thorp, 2009), whose periostracum was typically more 13C-enriched than soft tissue (about 0.21‰). Likewise, Feng et al. (2018) reported more than about 10‰ difference between shell CBOM and soft tissue d13C values. An opposite pattern was observed in the marine bivalve Mercanaria mercenaria (O’Donnell et al., 2003), according to which the d13C value of shell CBOM is about 0.1‰ lighter than that of soft tissue. To increase the robustness of shell periostracum to be used in a similar manner as soft tissues, underlying mechanisms driving differences in d13C records between shell periostracum and soft tissues needs to be better understood. An extremely complex assemblage of organic matrix components (proteins, carbohydrates, polysaccharides, etc.) has been identified in bivalve shells (Marin et al., 2008). In particular, the mineralization of the prismatic and nacreous shell layers is even regulated by two different sets of proteins (Marie et al., 2012). It is well known that different proteins (composed mainly of different amino acids such as glycine, proline, valine, lysine and alanine) are characterized by largely different d13C values, for exam-
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ple glycine typically showing higher d13C values in comparison to other amino acids (Abelson and Hoering, 1961). Thus, differences between the d13C value of shell periostracum and soft tissues can likely result from the existence of different amino acids (Cariolou and Morse, 1988). Further evidence in support of this assumption comes from the general recognition of tissue-specific patterns in isotopic fractionation stemming from differences in amino acids (Deudero et al., 2009; O’Donnell et al., 2003; Yokoyama et al., 2005). In A. islandica, for example, the isotopic offsets between shell periostracum, CBOM and adductor d15N values can be attributed to differences in amino acid compositions of the different tissue types (Whitney et al., 2019). It is also worth mentioning that the content of lipids may play a role. Lipids in consumers typically have lower d13C values by 3‰ relative to their diets. Bivalve soft tissues contain about 9 wt.% of lipids (O’Donnell et al., 2003), while only a trace amount has been reported in the shells (Cobabe and Ptak, 1999). Hence, the influence of lipids on d13C values is evident. In addition, acid-cleaned periostracum can likely lose part or even all of soluble organics (reviewed by Schlacher and Connolly, 2014), inducing shifts in d13Cperio values. Another plausible explanation for the observed differences between shell periostracum and soft tissue d13C values can be the result of time averaging. As shown in Fig. 4, soft tissues show much less d13C variability between individuals than observed in the periostracum. This can also likely be attributed to the fact that soft tissues integrate more time due to their relatively slow rates of metabolic turnover (Libby et al., 1964). In comparison to more metabolically active tissues (e.g., digestive gland, gonad and gill), adductor muscles measured in the present study have the lowest metabolic activity and turnover rate (Deudero et al., 2009), thereby only providing substantially time-averaged (over several weeks, months or even years) environmental records (Paulet et al., 2006). However, shell periostracum does not have any isotopic turnover, and once deposited, encoded d13C information are locked in place. It can therefore provide much less time-averaged but more highfrequency d13C values compared to soft tissues. The effect of time averaging can be particularly pronounced during the summer and autumn, during which mussels may allocate a considerable amount of energy to build their shells rapidly (Zhao et al., 2017). Hence, shell periostracum perhaps integrates relatively little time, thereby exhibiting finer temporal d13C variability than soft tissues (as shown in Fig. 4). Differences between shell periostracum and soft tissue d13C values may also explain why the plotted regression line does not fall exactly on the 1:1 line (Fig. 5). Indeed, there is also no such a priori expectation given the tissue-specific patterns in isotopic fractionation and the effect of time averaging discussed above. Regardless of the offset, the strong relationship (60% shared variance) lends support to the assumption that shell periostracum may provide the same or parallel information as the most widely used soft tissues which have been extensively used to track organic carbon sources and dynamics at the base of coastal food webs.
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4.3. Shell periostracum as an archive in carbon isotope studies As expected, mussel shell periostracum d13C values varied spatially and temporally in Otsuchi Bay (Fig. 4). In particular, mussels collected in the vicinity of river mouths had large seasonal d13Cperio variations, showing the lowest values recorded in summer at the site of Aka (close to the mouth of Otsuchi River and Kozuchi River) but in autumn at both sites of Northern Nebama and Southern Nebama (close to the Unosumai River mouth). However, these observations went beyond the initial expectations, i.e., mussels collected at all three river mouth sites had similar temporal variations in d13Cperio values during the rainy season of the East Asia summer monsoon (from early June through late September; Yihui and Chan, 2005). The latter is supported by the finding that the contribution of terrestrially derived carbon to the total OC pool dramatically decreased from 70–100% in the inner bay to 34–42% at the mouth of the bay (Wada et al., 1987). Mussels are highly selective feeders, assimilating their carbon primarily from phytoplankton in the OC pool, and in estuarine waters d13Ctissue (and hence d13Cperio) records can closely follow changes in d13CDIC records (Gillikin et al., 2006a, 2006b). Two different patterns observed at river mouth sites, therefore, suggest that aside from terrestrial carbon other carbon sources available to phytoplankton vary spatially and temporally. Seasonal fluctuations of d13Cperio values seen in the outer bay indicate that mussels assimilate most of their carbon from autochthonous phytoplankton, characterized by d13C values varying moderately throughout the year (Kojima and Ohta, 1989). Overall, mussel d13Cperio data assembled from specimens collected at multiple localities and time scales can potentially resolve spatiotemporal changes of primary production in Otsuchi Bay. Perhaps most promisingly, shell periostracum can provide high-resolution and temporally aligned snapshots of d13C variability at the base of coastal food webs. Mussels produce periodic growth increments in their shells (Richardson