Journal of Experimental Marine Biology and Ecology 522 (2020) 151249

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Journal of Experimental Marine Biology and Ecology

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Site and age discrimination using trace element fingerprints in the blue , edulis T ⁎ Aaron Honiga, , Ron Ettera, Kyle Peppermanb, Scott Morelloa, Robyn Hanniganc a Biology Department, University of Massachusetts, Boston, 100 Morrissey Blvd, Boston, MA 02125, United States of America b Downeast Institute for Applied Marine Research and Education, 39 Wildflower Lane, Beals, ME 04611, United States of America c School for the Environment, University of Massachusetts, Boston, 100 Morrissey Blvd, Boston, MA 02125, United States of America

ARTICLE INFO ABSTRACT

Keywords: The ability to accurately estimate population connectivity and larval dispersal among mussel populations in the Trace element fingerprinting Gulf of is important to better understand ongoing declines in blue mussel abundances. Such efforts are Bioincorporation crucial for crafting conservation strategies targeting important spawning and settlement sites necessary to Blue mussel support threatened local shellfisheries, and to mitigate the potential effects of rapid climate change on larval Population connectivity dispersal and survivorship. Trace element fingerprints can be used to infer larval dispersal and population connectivity, but require a reference map of geographic variation in elemental fingerprints to infer natal sites of settled . Previous work has suggested rearing mussel larvae in situ to create a reference map because biomineralization differs between larval and post-metamorphic mussels, which might lead to differences in trace element fingerprints. To test whether elemental fingerprints differed between larval and juvenile (post-meta- morphic) mussels (Mytilus edulis), we reared them in situ and under controlled laboratory conditions. Trace element concentrations in larval and juvenile shell matrices were quantified using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). The majority of trace element concentrations, normalized to calcium, differed between larval and juvenile mussels, resulting in elemental fingerprints that were consistently distinct between age classes. Larval and juvenile fingerprints differed consistently whether they were reared in the lab or field, probably reflecting age-specificdifferences in biomineralization. To use trace element finger- prints to estimate larval dispersal and population connectivity, our results suggest that a spatial map of ele- mental fingerprints should be created by rearing larval, not juvenile, mussels at potential source populations. Our results are consistent with what has been found for other mussels and thus may be true for all mussels, and likely true for any bivalves with age-specificdifferences in biomineralization.

1. Introduction et al., 2007; Cowen and Sponaugle, 2009; Lowe and Allendorf, 2010; Leis et al., 2011). Quantifying patterns of larval dispersal and population connectivity A widely used approach to empirically estimate dispersal and con- in marine ecosystems is important to further our understanding of nectivity in marine organisms relies on trace element fingerprints (TEF) ecology, evolution, conservation and management, and will be essential derived from the chemical signatures found in calcified structures for predicting how organisms might respond to anthropogenic stresses (Lopez-Duarte et al., 2012). TEFs take advantage of the chemical signal (Cowen et al., 2007; Gaines et al., 2007; Siegel et al., 2008; Carson incorporated into growing calcified structures such as shells (Becker et al., 2011; Puckett and Eggleston, 2016; Kroll et al., 2018). However, et al., 2007; Carson, 2010; Fodrie et al., 2011; Broadaway and tracking minute pelagic larvae that drift with the currents and Hannigan, 2012), statoliths (Zacherl et al., 2003; Zacherl, 2005; Warner potentially travel tens of kilometers during several weeks of develop- et al., 2009) or otoliths (Thorrold et al., 2001; Dorval et al., 2007; Leis ment is extremely difficult (DiBacco and Levin, 2000; Thorrold et al., et al., 2011). For example, as bivalves grow they deposit layers of

2002; Knights et al., 2006; Levin, 2006; Cathey et al., 2012). Because of CaCO3 (calcite and aragonite) on a matrix of proteins to form a shell its importance to ecology, evolution and conservation, a number of (Marin and Luquet, 2004). The calcite-aragonite shell chemistry reflects methods have been developed to estimate dispersal and population the elemental and isotopic chemistry of the surrounding water mass connectivity in marine ecosystems (reviewed in Levin, 2006; Thorrold through geochemically passive and biologically active transport to the