et al., 1990). If the date of collection is known, each shell portion can be placed into a precise temporal context using these growth patterns as a time gauge. The shell can also be temporally contextualized by aligning d18Oshell-reconstructed temperature with the instrumental temperature curve since mussels precipitate their shells in oxygen isotopic equilibrium with the ambient environment (Wanamaker et al., 2007). Moreover, mussels inhabiting the same locality grow highly synchronously, consequently showing similar isotopic patterns between individuals even at high resolution (Gillikin et al., 2017; Versteegh et al., 2012). Hence, a single shell periostracum can likely provide a glimpse of how d13C value at the base of food webs varies over time, and the temporal resolution is mainly dependent on the sampling resolution and rate of shell growth. As shown in Fig. 6, high-resolution d13Cperio time-series which were temporally aligned on a sub-seasonal time scale spanned the past one-and-a-half years and covered the entire lifespan of the mussel. There is a high degree of similarity between seasonally extracted d13Cperio values (at the site of Southern Nebama; Fig. 4) and high-resolution d13Cperio time-series, synchronously showing the lowest
values in autumn. Yet, the latter clearly provide several ephemeral scenarios of d13C dynamics at the base of food webs. For example, d13Cperio values displayed an overall decrease from June through September, yet during this period, the minimum record occurred in early summer. On the contrary, a striking decrease in d13Cperio value seen in late autumn was at odds with the autumn-winter increasing trend. These scenarios indicate the temporal heterogeneous nature of estuarine primary production in Otsuchi Bay. To elucidate the OC dynamics in coastal waters, it is critical to deepen our understanding of causes of these ephemeral scenarios. It has been well demonstrated that in coastal waters, large variations in d13CDIC value and hence salinity can be recorded by d13Cshell value (e.g., Gillikin et al., 2006a, 2006b; Poulain et al., 2010). A statistically significant correlation between periostracum and shell d13C values shown in Fig. 6 demonstrates that there is indeed a substantial input of terrestrial carbon into the total OC pool at the river mouth of the bay. Yet, only 20% of d13Cperio variability can be explained by large changes in d13Cshell records caused by the riverine input of freshwater, indicating that mussels may assimilate carbon from not only phytoplankton but also other sources such as dissolved organic carbon (Roditi et al., 2000) and particulate organic materials such as microalgae detritus (Levinton et al., 2002). Evidence in support of this assumption also comes from the observations of Gillikin et al. (2006a, 2006b), according to which on a seasonal scale, d13Ctissue value varied differentially than those of d13CDIC and salinity, despite a strong correlation between d13Ctissue and d13CDIC records seen in March. Overall, it seems more reasonable to conclude that mussel d13Cperio record can be used as a tracer of assimilated carbon out of the total OC pool in coastal ecosystems. 4.4. Future research needs Methodologically, it is critical that shell periostracum materials should be acidified thoroughly to remove any shell carbonate. Yet, before acidification is used as a routine pretreatment step, the effects of acid treatment (e.g., duration and concentration) on d13C values need to be elucidated, because acidification may fractionate stable isotope ratios if part of organic matter is chemically transformed or lost (Schlacher and Connolly, 2014). The predictability of the changes in d13C values caused by acid treatment can enhance the robustness of shell periostracum as an archive in isotopic studies. It is promising that shell periostracum may represent a viable alternative to the soft tissues for tracking organic carbon sources and dynamics in modern coastal ecosystems, thus sparking the interest in utilizing fossil shells to assess baseline changes over long-time scales (e.g., Black et al., 2017; Darrow et al., 2017). Shells which are stored dry in museum collections can remain pristine (Versteegh et al., 2011). However, it is unknown whether and to what extent diagenesis alters d13Cperio records in midden shells after burial in sediments. Potential indicators of organic matter diagenesis include shift in stable isotope ratios (Fogel et al., 1989) and fractionation of amino acids (Qian et al., 1992). In particular, compound specific isotope
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analysis (CSIA) holds great potential for identifying amino acids that are more robust against diagenesis (Engel et al., 1994) and pinpointing specific components that record baseline d13C values. Given that some amino acids in well-preserved shells remain indigenous even over time scales of more than 100,000 years (Engel et al., 1994), CSIA of d13C values in bivalve shell periostracum may provide deeper insights into food web dynamics over various time scales in coastal ecosystems. 5. CONCLUSIONS Acidification is needed for d13C analysis in shell periostracum, but isotopic variability caused by acid treatment should be further tested. The latter may partially explain large inter-individual variability in d13Cperio records between mussels growing at the same site and time. Likewise, differences between shell periostracum and soft tissue d13C values are large in summer and autumn, which can likely be attributed to metabolically induced differences in organic components and effects of time averaging more pronounced in soft tissues. On a seasonal scale, nevertheless, d13Cperio values follow d13Ctisue values with 60% shared variance, indicating that shell periostracum can be utilized as a viable alternative to soft tissues for tackling important ecological questions. In comparison to soft tissues which usually integrate more time, shell periostracum archives d13C variations at higher resolution, thereby tracking rapid and unpredictable ephemeral scenarios of food web dynamics. Perhaps most promisingly, by means of CSIA as proposed above, it is possible to extend shell periostracum d13C records back in time to understand baseline changes in coastal ecosystems. ACKNOWLEDGEMENTS We gratefully acknowledge Noriko Izumoto for shell and seawater oxygen isotope analysis. This study was financially supported by Japan Society for the Promotion Science (JSPS) Kakenhi Grants 17F17333 to L. Zhao and 16K13912 and 18H01324 to K. Shirai, and the research program ‘‘Tohoku Ecosystem-Associated Marine Sciences” of the Ministry of Education, Culture, Sports, Science and Technology in Japan.
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Mussel periostracum as a high-resolution archive of soft tissue d13C records in coastal ecosystems Liqiang Zhao a,b,⇑, Kotaro Shirai a, Naoko Murakami-Sugihara a, Tomihiko Higuchi a, Kiyoshi Tanaka a a
Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba 277-8564, Japan b Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China Received 2 July 2018; accepted in revised form 23 June 2019; available online 2 July 2019
Abstract Stable carbon isotope ratios (d13C) of mussel soft tissues have been widely used to characterize baseline d13C isoscapes and identify carbon sources at the base of coastal food webs. Extending soft tissue d13C records back in time, however, is extremely challenging due to very limited sample availability. Here, we test if the stable carbon isotopic composition of periostracum (the outermost organic layer of the shell) in the Mediterranean mussel (Mytilus galloprovincialis) can be used as an environmental archive, similar to soft tissue records. In general, spatial and seasonal variations of periostracum d13C values are comparable to those of soft tissues, but apparently the latter are more time-averaged and smoothed. Irrespective of such offset, there is a significant linear correlation between mussel periostracum and soft tissue d13C values (R2 = 0.608, p < 0.001). Moreover, high-resolution d13C analysis of mussel periostracum indicates that it integrates much less time than soft tissue due to metabolically inert nature, consequently being able to record predictable events such as tidal changes and also unpredictable ephemeral events in coastal ecosystems. The present study demonstrates the potential of the periostracum as a viable alternative to the most widely used soft tissues in isotopic studies. Most promisingly, given ample collections over time scales up to hundreds of years and usually stored dry, this technique could be used to extend coastal organic carbon d13C records back in time. Ó 2019 Elsevier Ltd. All rights reserved. Keywords: Stable carbon isotope; Bivalve mollusks; Periostracum; Coastal food web; Otsuchi Bay
1. INTRODUCTION Coastal zones represent one of the most highly diverse, biologically productive and ecologically valuable areas of the world’s oceans (Costanza et al., 1997). However, global coastal ecosystems have degraded at unprecedent rates during recent decades, due to increasing anthropogenic threats such as overharvesting, habitat destruction, eutrophication, hypoxia, pollution, ocean warming and acidification ⇑ Corresponding author at: Atmosphere and Ocean Research
Institute, The University of Tokyo, Chiba 277-8564, Japan. E-mail address: [email protected] (L. Zhao). https://doi.org/10.1016/j.gca.2019.06.038 0016-7037/Ó 2019 Elsevier Ltd. All rights reserved.
(Dutkiewicz et al., 2015; Kirwan and Megonigal, 2013; Temmerman et al., 2013; Wu et al., 2017). Coastal deterioration stems primarily from changes at the base of food webs (Bauer et al., 2013; Hoegh-Guldberg and Bruno, 2010; Middelburg, 2014), fueled by diverse sources and high concentrations of organic carbon (OC) originating from local pelagic and benthic primary production and/or marine and terrestrial import (Christianen et al, 2017; Vlahos and Whitney, 2017; Yokoyama et al., 2005). Consequently, real-time and retrospective monitoring of sources and spatiotemporal dynamics of OC represents a critical step forward in predicting how coastal ecosystems may respond to anthropogenic perturbation.
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Stable carbon isotope analysis serves as a useful tool for inferring sources and flows of OC in aquatic and terrestrial ecosystems (Middelburg, 2014). Stable carbon isotope ratios (d13C) of primary producers in marine, freshwater and terrestrial food webs differ distinctly due to differences in d13C values of carbon substrate assimilated, i.e., dissolved inorganic carbon (DIC) or atmosphere carbon dioxide, and isotopic fractionation during photosynthetic carbon fixation (Fry, 2006). Marine benthic primary producers have d13C values between 18 and 13‰ (Currin et al., 1995; Riera et al., 1999), while marine pelagic primary producers often display a narrow range of 22 to 20‰ (Creach et al., 1997; Currin et al., 1995). The average d13C value of carbon transferred from terrestrial sources to the oceans is rather similar to that found in C3 plants varying slightly from 27 to 26‰ (Peterson and Fry, 1987). Riverine phytoplankton and their detritus are typically characterized by d13C values (40 to 30‰) much lower than those of marine and terrestrial derived carbon (Hamilton and Lewis, 1992; Hellings et al., 1999). Values of OC d13C falling between these extremes are often interpreted as indicating mixing of differential carbon components, and the relative contribution of each source can be estimated through end-member mixing analysis (Fry, 2006). However, documenting the isotopic composition of OC sources at the base of the coastal food web (i.e., isotopic baseline) is still fraught with difficulty, due largely to its heterogeneous nature (Compton et al., 2013). Hence, exploring proxies of this isotopic baseline is becoming imperative. d13C records of bivalve soft tissues have been used to decipher sources and dynamics of OC in coastal food webs with great success (e.g., Briant et al., 2018; Dubois et al., 2014; Gillikin et al., 2006a, 2006b; Oczkowski et al., 2014; Yokoyama et al., 2005; Zhao et al., 2013a, 2013b), because d13C values typically exhibit little (<1‰) or no trophic enrichment (Fry, 2006). Through d13C analyses of multiple bivalve species at the landscape scale, for example, Christianen et al. (2017) found that the Dutch Wadden Sea intertidal food webs were sustained by OC originating from microphytobenthos (i.e., benthic primary producers). Therefore, a retrospective analysis of archived soft tissues may offer the potential to track OC baselines and food web dynamics, and document long-term coastal ecosystem changes increasingly modified by human activities (e.g., Chasar et al., 2005; Syva¨ranta et al., 2006; Vander Zanden et al., 2003). Despite such promise, however, this approach has not been routinely applied, due largely to concerns that preservation of soft tissues in formalin (and/or ethanol) can potentially induce shifts in d13C values through the incorporation of isotopically light carbon from preservatives (e.g., Carabel et al., 2009; Delong and Thorp, 2009; Kaehler and Pakhomov, 2001; Liu et al., 2013). Another constraint comes from the limited availability of long-term preserved soft tissues, with most only dating back over few decades (Delong and Thorp, 2009). On the contrary, skeletal hard parts of primary consumers, especially bivalve shells, are likely collected over time scales up to hundreds of years and usually stored dry, such as