⁎ Corresponding author. E-mail address: [email protected] (A. Honig). https://doi.org/10.1016/j.jembe.2019.151249 Received 11 April 2018; Received in revised form 26 April 2019; Accepted 12 October 2019 0022-0981/ © 2019 Elsevier B.V. All rights reserved. A. Honig, et al. Journal of Experimental Marine Biology and Ecology 522 (2020) 151249 site of shell deposition (Klein et al., 1996; Carson et al., 2013). Trace abundance allows us to identify potential sources for declines within elements in the shell can be quantified using laser ablation inductively the Gulf of Maine, provide important insights into the ecological forces coupled plasma mass spectrometry (LA-ICPMS; Liu et al., 2013). Re- shaping regional dynamics and contribute to more effective manage- sulting trace element fingerprints (e.g., Mg/Ca, Mn/Ca, Ba/Ca, La/Ca, ment and conservation strategies for this ecologically and economically Pb/Ca), vary spatially (< 50 km; Becker et al., 2007); and temporally important species (Botsford et al., 2001, 2009; Bode et al., 2008; (monthly and annual periodicity; Miller et al., 2013, Carson et al., Hayhurst and Rawson, 2009; Sorte et al., 2013; Bryson et al., 2014; 2013), characterize different water masses, and can be used to identify Morello and Etter, 2017). the location where shell material was deposited (Becker et al., 2005; The potential importance of using TEFs in quantifying population Levin, 2006; Broadaway and Hannigan, 2012; Carson et al., 2013). connectivity, and the likely impact of environmental chemistry on shell Because many adult bivalves retain their larval shell even after meta- TEFs has led to the suggestion that larvae should be outplanted and morphosis and settlement, TEFs of the larval shell can be used to assign reared in all potential source populations to generate an appropriate settlers to likely natal sites, through a reference map of site-specific reference map of fingerprints (Becker et al., 2007). Although TEFs ap- TEFs generated during natural spawning events (Becker et al., 2005). pear to differ between larval and adult shells for some bivalves (Becker Temporal changes in host water mass and shell geochemistry necessi- et al., 2007; Strasser et al., 2007, 2008), this has not been tested in tate TEF reference maps be generated through in-situ rearing when Mytilus edulis. Because outplanting larvae and rearing them at potential adults are spawning (Putten et al., 2000; Carré et al., 2006; Lazareth source populations requires considerable time and resources, it is im- et al., 2013, Kroll et al., 2018). High seasonal and annual variability of portant to test whether TEFs differ between larval and post-meta- element ratios (particularly Mn, Co relative to Ca) in outplanted Mytilus morphic (juvenile) shell material. Here we test whether TEFs differ sp. shells was observed by Carson et al. (2013), likely in part due to between age classes of Mytilus edulis and whether any age-specific dif- interannual variability in site-specific environmental factors that im- ferences exceed geographic variation in natural populations in the GOM pact biomineralization (geology, freshwater input, upwelling, anthro- that would preclude accurately inferring natal sites. pogenic impact, e.g., Miller et al., 2013). Estimating the variability among site-specific TEFs is therefore critical to evaluate the analytical 2. Material and methods utility of TEF reference maps, and at what spatial and temporal scale TEFs should be effectively used to estimate patterns of dispersal and 2.1. Laboratory experiment population connectivity (site vs region, week vs month; Becker et al., 2005, 2007). Both laboratory and in-situ field experiments were conducted to To estimate dispersal and population connectivity using TEFs, a determine if TEFs differed between larvae and juvenile mussels. Initial reference map of geographic differences in fingerprints is necessary to laboratory experiments were conducted at the Downeast Institute's infer natal sites (reviewed in: Levin, 2006; Becker et al., 2007; Carson shellfish hatchery on Beals Island, ME (DEI, Fig. 1). Adult mussels et al., 2013). A reference map is typically developed from TEFs of se- (≥5 cm), representing multigenerational brood stock originally locally dentary adults collected from potential source populations (DiBacco collected on Beal's Island, were conditioned during the spring of 2015 and Levin, 2000). However, larval and adult mussels precipitate dif- and spawned in June. Resulting larvae (70 μm diameter, n = 100,000) ferent calcium carbonate polymorphs during shell growth, which might were reared alongside juvenile mussels (2–4 mm shell length, n = 100) influence TEFs (Medaković, 2000; Weiss et al., 2002; Dalbeck et al., in large, shallow circular tanks (~1000 L) within an open-circulation 2006; Strasser et al., 2008). The larval shell () of Mytilus system supplied with 1 μm filtered local seawater. Mussels were fed edulis first appears toward the end of gastrulation (24–48 h. post ferti- daily with a multi-species mix of live microalgae including Isochrysis lization) and is initially precipitated as amorphous calcium carbonate galbana, Chaetoceros calcitrans and C. mulleri. After 4 weeks, juvenile and later as aragonite (Marin and Luquet, 2004). Post-metamorphosis, and larval mussels were removed, rinsed in deionized water, tissues the initial juvenile shell (dissoconch) forms beyond the retained pro- extracted (juveniles) and shells dried. dissoconch and consists of a lower nacreous layer composed of arago- Additional laboratory trials were conducted at the University of nite overlain by a prismatic layer of both aragonite and calcite (re- Massachusetts, Boston (UMB; Fig. 1) where adult mussels (≥ 5 cm) and viewed in Gazeau et al., 2013). Potential differences in TEFs across juvenile mussels (2–4 mm shell length) were collected from Savin Hill developmental stages may preclude using post-metamorphic shells for Cove near UMass, Boston in May 2015. Adult mussels were spawned creating a reference map for inferring natal origins (Fodrie et al., 2011; and resulting larvae (70 μm diameter, n = 20,000) were reared with Cathey et al., 2012; Carson et al., 2013). locally collected juveniles (n = 50) in three replicate 1 L glass jars, Quantifying population connectivity of the blue mussel (Mytilus using 5 μm filtered local seawater. Mussels were reared at 20 °C and fed edulis) through trace element fingerprinting is critically important as daily with a multi-species mix of dead microalgae including Isochrysis mussels play pivotal ecological roles in structuring hard and soft bottom galbana, Chaetocerous sp. and Thalassiosira sp. (Reed Mariculture intertidal communities (Lubchenco and Menge, 1978; Seed, 1996; Shellfish Diet 1800). After 4 weeks, juvenile and larval mussels were Snover and Commito, 1998; Dudgeon and Petraitis, 2001) and have removed, rinsed in deionized water, tissues extracted (juveniles) and significantly declined in abundance in recent decades (Sorte et al., shells dried. 2017), possibly due to reduced larval supply (Petraitis and Dudgeon, 2015) but also attributed to recent species invasions, overfishing, 2.2. Field experiment trawling-associated benthic degradation and regional climate change (Jackson et al., 2001; Harris and Tyrrell, 2001; Thrush and Dayton, Even if TEFs differ between larvae and juveniles, age differences 2002; Petraitis and Dudgeon, 2015). Such population losses may also may be small relative to spatial variation, enabling the generation of a severely reduce wild-seed availability in a region with domestic mussel site-specific elemental reference map regardless of age. To test how age- mariculture that inputs $14 million dollars annually (projected to $30 specificdifferences in TEFs compared to spatial variation of TEFs, we million by 2030; GMRI, 2016) to economically poor coastal commu- reared larvae and juveniles in situ at 8 sites within the GOM (Fig. 1). To nities in the Gulf of Maine (Newell, 2001; Alden, 2011). generate larval mussels for in situ experiments, adult mussels (brood Thus, there is great interest in understanding patterns of local stock at the Downeast Institute) were artificially spawned to coincide spawning and settlement, regional larval dispersal, connectivity and with natural spawning events, indicated by sharp drops in gonadal in- metapopulation dynamics (Auker et al., 2014; Bertness et al., 2002; dices (e.g., Newell and Campbell, 1992) in mussels sampled from target Dobretsov and Wahl, 2008; Hayhurst and Rawson, 2009; Sorte et al., field sites (Fig. 1). Resulting larvae were reared to stage (to 2013). A better understanding of the forces that drive mussel reduce high mortality rates of blastulae during transport) and deployed