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in museums, institutes, and personal collections, thereby sparking interest in exploring their potential as archives of d13C in coastal ecosystems. Bivalve shells precipitate in the extrapallial fluid (EPF), a thin liquid film located between the outer mantle epithelium (OME) and inner shell surface (Wheeler, 1992). In the EPF, the crystallization of inorganic calcium carbonate is mediated by organic matrix secreted from the OME (Wheeler, 1992), the latter referred to as carbonate-bound organic matter (CBOM). Individual crystals are subsequently deposited on the inner surface of periostracum, a protective organic layer covering the shell. It is, therefore, reasonable to assume that during shell building, isotopic characteristics of the OME (and hence whole soft tissue) can be potentially archived and retained by the CBOM and periostracum. Unlike soft tissues which are continuously degrading and accruing new material consequently resulting in temporal integration of stable isotope records (Gillikin et al., 2017), shell organics are metabolically inert (Saleuddin and Petit, 1983), i.e., once deposited the isotopic information is locked in place. An increasing number of studies have recently tested the use of bivalve shell CBOM as an archive of stable nitrogen isotopes (d15N) in coastal and estuarine ecosystems with promising results (e.g., Carmichael et al., 2008; Gillikin et al., 2017; Graniero et al., 2016; Kovacs et al., 2010; O’Donnell et al., 2003; Versteegh et al., 2011; Whitney et al., 2019), and d15N records have been extended back over long-time scales (Black et al., 2017; Darrow et al., 2017). In particular, Gillikin et al. (2017) demonstrated the feasibility of direct combustion with minimal processing for CBOM d15N measurement, paving the way for high resolution d15N reconstructions. However, determining d13C values in shell CBOM by means of direct combustion is clearly not feasible given an extremely large CaCO3 percent (typically 95–99% in the bulk shell; Marin et al., 2008). Hence, the study of d13C records in shell periostracum has recently attracted renewed attention. For example, Delong and Thorp (2009) reported that d13C values derived from the periostracum of freshwater mussel (Amblema plicata) were comparable to those of soft tissues, and longterm (>110 years) d13C records from A. plicata periostracum have been recently developed, demonstrating the promise of this technique in tracking OC dynamics and developing baseline isoscapes in freshwater ecosystems. In a pilot study to date to explore the periostracum as a potential environmental proxy in marine bivalves (Arctica islandica), Wanamaker and Luzier (2014) demonstrated that d13C and d15N values varied with shell growth, indicating that the periostracum may record ambient environmental conditions during the lifetime of A. islandica. Recently, Whitney et al. (2019) reported that the significant correlation of periostracum d15N values with contemporaneous CBOM d15N values of the same A. islandica shell, which suggests that shell periostracum preserves a similar d15N record to that encoded into the CBOM. More promisingly, source amino acid d15N analysis of A. islandica periostracum reveals that bulk periostracum d15N values can reflect the d15N values of the clam’s food source (Whitney
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et al., 2019). However, whether shell periostracum and soft tissue d13C values are strongly correlated on spatial and temporal scales remains to be tested. The present study aims to explore the potential of marine bivalve shell periostracum as an archive for d13C studies in coastal ecosystems. Firstly, to investigate if shell periostracum can provide the same or parallel information as soft tissues, a comparison between their d13C values will be conducted over a wide range of spatial and temporal scales. Then, d13C time-series extracted from one single periostracum will be analyzed to investigate if shell periostracum can be used to reconstruct d13C changes at a highresolution. These results may promote the routine use of d13C records in marine shell periostracum as a tool for studying important ecological questions, especially regarding OC sources and dynamics at the base of food webs in coastal ecosystems. 2. MATERIALS AND METHODS 2.1. Study area Otsuchi Bay (39°210 N, 141°580 E) is a semi-enclosed bay located on the Sanriku Ria coast of Northern Japan (Fig. 1). The bay opens northeast toward the North Pacific Ocean, with a length of approximately 8 km and width of 3 km. Three rivers, the Otsuchi River, the Kozuchi River and the Unosumai River, flow into the bay, with the highest discharge (about 30 m3 s1) occurring in summer and lowest (6 m3 s1) in winter (Anbo et al., 2005). Freshwater inflow is also one of the primary driving forces resulting in highly stratified water column in the bay from spring through to early autumn. During the stratification period, low saline surface water mass with an average depth of 10 m flows out of the bay and a dense bottom water mass moves in an opposite direction (Otobe et al., 2009). The pelagic food webs in the Bay are supported not only by local primary production but also high riverine inputs of
Fig. 1. Bathymetry map showing sampling sites in Otsuchi Bay and one site designated as the control in Funakoshi Bay.
organic matter, the relative contribution of the latter decreasing gradually with distance from the mouth of the river (Wada et al., 1987). Evidently, the ecosystem biogeochemistry in Otsuchi Bay is dynamic and multiple carbon sources showing distinct isotopic values vary temporally and spatially. Accordingly, a large d13C gradient of particulate organic carbon, which is an essential prerequisite to fulfill the task in the present study, can be expected along the coastal of Otsuchi Bay. As shown in Fig. 1, seven sites in Otsuchi bay which are affected by riverine discharge to different degrees were deliberately selected. One control site in Funakoshi Bay which is not affected by freshwater runoff was designated. 2.2. Study species The Mediterranean mussel, Mytilus galloprovincialis, is an invasive bivalve species along the Pacific coast of Japan (Wilkins et al., 1983). In Otsuchi Bay, M. galloprovincialis is a dominant inhabitant in rocky intertidal and shallow subtidal zones. Its shell is subdivided into three major layers: protective organic layer (periostracum), calcitic prismatic layer and aragonitic nacreous layer (Fig. 2). Generally, the thickness of the latter two layers varies significantly over the whole lifespan, while the thickness of the periostracum remains relatively constant (Fig. 2). Shells of M. galloprovincialis grow by periodic deposition of calcium carbonate on the inner surface of the periostracum, accordingly forming periodic growth increments and lines in the prismatic layers (Richardson et al., 1990). 2.3. Mussel collection and preparation At each sampling site, more than 10 adult mussels of similar shell length (5–6 cm) were randomly collected from the quay walls in Otsuchi Bay during low tide on 23 April, 06 June and 29 September in 2016 and 6 January 2017, corresponding to spring, summer, autumn and winter, respectively. Water samples were collected simultaneously for the measurements of oxygen isotopes, which can be used to indicate the intensity of riverine discharge (Zhao et al., 2019). Upon arrival in the laboratory, mussels were immediately stored at 20 °C for further analysis. After thawing at room temperature, the adductor muscle and most recently formed shell periostracum (as illustrated in Fig. 2) were carefully dissected using a scalpel, freeze-dried and homogenized. One mussel shell collected on 6 January 2017 at the site of Southern Nebama (in the vicinity of the mouth of the Unosumai River) was used for high-resolution carbon isotopic analysis. More specifically, prior to taking samples, the periostracum was cleaned thoroughly by lightly brushing to remove any surface contamination. Afterward, periostracum (>200 lg) was scraped off equidistantly (ca. 3 mm) along the axis of maximum shell growth. To constrain the timing and duration of shell formation, shell carbonate materials (>100 lg) were sequentially taken at approximately 0.4–0.5 mm resolution along the axis of maximum shell growth (Fig. 2) for oxygen isotopic analysis. Finally, periostracum materials were placed into open silver capsules and acid-cleaned (1 ml of
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Fig. 2. Shells of the Mediterranean mussel Mytilus galloprovincialis. (a) High-resolution periostracum sampling following the axis of maximum shell growth. (b) Schematic of the shell cross section showing major shell structures. (c) Magnification of the shell ventral margin indicating the most newly formed shell periostracum.
0.5 mol HCl) overnight to prevent potential contamination from shell carbonate (Delong and Thorp, 2009). It should be emphasized here that to reduce the loss of soluble organic components, all materials were not rinsed after acid treatment (Schlacher and Connolly, 2014). These silver capsules were subsequently dried on a hot plate at 50 °C for 48 h, sealed and placed in a desiccator. In addition, 30 specimens were randomly selected from different sampling sites to assess the differences in d13C values between acid-cleaned and uncleaned periostracum materials. 2.4. Carbon and oxygen isotopic analysis via EA-IRMS and CF-IRMS Approximately 1–2 mg homogenized soft tissue and periostracum materials were weighed into tin capsules, which were then folded and subsequently measured with an elemental analyzer–isotope ratio mass spectrometer (EA-IRMS; FLASH 2000/Conflo IV/DELTA V Advantage, Thermo Fisher Scientific) at Atmosphere and Ocean Research Institute, The University of Tokyo. The daily calibration was conducted based on standard material Ala (L-Alanine powder, SI Science, Ltd., Saitama, Japan), of which the d13C value is traceable against the international reference materials (e.g., NBS19 and Sucrose ANU). The instrumental analytical error was estimated based on a daily reproducibility of the standard analyses (n > 7) and on average, the value was better than ±0.2‰ (1 standard deviation). Measured carbon isotopic ratios were reported relative to the Vienna Pee Dee Belemnite (VPDB) scale and expressed in the conventional delta (d) notation in per mil (‰). Regarding oxygen and carbon isotopic analysis of the shell, 100–200 lg carbonate materials were weighed into glass vials, flushed with pure helium and dissolved with 100% phosphoric acid at 72 °C for one hour. Liberated carbon dioxide (CO2) gas was analyzed using a continuous
flow-isotope ratio mass spectrometer (CF-IRMS; DELTA V Plus, Thermo Fisher Scientific) coupled to an automated carbonate reaction device (GasBench II, Thermo Fisher Scientific) housed at the same institute. Measured isotopic data were then calibrated against NBS-19 (d18O = 2.20‰, d13C = +1.95‰). The external reproducibility was better than ±0.23‰ and ±0.05‰, respectively for d18O and d13C (1 standard deviation) based on repeated analysis of an in-house standard. Shell carbonate d18O and d13C values were calculated against the VPDB and expressed in the same manner as d13C value of organic materials. Detailed analytical conditions were previously reported (Shirai et al., 2018). Seawater samples were analyzed using a L2120-I wavelength-scanned cavity ring-down spectroscopy isotopic water analyzer Isotopic (Picarro, Sunnyvale, CA, USA). Before introduction into the analyzer, samples were filtered using a membrane filter (pore size: 0.45 lm, Toyo Roshi Kaisha) to reduce suspended particles and prevent blocking of the sampling line. The analytical uncertainty was better than ±0.10‰ (1 standard deviation) based on repeated analyses of Milli-Q water. d18Owater values were calibrated against the Vienna Standard Mean Ocean Water (V-SMOW). 2.5. Statistical analysis All data were statistically analyzed using SPSS 19.0 software. The Shapiro-Wilk’s test and Levene’s F-test were used to examine whether measured data are normally distributed and vary equally across groups. Then, two-way analysis of variance (Two-way ANOVA) followed by the LSD post-hoc was performed to test the combined effects of sampling site and season on d13C values of mussel soft tissue and periostracum, respectively. The correlation between the d13C values of mussel soft tissue and correspondingly most recently formed portion of the perios-
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tracum was computed by means of Pearson correlation analysis. Statistically significant differences were set at p values < 0.05. 3. RESULTS 3.1. Comparison of d13C data between acid-cleaned and uncleaned mussels Without exception, acid-cleaned mussels showed lower d13Cperio values in compared to those in uncleaned specimens, exhibiting large offsets ranging from 0.35 to 5.13‰ (Fig. 3). A paired Student’s t-test confirmed that there is a statistically significant difference in d13C values between acid-cleaned and uncleaned periostracum materials.