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Fig. 1. Lab and field-rearing sites. Lab sites: UMB - UMass, Boston (blue circle), DEI - Downeast Institute (pink circle); Field sites: FBC - Frenchman Bay Central (red triangle), FBE - Frenchman Bay East (orange square), GB - Gouldsboro Bay (yellow diamond), PHB - Pigeon Hill Bay (green pentagon), WRL - West River Landing (teal downward triangle), MBR - Moosabec Reach (light blue star), CHR - Chandler River (dark blue diagonal cross) and MB - Machias Bay (fuchsia hourglass) in the northern and southeastern Gulf of Maine (see inset). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) in suspended chambers (400 mL plastic canisters with five longitudinal normalized to total calcium as calculated from 46Ca abundance. 35 μm nylon mesh windows; Fig. S1) as prior to the first calcified larval stage (~ 80 μm diameter). Hatchery-reared ju- 2.4. Data analysis venile mussels (2–4 mm shell length) were jointly deployed in similar chambers, suspended adjacent to in situ larval chambers. Chambers Trace element concentrations relative to Ca (μmol element/mol Ca) were attached to mooring lines at 2-5 m below the surface. Although were quantified using GeoPro™ software (Cetac Technologies). multiple chambers were deployed at each site, loss of some canisters Concentrations were log transformed (log10 + 1), outliers were re- due to storm damage or interference from local lobsterpot buoys pre- moved (identified through the Grubb's test α = 0.05; Tietjen and cluded analysis of within-site elemental variation in outplanted in- Moore, 1972) and normalized using Z-score standardization to account dividuals. However, all canisters were suspended over bottom depths for consistently high concentrations of specific elements (Mg, Sr, e.g.) of < 15 m (mean low water; NOAA ENC charts and confirmed on-site) relative to trace elements (La, Pb, e.g.) across all ages within sites (for in hydrologically well-mixed bays and inlets (Pettigrew et al., 2005; tests where age was the variable of interest; Tables S8, S9) and across Xue et al., 2000), likely resulting in similar environmental conditions all ages and sites (for tests where age and sites were variables of in- between each canister, and between canisters and local benthic mussel terest; Tables S10, S11). Resulting TEFs in lab and field-reared mussels beds. After four weeks, larval and juvenile mussels were removed from were compared with MANOVA followed by Tukey's HSD tests in situ chambers, tissues were extracted (juveniles), shells were rinsed in (α ≤ 0.05) when differences were detected. Final sample numbers deionized water and dried. varied from 31 to 41 individuals in lab experiments and 10 to 66 in field experiments (see Figs. 3, 5). Quadratic discriminant function analyses 2.3. Trace elemental analysis were used to determine the strength of age class assignment by TEFs within deployment sites (by quantifying the percent of correctly as- Larval and juvenile shells from all experiments were mounted on signed individuals by age; JMP Pro 14 statistical software, SAS Institute petrographic slides using double-sided Scotch tape. Elemental compo- Inc, 2018). sitions were quantified by external calibration (USGS MACS-3) and To determine if larvae alone can be used to generate the site-specific internal standardization. We quantified elemental compositions from element fingerprints required to accurately assign settlers to likely count per second (cps) measurements of 24Mg, 55Mn, 59Co, 88Sr, 138Ba, spawning sites using the geochemistry of their retained larval shell, we 139La, 208Pb, 43Ca, and 46Ca using laser ablation inductively coupled analyzed elemental differences and discrimination between sites in mass spectrometry (LA-ICPMS; 213 Nd:YAG laser coupled with Elan field-reared larvae and juveniles separately. To estimate the strength of DRC II quadrupole mass spectrometer) at the University of assignments relative to random chance, the probability of random as- Massachusetts, Boston (Craig et al., 2000; D'Oriano et al., 2008; Kaimal signment was estimated as 1/# groups (1/4 = 25% in laboratory and et al., 2009; Broadaway and Hannigan, 2012; Sorte et al., 2013; Darrah 1/16 = 6.3% in field age and site assignments, and 1/8 = 12.5% in site et al., 2013). Elemental abundances are corrected for drift using 43Ca as assignments for each age class). All statistical tests were performed at the internal standard. We performed a one-point calibration using gas 95% confidence levels. blank and MACS-3, and ran MACS-1 as a check standard every ten samples. Analytical accuracy and precision, based on repeated analyses 3. Results of MACS-1 (n = 50) averaged 96 ± 4.4%. Elemental concentrations were determined from mean internal standard normalized concentra- 3.1. Laboratory experiments tions of unique line ablations (n = 3) along the growing edge in juve- nile shells and standards (55% energy, 20 hz, 50 μm spot width at 50 3.1.1. Trace element concentrations − μms 1) and single spot ablations in larval shells and standards (85% Hatchery-reared larval mussels at the Downeast Institute, Maine energy, 10 hz, 200 shots, 25 μm spot diameter). Elemental counts per reached a slightly greater shell length than those reared at UMass, second were acquired as 3 sweeps per replicate allowing for counting Boston, Massachusetts (DEI: n = 21, shell diam. = 243 ± 40 μm; standards to yield relative standard deviations for each analyte below UMB: n = 40; shell diam. = 180 ± 32 μm) while juvenile mussels 10%. Analyte concentrations of samples and standards are reported reared alongside larvae were slightly larger at UMass, Boston (DEI:

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Fig. 2. Trace element concentrations in laboratory reared mussels.Mean ( ± SE) trace element concentrations of log transformed (log10 (x + 1)) elements within larval (filled) and juvenile (unfilled) mussels reared in laboratory experiments at UMass, Boston (A, UMB, blue) and the Downeast Institute ( B, DEI, pink). Significant differences in element concentration between age classes at each site, determined by MANOVA, performed on Z-score standardized data at α = 0.05, are starred. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Age and Site Discrimination in Laboratory Reared Mussels. Age discrimination (A, B) and age and site discrimination (C) of larval (filled) and juvenile (unfilled) mussels reared at UMB (blue) and DEI (pink). First two canonical axes generated through quadratic discriminate function analyses (QDFA). Group centroids and 90% confidence intervals are shown. Classification accuracies are inset, with actual rows and assigned columns. Direction and length of biplot rays represent elemental correlation coefficients with respect to the included canonical axes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) n = 18, shell length = 2.6 ± 0.3 mm; UMB: n = 28, shell in each laboratory experiment was very accurate (> 90%), reflecting length = 3.7 ± 0.8 mm). Differences in sample sizes among experi- differences in trace element chemistry between larval and juvenile mental tanks are due to variation in larval mortality within canisters. shells (Fig. 3A–C). In combined age and site assignments, mussels were In trials, Mg/Ca and Sr/Ca shell concentrations were highest fol- assigned to their actual age class and site with similarly high accuracy lowed by Mn/Ca, Ba/Ca and Co/Ca. Pb/Ca and La/Ca were consistently (> 70%; 3C), and consistently greater accuracy than random assign- found in low concentrations (Figs. 2, 4). Shell concentrations of several ment accuracies (25%, Table S3). Such discrimination patterns reflect elemental ratios differed significantly by age class in laboratory trials greater chemical differences in age class than between experimental conducted at UMass, Boston (Mg/Ca, Co/Ca, La/Ca; NLarv = 41, sites. All assignment accuracies were at least 3 times greater than those NJuv = 37; MANOVA at α = 0.05; Fig. 2, Table S1) and at the Downeast estimated by random chance in laboratory trials (Table S4), demon- Institute (Mg/Ca, Mn/Ca, Co/Ca, Sr/Ca, La/Ca; NLarv = 31, NJuv = 37; strating strong QDFA age and site discrimination using shell element MANOVA: α = 0.05; Fig. 2, Table S2). Reared juvenile shells exhibited concentrations. greater Mg/Ca in both experiments, reduced Mn/Ca, Sr/Ca, Ba/Ca and La/Ca at DEI and greater Co/Ca and La/Ca at UMB compared to larval shells. 3.2. Field experiments