lected near the mouths of all three rivers in Otsuchi Bay. It is worth noting here that in comparison to contemporaneous d13Ctissue records, some d13Cperio values are much more scattered displaying larger standard deviations, as illustrated in Fig. 4. Likewise, significant offsets between periostracum and soft tissue d13C values occurred occasionally (Fig. 4). Without exception, these offsets occurred in summer and autumn. On average, nevertheless, d13Cperio and d13Ctissue records compared reasonably to each other, thereby yielding a high degree of similarity. A significant linear relationship between mussel periostracum and soft tissue d13C values in all contemporaneous specimens (R2 = 0.608, p < 0.001) was subsequently computed (Fig. 5), with a slope of 1.335 and intercept of 6.292. 3.3. High-resolution shell periostracum d13C profiles
3.2. Spatial and seasonal variations of d13C values 13
Spatial and seasonal variations in mussel soft tissue d C records are shown in Fig. 4. d13Ctissue values displayed significant differences in the effects of sampling site and season as well as interaction between these two factors (Table 1). Mussels inhabiting the river mouths in Otsuchi Bay exhibited evident seasonality in d13Ctissue records. Specifically, mussels collected at the site of Aka in the vicinity of the Otsuchi River and Kozuchi River mouths showed the lowest d13Ctissue values in summer, while at sites of Northern Nebama and Southern Nebama close to the Unosumai River mouth, maximum and minimum d13Ctissue values were recorded in spring and autumn, respectively. No evident seasonality was shown in mussels collected in the outer part of the bay (e.g., at sites of Shirahama and Keisen). For better comparison of mussel soft tissue and periostracum d13C values, the latter was plotted in the same diagram (Fig. 4). As in the case of soft tissue, d13Cperio values extracted from the ventral margin of the shell (representing the most recently deposited periostracum portion) varied significantly in space and time (Table 1), with striking seasonal fluctuations being most pronounced in specimens col-
Fig. 6 depicts high-resolution d13C analysis of shell periostracum in one specimen randomly collected at the site of Southern Nebama on 6 January 2017. Its ontogenetic age was estimated according to major growth lines visible on the outer periostracum (forming exclusively during winter; Richardson et al., 1990) and then verified by d18Oshell oscillations. As shown in Fig. 6a, the individual chosen herein was two-years old. It has been shown that blue mussels (Mytilus edulis) precipitate their shells in oxygen isotopic equilibrium with the ambient seawater (Wanamaker et al., 2007). Therefore, d18Oshell values reconstructed from d18Owater and temperature recorded on the day of collection can function as a time gauge, by which d18Oshell profiles and corresponding shell portion can be contextualized (Zhao et al., 2019). At the site of Southern Nebama, d18Owater values varied seasonally (0.75‰ on 23 April, 1.75‰ on 06 June, 4.95‰ on 29 September in 2016 and 0.14‰ on 6 January 2017, respectively). As shown in Fig. 6b, the onset and end of shell growth were constrained to mid-March and late-November, according to reconstructed seawater temperatures of 8.3 °C and 13.8 °C using the Eq. (1) calibrated by Wanamaker et al. (2007). To place the shell
Fig. 3. Differences in d13C values between acid-cleaned and uncleaned shell periostracum materials.
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Fig. 4. Spatial and seasonal variations of mussel soft tissue and the most newly formed shell periostracum d13C records. Values were expressed as average ± standard deviation (n = 2–5). At each locality, significant differences between contemporaneous soft tissue and periostracum d13C values were indicated by asterisk (p < 0.05).
Table 1 Summary of two-way ANOVA analyses on the interactive effects of sampling season and site on mussel soft tissue and periostracum d13C signatures. Source of variations
df
MS
F
p
Soft tissue Season Site Season site
3 7 21
0.735 0.394 0.247
11.287 6.052 3.794
<0.001 <0.001 <0.001
portion into a precise temporal context, an interpolated method was applied. Specifically, d18Oshell data with known date of sampling (0.90‰ on 23 April, 0.77‰ on 06 June, 3.48‰ on 29 September in 2016 and 1.44‰ on 6 January 2017, respectively) were measured and inserted into the d18Oshell time-series, by which the shell can be temporally contextualized on sub-seasonal scales. Td18 O ð CÞ¼ 16:33 4:48 ðd18 Oshell d18 Owater Þ 13
Periostracum Season Site Season site
3 7 21
1.465 1.125 0.794
7.105 5.365 3.801
<0.001 <0.001 <0.001
ð1Þ
In comparison to the seasonality in d Cperio records demonstrated in Fig. 4, high-resolution d13Cperio profiles showed that, despite exhibiting an overall decreasing trend in warmer months, 13Cperio values increased gradually over a certain period of time from June through September,
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Given that mussel d13Cshell record can likely be used as an indicator of large d13CDIC (and hence salinity) differences (Gillikin et al., 2006a, 2006b), the degree to which d13Cperio value correlates with that of d13Cshell was evaluated. As shown in Fig. 6c, d13Cperio and d13Cshell profiles were significantly correlated (p < 0.05). However, only up to 19% variations in d13Cperio were explained by d13Cshell changes. 4. DISCUSSION 4.1. Inter-individual variability in periostracum d13C values
Fig. 5. Simple linear regression of mussel periostracum d13C values (obtained from the most newly formed portion) plotted against corresponding soft tissue d13C values. The grey dashed line is the line of identity (i.e., the 1:1 line).
beyond which decreases occurred between October and November (Fig. 6a). A similar decrease in d13Cperio value was also evident from April to June.
As shown in Fig. 4, d13Cperio values observed at the same site and time show variability between individuals, even larger than seen in soft tissues. Such individual variability could be ascribed to serval potential causes. While d13C value typically exhibits little (<1‰) trophic enrichment (Fry, 2006), factors such as food quality and quantity, ontogenetic age and physiological condition can affect carbon isotope fractionation (reviewed by Fry, 2006; Caut et al., 2009). In the present study, effects of food and age can be ruled out, given that mussels inhabited the same area and were practically the same age (two-years old according to major growth lines clearly visible in the periostracum). On the other hand, mussels which were randomly selected for d13C analysis had different shell length. It is, therefore, reasonable to assume that these individuals could have
Fig. 6. High-resolution isotopic analysis of the shell. (a) d18O and d13C values measured from shell periostracum and corresponding carbonate materials. The grey solid line demonstrates the position of the major growth lines visible on the periostracum. d18Oshell and d13Cperio data with known date were used to place the shell portion into a temporal context. (b) Instrumental temperature, salinity and precipitation time-series recorded in the middle of Otsuchi Bay were shown. Seasonal changes of d18Oseawater at the site of Sothern Nebama were plotted together. d18Oshell-reconstructed temperatures (8.3 °C and 13.8 °C) were used to constrain the timing of seasonal shell growth which was indicated by grey arrows. (c) Simple linear regression of mussel periostracum d13C values plotted against corresponding shell carbonate d13C values.
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different metabolic rates. Metabolic fractionation of carbon isotope has been well documented in aquatic animals (Blair et al., 1985; Focken and Becker, 1998), as the lighter 12C is preferred in metabolism (Fry, 2006). Variations in carbon assimilation and excretion caused by metabolic activities can therefore partially explain the inter-individual variability seen in d13Cperio records. The contamination resulting from shell carbonate may also play a role. Shell carbonate is considerably enriched in 13C. In the case of M. galloprovincialis, d13Cshell values varied between 3.5 and 0.8‰ (Fig. 5a). While scrapping off periostracum, the presence of even small amounts of carbonate that flaked off the shell may increase the value of d13Cperio, as exemplified in Fig. 3. A detailed examination of the data also confirmed that some specimens showed unreasonably high d13Cperio data. This finding suggests that the duration of acidification (e.g., 24 h in the present study) may not be sufficient to remove all carbonate. Further evidence in support of such assumption comes from similar observations from Delong and Thorp (2009), according to which several A. plicata individuals still showed anomalously high d13Cperio values following 48-h acidification. Therefore, it is paramount to thoroughly clean the periostracum materials with acid of sufficient duration to eliminate the contamination of intracrystalline organic coating carbonates (Delong and Thorp, 2009). 4.2. Offsets between shell periostracum and soft tissue d13C values In general, d13C records extracted from the most recently deposited shell periostracum exhibited similar variations in space and time compared to those of soft tissues (Fig. 4). Nevertheless, mussels that grew at the same site and time exhibited significant differences in d13C value between these two tissues (up to 0.92‰), occurring exclusively in summer and/or autumn (Fig. 4). The finding is in line with the observation in the freshwater mussel A. plicata (Delong and Thorp, 2009), whose periostracum was typically more 13C-enriched than soft tissue (about 0.21‰). Likewise, Feng et al. (2018) reported more than about 10‰ difference between shell CBOM and soft tissue d13C values. An opposite pattern was observed in the marine bivalve Mercanaria mercenaria (O’Donnell et al., 2003), according to which the d13C value of shell CBOM is about 0.1‰ lighter than that of soft tissue. To increase the robustness of shell periostracum to be used in a similar manner as soft tissues, underlying mechanisms driving differences in d13C records between shell periostracum and soft tissues needs to be better understood. An extremely complex assemblage of organic matrix components (proteins, carbohydrates, polysaccharides, etc.) has been identified in bivalve shells (Marin et al., 2008). In particular, the mineralization of the prismatic and nacreous shell layers is even regulated by two different sets of proteins (Marie et al., 2012). It is well known that different proteins (composed mainly of different amino acids such as glycine, proline, valine, lysine and alanine) are characterized by largely different d13C values, for exam-
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ple glycine typically showing higher d13C values in comparison to other amino acids (Abelson and Hoering, 1961). Thus, differences between the d13C value of shell periostracum and soft tissues can likely result from the existence of different amino acids (Cariolou and Morse, 1988). Further evidence in support of this assumption comes from the general recognition of tissue-specific patterns in isotopic fractionation stemming from differences in amino acids (Deudero et al., 2009; O’Donnell et al., 2003; Yokoyama et al., 2005). In A. islandica, for example, the isotopic offsets between shell periostracum, CBOM and adductor d15N values can be attributed to differences in amino acid compositions of the different tissue types (Whitney et al., 2019). It is also worth mentioning that the content of lipids may play a role. Lipids in consumers typically have lower d13C values by 3‰ relative to their diets. Bivalve soft tissues contain about 9 wt.% of lipids (O’Donnell et al., 2003), while only a trace amount has been reported in the shells (Cobabe and Ptak, 1999). Hence, the influence of lipids on d13C values is evident. In addition, acid-cleaned periostracum can likely lose part or even all of soluble organics (reviewed by Schlacher and Connolly, 2014), inducing shifts in d13Cperio values. Another plausible explanation for the observed differences between shell periostracum and soft tissue d13C values can be the result of time averaging. As shown in Fig. 4, soft tissues show much less d13C variability between individuals than observed in the periostracum. This can also likely be attributed to the fact that soft tissues integrate more time due to their relatively slow rates of metabolic turnover (Libby et al., 1964). In comparison to more metabolically active tissues (e.g., digestive gland, gonad and gill), adductor muscles measured in the present study have the lowest metabolic activity and turnover rate (Deudero et al., 2009), thereby only providing substantially time-averaged (over several weeks, months or even years) environmental records (Paulet et al., 2006). However, shell periostracum does not have any isotopic turnover, and once deposited, encoded d13C information are locked in place. It can therefore provide much less time-averaged but more highfrequency d13C values compared to soft tissues. The effect of time averaging can be particularly pronounced during the summer and autumn, during which mussels may allocate a considerable amount of energy to build their shells rapidly (Zhao et al., 2017). Hence, shell periostracum perhaps integrates relatively little time, thereby exhibiting finer temporal d13C variability than soft tissues (as shown in Fig. 4). Differences between shell periostracum and soft tissue d13C values may also explain why the plotted regression line does not fall exactly on the 1:1 line (Fig. 5). Indeed, there is also no such a priori expectation given the tissue-specific patterns in isotopic fractionation and the effect of time averaging discussed above. Regardless of the offset, the strong relationship (60% shared variance) lends support to the assumption that shell periostracum may provide the same or parallel information as the most widely used soft tissues which have been extensively used to track organic carbon sources and dynamics at the base of coastal food webs.