3.2.1. Trace element concentrations 3.1.2. Discrimination using trace element fingerprints Veliger larvae collected from deployed chambers ranged in mean Statistical assignment of larval and juvenile age classes using TEFs size from 110 ± 20 (GB) to 160 ± 50 μm (FBE) while collected

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Fig. 4. Trace element concentrations in deployed mussels. Mean ( ± SE) trace element concentrations of log transformed (log10 (x + 1)) elements within larval (filled) and juvenile (unfilled) mussels reared at FBC, FBE, GB, PHB, WRL, MBR, CHR and MB (see Fig. 1, text for site details). Significant differences in trace element concentration between larval and juvenile mussels at each site, determined through MANOVA, determined by MANOVA, performed on Z-score standardized data at α = 0.05, are denoted by the letter “S”. juvenile mussels ranged from 2.3 ± 0.4 (PHB) to 2.8 ± 0.7 mm (MB). 4. Discussion Juvenile mussels deployed at FBE were slightly larger (3.3 ± 1.4 mm). Similar to laboratory experiments, several trace elemental ratios 4.1. Trace elements in larval and juvenile mussels differed significantly between age classes (Fig. 4, Table S2). Interest- ingly, the magnitude and direction of the differences between larval The concentration of several trace elements differed between larval and juvenile mussels varied among deployment sites, perhaps reflecting and juvenile mussel shells reared in either controlled laboratory con- large differences in marine geochemistry among bays. Among deployed ditions or in situ chambers at several field sites in the northern Gulf of mussels, Mg/Ca and Pb/Ca ratios differed by age class in four sites, Mn/ Maine (Figs. 2, 4). There are likely several interacting factors influen- Ca and Co/Ca in six sites, Sr/Ca and La/Ca in five sites, Ba/Ca in three cing structures and processes of biomineralization, such as differences sites, La/Ca in seven sites of eight total sites (Fig. 4; Table S2; sample N in the crystal structure of CaCO3 polymorphs (de Leeuw and Parker, subset in Fig. 5A–H; MANOVA at α = 0.05). Mg/Ca concentrations 1998; Medaković, 2000; Dorval et al., 2005; Dalbeck et al., 2006), were greater than all other recorded elemental ratios in deployed larval ontogenic shifts in ion transport pathways (Weiner and Dove, 2003) and juvenile mussels. Mg/Ca concentrations were greater in juveniles and the relative frequency of element insertion within aragonitic, cal- than larvae in all but two sites (FBC, GB), Mn/Ca concentrations were citic and proteinaceous shell structures (Lorens and Bender, 1980; greater in larvae in all sites except for FBE, Co/Ca in all sites but FBC Addadi et al., 2003; Schöne et al., 2010; Littlewood et al., 2017; and FBE, Sr/Ca in all sites but PHB and MBR, Ba/Ca in all sites but FBC Dauphin et al., 2018). and MBR, La/Ca in all sites but FBE and Pb/Ca in all sites but FBC, FBE The observed differences in TEFs between larval and juvenile shells and GB, of eight total sites (Fig. 4). reared under similar environmental conditions complicate efforts to Among sites, larval shells significantly differed in Mg/Ca, Co/Ca, generate unique site-specific fingerprints for estimating natal sites of Sr/Ca, Ba/Ca and Pb/Ca concentrations (particularly among midcoast settled mussels (Fodrie et al., 2011; Carson et al., 2013). A reference sites (GB, PHB, WRL), and juvenile shells differed in all element con- map of spatial variation in TEFs among potential source populations is centrations (Table S12A, B; MANOVA at α = 0.05). Among juveniles, required to estimate dispersal pathways and population connectivity FBC exhibited higher concentrations of every element except Sr/Ca, from the larval shell of recently settled bivalves. Since we found dif- which was significantly reduced compared to juveniles at all other sites. ferences in TEFs between larval and juvenile shells, a reference map must be derived from the in situ reared larval signatures which is con- sistent with what others have suggested for Mytilid mussels (sensu 3.2.2. Discrimination using TEF Becker et al., 2007; Table S13). Outplanted mussels were assigned to age classes with over 90% accuracy at all sites, and 100% accuracy at five sites, reflecting clear 4.2. Potential causes of age and temporal differences in TEFs differences in trace element fingerprints between larval and juvenile mussels reared in the same bay (Fig. 5A–H). In combined age and site Understanding differences in likely transport mechanisms (Weiner assignment, juvenile mussels were assigned to their actual age class and and Dove, 2003) and trace element incorporation within unique cal- site with greater accuracy than larval mussels (87% vs 47%; Fig. 5I). cium carbonate polymorphs crystallized and phase transformed across Such discrimination patterns reflect greater chemical differences be- successive ontogenetic stages (Furuhasi et al., 2009) is key to explaining tween age classes than between deployment sites, similar to our find- why TEFs vary spatially and temporally. Intense enzymatic activity ings within laboratory experiments. Across all trials, assignment ac- occurs at the site of crystallization, contributing to ontogenetic changes curacies were at least 5 times greater than those estimated by random in calcium carbonate polymorph, size and crystal shape (Medaković, chance in laboratory trials (Table S5), demonstrating strong QDFA age 2000; Marin and Luquet, 2004; Barthelat et al., 2009; Cartwright et al., and site discrimination using shell element concentrations. 2009). Larval aragonitic shell content increases over time from amor- Elemental differences in larval and juvenile shells between sites phous calcium carbonate (ACC; Medaković, 2000), while newly settled contributed to strong site discrimination in each age class, even when dissoconch and juvenile shells are composed of surface trilayered removing Mg/Ca and Sr/Ca (elements corresponding to observed age- (Harper, 1997) underlain by prismatic calcite and nacr- class differences) from QDFA analyses (Table S13A, B). eous aragonite (Marin and Luquet, 2004). The recent observation that both Mg and Sr may also play a key role in stabilizing ACC as a pre- cursor to subsequent aragonite or calcite precipitation (Addadi et al.,

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Fig. 5. Age and Site Discrimination in Deployed Mussels. Age discrimination (A-H) and age and site discrimination (I) of larval (filled) and juvenile (unfilled) mussels reared at FBC, FBE, GB, PHB, WRL, MBR, CHR and MB (see text for site details). First two canonical axes generated through quadratic discriminate function analyses (QDFA). Group centroids and 90% confidence intervals are shown. Classification accuracies are inset, with actual rows and assigned columns. Direction and length of biplot rays represent elemental correlation coefficients with respect to the included canonical axes.

2003; Littlewood et al., 2017), and previous work indicating that M. 2007). Age-specificdifferences in Mg utilization, insertion and trace edulis exerts control over Mg activity in the extrapallial fluid (Lorens element incorporation may have contributed to observed differences in and Bender, 1980), suggest that Mg-driven ACC stabilization in bivalve TEFs recorded in this study. larvae may have contributed to significant differences in Mg/Ca con- Mechanisms of ion transport and cation specificity during calcifi- centration between larval (high ACC) and juvenile (aragonitic and cation may have also impacted bioincorporation and contributed to calcitic) shells (Figs. 2, 4), and may help explain the high importance of elemental differences between larval and juvenile shells. Divalent ca- Mg/Ca in age class discrimination during both lab and field experi- tions similar in size to Ca2+ likely compete with calcium during bio- ments (Figs. 3, 5). mineralization, resulting in non-target ion insertion within calcium The strong relationship between shell mineralogy and Mg con- carbonate crystal matrices (Simkiss and Taylor, 1989; Depledge and centration, and cascading impacts on other trace elements have long Rainbow, 1990). been suspected (Harriss, 1965) and are supported by subsequent re- Specific transport mechanisms among cations may impact the fre- search showing crystal distortion during Mg and Sr insertion, resulting quency of this insertion. When calcium channels were experimentally in trace element replacement within biogenic shells (Finch and Allison, blocked (Zhao et al., 2017), Mn and Pb concentrations decreased while