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4.3. Shell periostracum as an archive in carbon isotope studies As expected, mussel shell periostracum d13C values varied spatially and temporally in Otsuchi Bay (Fig. 4). In particular, mussels collected in the vicinity of river mouths had large seasonal d13Cperio variations, showing the lowest values recorded in summer at the site of Aka (close to the mouth of Otsuchi River and Kozuchi River) but in autumn at both sites of Northern Nebama and Southern Nebama (close to the Unosumai River mouth). However, these observations went beyond the initial expectations, i.e., mussels collected at all three river mouth sites had similar temporal variations in d13Cperio values during the rainy season of the East Asia summer monsoon (from early June through late September; Yihui and Chan, 2005). The latter is supported by the finding that the contribution of terrestrially derived carbon to the total OC pool dramatically decreased from 70–100% in the inner bay to 34–42% at the mouth of the bay (Wada et al., 1987). Mussels are highly selective feeders, assimilating their carbon primarily from phytoplankton in the OC pool, and in estuarine waters d13Ctissue (and hence d13Cperio) records can closely follow changes in d13CDIC records (Gillikin et al., 2006a, 2006b). Two different patterns observed at river mouth sites, therefore, suggest that aside from terrestrial carbon other carbon sources available to phytoplankton vary spatially and temporally. Seasonal fluctuations of d13Cperio values seen in the outer bay indicate that mussels assimilate most of their carbon from autochthonous phytoplankton, characterized by d13C values varying moderately throughout the year (Kojima and Ohta, 1989). Overall, mussel d13Cperio data assembled from specimens collected at multiple localities and time scales can potentially resolve spatiotemporal changes of primary production in Otsuchi Bay. Perhaps most promisingly, shell periostracum can provide high-resolution and temporally aligned snapshots of d13C variability at the base of coastal food webs. Mussels produce periodic growth increments in their shells (Richardson et al., 1990). If the date of collection is known, each shell portion can be placed into a precise temporal context using these growth patterns as a time gauge. The shell can also be temporally contextualized by aligning d18Oshell-reconstructed temperature with the instrumental temperature curve since mussels precipitate their shells in oxygen isotopic equilibrium with the ambient environment (Wanamaker et al., 2007). Moreover, mussels inhabiting the same locality grow highly synchronously, consequently showing similar isotopic patterns between individuals even at high resolution (Gillikin et al., 2017; Versteegh et al., 2012). Hence, a single shell periostracum can likely provide a glimpse of how d13C value at the base of food webs varies over time, and the temporal resolution is mainly dependent on the sampling resolution and rate of shell growth. As shown in Fig. 6, high-resolution d13Cperio time-series which were temporally aligned on a sub-seasonal time scale spanned the past one-and-a-half years and covered the entire lifespan of the mussel. There is a high degree of similarity between seasonally extracted d13Cperio values (at the site of Southern Nebama; Fig. 4) and high-resolution d13Cperio time-series, synchronously showing the lowest
values in autumn. Yet, the latter clearly provide several ephemeral scenarios of d13C dynamics at the base of food webs. For example, d13Cperio values displayed an overall decrease from June through September, yet during this period, the minimum record occurred in early summer. On the contrary, a striking decrease in d13Cperio value seen in late autumn was at odds with the autumn-winter increasing trend. These scenarios indicate the temporal heterogeneous nature of estuarine primary production in Otsuchi Bay. To elucidate the OC dynamics in coastal waters, it is critical to deepen our understanding of causes of these ephemeral scenarios. It has been well demonstrated that in coastal waters, large variations in d13CDIC value and hence salinity can be recorded by d13Cshell value (e.g., Gillikin et al., 2006a, 2006b; Poulain et al., 2010). A statistically significant correlation between periostracum and shell d13C values shown in Fig. 6 demonstrates that there is indeed a substantial input of terrestrial carbon into the total OC pool at the river mouth of the bay. Yet, only 20% of d13Cperio variability can be explained by large changes in d13Cshell records caused by the riverine input of freshwater, indicating that mussels may assimilate carbon from not only phytoplankton but also other sources such as dissolved organic carbon (Roditi et al., 2000) and particulate organic materials such as microalgae detritus (Levinton et al., 2002). Evidence in support of this assumption also comes from the observations of Gillikin et al. (2006a, 2006b), according to which on a seasonal scale, d13Ctissue value varied differentially than those of d13CDIC and salinity, despite a strong correlation between d13Ctissue and d13CDIC records seen in March. Overall, it seems more reasonable to conclude that mussel d13Cperio record can be used as a tracer of assimilated carbon out of the total OC pool in coastal ecosystems. 4.4. Future research needs Methodologically, it is critical that shell periostracum materials should be acidified thoroughly to remove any shell carbonate. Yet, before acidification is used as a routine pretreatment step, the effects of acid treatment (e.g., duration and concentration) on d13C values need to be elucidated, because acidification may fractionate stable isotope ratios if part of organic matter is chemically transformed or lost (Schlacher and Connolly, 2014). The predictability of the changes in d13C values caused by acid treatment can enhance the robustness of shell periostracum as an archive in isotopic studies. It is promising that shell periostracum may represent a viable alternative to the soft tissues for tracking organic carbon sources and dynamics in modern coastal ecosystems, thus sparking the interest in utilizing fossil shells to assess baseline changes over long-time scales (e.g., Black et al., 2017; Darrow et al., 2017). Shells which are stored dry in museum collections can remain pristine (Versteegh et al., 2011). However, it is unknown whether and to what extent diagenesis alters d13Cperio records in midden shells after burial in sediments. Potential indicators of organic matter diagenesis include shift in stable isotope ratios (Fogel et al., 1989) and fractionation of amino acids (Qian et al., 1992). In particular, compound specific isotope
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analysis (CSIA) holds great potential for identifying amino acids that are more robust against diagenesis (Engel et al., 1994) and pinpointing specific components that record baseline d13C values. Given that some amino acids in well-preserved shells remain indigenous even over time scales of more than 100,000 years (Engel et al., 1994), CSIA of d13C values in bivalve shell periostracum may provide deeper insights into food web dynamics over various time scales in coastal ecosystems. 5. CONCLUSIONS Acidification is needed for d13C analysis in shell periostracum, but isotopic variability caused by acid treatment should be further tested. The latter may partially explain large inter-individual variability in d13Cperio records between mussels growing at the same site and time. Likewise, differences between shell periostracum and soft tissue d13C values are large in summer and autumn, which can likely be attributed to metabolically induced differences in organic components and effects of time averaging more pronounced in soft tissues. On a seasonal scale, nevertheless, d13Cperio values follow d13Ctisue values with 60% shared variance, indicating that shell periostracum can be utilized as a viable alternative to soft tissues for tackling important ecological questions. In comparison to soft tissues which usually integrate more time, shell periostracum archives d13C variations at higher resolution, thereby tracking rapid and unpredictable ephemeral scenarios of food web dynamics. Perhaps most promisingly, by means of CSIA as proposed above, it is possible to extend shell periostracum d13C records back in time to understand baseline changes in coastal ecosystems. ACKNOWLEDGEMENTS We gratefully acknowledge Noriko Izumoto for shell and seawater oxygen isotope analysis. This study was financially supported by Japan Society for the Promotion Science (JSPS) Kakenhi Grants 17F17333 to L. Zhao and 16K13912 and 18H01324 to K. Shirai, and the research program ‘‘Tohoku Ecosystem-Associated Marine Sciences” of the Ministry of Education, Culture, Sports, Science and Technology in Japan.
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