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Mg, Sr and Ba concentrations remained unaffected, suggesting that laboratory and field-reared biogenic shells. The efficacy of these tech- some microelemental concentrations may covary with calcium due to niques is supported by confirmation of ablation pit location and depth similar transport mechanisms, while other cations may be unaffected by through scanning electron microscopy (SEM) and observed elemental Ca transport and calcification rates. Differences in calcification rate differences between shell and underlying mounting tape and slide may impact trace element incorporation (Lorens and Bender, 1980), materials (e.g. elevated Ca, Mg, Sr, minimal Si in larval shells). particularly across developmental stages (Bayne et al., 1989; Widdows, 1991), potentially impacting elemental ratios observed in this study. 4.3. Trace element variation in the Gulf of Maine Prior to the transformation to aragonitic shells, larval mussels, un- dergoing rapid shell growth, are unlikely to have the physiological The TEFs of mussels reared in situ differed greatly among popula- capacity to actively regulate trace element incorporation and prevent tions across the northern Gulf of Maine. Numerous studies have docu- non-target ions from insertion within calcium carbonate crystal lattices, mented that chemistry is correlated with a suite of en- possibly due to their relatively greater surface area to body ratio, vironmental proxies. For example, Mg/Ca has been correlated with greater amorphous calcium carbonate, and greater investment in temperature (Levin, 2006; Heinemann et al., 2011), Mn/Ca with dis- growth and metamorphosis rather than homeostatic mechanisms (Klein solved oxygen (Tynan et al., 2005; Broadaway and Hannigan, 2012), et al., 1996). During rapid calcification, intracellular transport favoring Sr/Ca with temperature (Carré et al., 2006; Barats et al., 2007) and calcium over strontium was found to increase relative to intercellular salinity (Dorval et al., 2005; Strasser et al., 2008; Heinemann et al., transport, driving down Sr/Ca concentrations (Klein et al., 1996). 2011), Ba/Ca with dissolved oxygen (Broadaway and Hannigan, 2012) Reduced physiological control and shifts in elemental transport may and temperature (Strasser et al., 2008; Carson et al., 2013; Chan et al., explain why Sr/Ca concentrations, and those of other elements that 2013) while Pb/Ca concentrations were associated with terrestrial likely do not compete with calcium transport, were generally greater in input (Levin, 2006), anthropogenic pollution (Barats et al., 2007; both lab and field-reared larvae. This inability to selectively filter non- Carson et al., 2013) and pH (Broadaway and Hannigan, 2012). target ions from inside the epipallial cavity and subsequent shell was The geographic variation in TEFs we observed among mussels proposed as an explanation for large differences in Mg and Sr con- reared in different bays likely reflects differences in local environmental centrations between ambient water and M. edulis shells, only at high conditions, exacerbated by nonuniform coastal warming and acid- salinity (> 28 ppt; Wanamaker et al., 2008). During this study, out- ification, and subsequent impacts on inorganic solubility, organic planted larval mussels may have experienced greater vulnerability to complexation, redox chemistry and metal biouptake (Cao et al., 2007; salinity variation within estuarine (Xue et al., 2000) and coastal waters Hoffman et al., 2012; Range et al., 2012). In a study of physical, che- in the Gulf of Maine (Mountain, 2004; Smith, 1983) than juveniles due mical and biological variation in the southern Gulf of Maine (Seip, to physiological and structural differences (Berger and Kharazova, 2015), regions 20 km apart differed in salinity, temperature, water 1997; May et al., 2017), which might have reduced element selectivity chemistry and phytoplankton abundance. Sites adjacent to river outputs during biomineralization and contributed to greater and more variable in the northern Gulf of Maine may be impacted by seasonal freshening, trace element concentrations within larval shells (Fig. 3). reductions in dissolved inorganic carbon and lower aragonite saturation Despite apparent competition with Ca2+ ions during biominer- states (Salisbury et al., 2008), with significant impacts on trace element alization, Mn and Co are biologically essential elements (Bellotto and availability and incorporation into mussel shells (Fitzer et al., 2014). Miekeley, 2007) that may have been incorporated within the organic Trace element concentrations differed slightly between sites in close matrix of precipitated aragonite (Thomas et al., 2003) during rapid proximity to significant freshwater inputs (MB: Machias River; CHR: shell growth in reared larvae. Barium may be uptaken through inges- Chandler River; WRL: Indian River; PHB: Narraguagus River; GB: Tunk tion of Ba-rich phytoplankton (Stecher et al., 1996) or directly ingested Stream) and those located along the coast or within enclosed tidal as barite associated with decaying algal flocs (Stecher et al., 1996; embayments (FBC, FBE, MBR). Juveniles outplanted near freshwater Brannon and Rao, 1979). Differences in preferred diet between larval inputs exhibited reduced concentration of all elements relative to Ca and juvenile mussels may partially explain why Ba/Ca concentrations except Sr/Ca, which was significantly elevated, while similarly out- only differed by age class in outplanted mussels, where phytoplankton planted larvae further from freshwater sources exhibited lower Mg/Ca diversity and availability were likely more variable (Thomas et al., and greater Co/Ca, Ba/Ca and Pb/Ca (Table S5). Such differences may 2003). As well, bivalves may be able to concentrate metals within shells have been the result of variation in both the volume and water chem- during toxic exposure (Carriker et al., 1980), contributing to relatively istry of upstream waters, particularly with respect to pH, Ca and Mg greater Mn, La, and Pb concentrations observed within relatively more concentrations, acid neutralizing capacity and dissolved organic vulnerable larval shells during this study. carbon, all of which were previously found to vary across streams in In addition, structural differences in the distribution of insoluble Downeast Maine (Johnson and Kahl, 2005). and soluble organic matrices (IOM, SOM; Furuhasi et al., 2009) be- Elemental bioavailability in the Gulf of Maine can also be impacted tween larval and juvenile shells may have contributed to elemental by interrelationships among inorganic solubility, organic complexation, differences recorded using LA-ICPMS in this study. Concentrations of redox chemistry and metal uptake by dietary plankton within deploy- Mg, Sr, and likely trace elements, were mischaracterized (200× Mg/Ca, ment sites increasingly impacted by regional climate change (Cao et al., 2× Sr/Ca than average shell values) through ablation of shell enriched 2007; Hoffman et al., 2012; Range et al., 2012). Within the Gulf of in IOM (compared to values obtained through optical emission spec- Maine, soft-tissue concentrations of heavy metals appear to be greater trometry: ICP-OES; Schöne et al., 2010). However, removal of the IOM in mussels collected in the south, closer to urban and commercial is difficult prior to LA-ICPMS analysis, and key sampling resolution is fishing communities (Chase et al., 2001), although this was not re- lost in ICP-OES techniques. Such challenges to quantifying elemental flected in our study of biomineralization within shell matrices. Elevated concentrations within complex biostructures that greatly differ by age concentrations of Pb were previously found in mussels collected near class emphasize the importance of using age-specific individuals to Frenchman Bay (GN; Sorte et al., 2013), potentially due to historic develop geochemical reference maps to quantify population con- mining operations (Osher et al., 2006). Similar to previous studies nectivity using TEFs. where TEFs varied across diverse environmental conditions (Carson Despite several challenges to accurately quantifying TEFs through et al., 2013; Miller et al., 2013), likely spatial and temporal variation in LA-ICPMS, including potential impacts of sample preparation and ab- trace element availability across sites sampled in this study suggest that lation methods (Génio et al., 2015; Strasser et al., 2007) on relatively TEFs should be quantified by rearing larvae in close temporal and thin larval shells (~ 4um thickness; Gazeau et al., 2010), we believe spatial proximity to naturally spawning populations to generate the that TEFs recorded in this study, accurately reflect the geochemistry of most accurate TEF reference map.

7 A. Honig, et al. Journal of Experimental Marine Biology and Ecology 522 (2020) 151249

4.4. Implications for trace element fingerprinting Acknowledgements

The TEFs of mussel shells differed strongly among ages and sites We thank the Downeast Institute shellfish hatchery for assistance in across the northern Gulf of Maine (Fig. 4; Table S4), and among sites in spawning and rearing laboratory mussels, Phil Yund for help identifying each separate age class (Tables S12, S13) indicating elemental finger- significant mussel beds and in mussel outplanting, and the Northeastern prints should allow us to reliably identify source populations. Overall University Marine Science Center and the Massachusetts Division of site assignment accuracy of larval shells was lower than that of juvenile Marine Fisheries for help accessing field sites. We would also like to shells, although accuracies differed greatly among sites. Many of the thank Casey McCabe, Angelo Christophe, Chris Bukowski, Joel Rosen misclassified individuals were assigned to nearby field sites suggesting for contributions to sample preparation, and Alan Stebbins and Amy that trace element signatures of nearby sites may correspond to similar Johnson for assistance with ICPMS instrumentation and geochemical marine hydrodynamic conditions. Exceptions included mussels reared analyses. We thank the two anonymous reviewers for their helpful at Pigeon Hill Bay and Machias Bay, which were frequently misassigned comments. The authors declare no conflict of interest. Funding for this to non-adjacent study sites separated by > 50 km. This may be due to work was provided by the National Science Foundation, USA (OCE – patchy coastal marine chemistry, where non-adjacent water bodies may 1334022, 1458154). be more similar to each other than those along contiguous areas of coastline. Similarly, large differences in assignment accuracy among References field sites in both larval and juvenile mussels, and in the identity of trace elements most important to accurate discrimination, likely reflect Addadi, L., Raz, S., Weiner, S., 2003. Taking advantage of disorder: amorphous calcium great natural variation in the interaction between local trace element carbonate and its roles in biomineralization. Adv. Mater. 15, 959–970. Alden, R., 2011. Building a sustainable system for maine. Maine Pol. Rev. 20 (1), availability and shell incorporation, mediated through highly variable 87–95. environmental conditions. Auker, L., Majkut, A., Harris, L., 2014. Exploring biotic impacts from Carcinus maenas Our assignment accuracies are similar to those based on recent es- predation and didemnum vexillum epibiosis on Mytlius edulis in the Gulf of maine. Northeast. Nat. 21 (3), 479–494. timates of geographic variation in TEFs of mussels. 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