Mercury isotope clocks predict coastal residency and migration timing of hammerhead sharks

1. The management of migratory taxa relies on the knowledge of their movements. Among them, ontogenetic habitat shift, from nurseries to adult habitats, is a behavioral trait shared across marine taxa allowing resource partitioning between life stages and reducing predation risk. As this movement is consistent over time, characterizing its timing is critical to implement efficient management plans, notably in coastal areas to mitigate the impact of fisheries on juvenile stocks.

2. In the Mexican Pacific, habitat use of the smooth hammerhead shark (Sphyrna zygaena) is poorly described, while the species is heavily harvested. Given the large uncertainties associated with the timing of out-migration from coastal nursery grounds to offshore waters prior to reproductive maturity, a more precise assessment of smooth hammerhead shark movements is needed. 

3. Photochemical degradation of mercury imparts mass-independent isotope fractionation (Δ199Hg) which can be used to discriminate between neonate coastal shallow habitats and the offshore deep foraging patterns of late juveniles. Here, we present the application of muscle Δ199Hg as molecular clocks to predict the timing of ontogenetic habitat shifts by smooth hammerhead sharks, based on their isotopic compositions at the initial and arrival habitats and on muscle isotopic turnover rate.

4. We observed decreases in Δ199Hg values with shark body length, reflecting increasing reliance on offshore mesopelagic prey with age. Coastal residency estimates indicated that smooth hammerhead sharks utilize coastal resources for two years prior to offshore migration, suggesting a prolonged residency in these ecosystems.

Policy implications: This study demonstrates how mercury stable isotopes and isotopic clocks can be implemented as a complementary tool for stock management by predicting the timing of animal migration—a key aspect in the conservation of marine taxa. In the Mexican Pacific, fishing pressure on shark species occurs in coastal habitats depleting juvenile stocks. Consequently, management decision support tools are imperative for effectively maintaining early life stage population levels over time. The finding that smooth hammerhead sharks extensively rely on highly fished habitats for two years after parturition supports the relevance of establishing a size limit in coastal fisheries and demonstrates how the current temporal shark fishing closure could lack efficiency for the species.

Methods
Sharks were collected along the western coast of Baja California Sur (Mexico). Juvenile smooth hammerhead sharks were caught by gillnets and longlines in 2009 and from 2014 to 2018. Sharks (n=102) were sampled at four locations in artisanal fishing camps: Bahía Tortugas, Las Barrancas, San Lázaro and Punta Lobos. For each individual, approximately 1 g of dorsal white muscle tissue was sampled. Samples were transported on ice, stored at -20°C at the laboratory and ultimately freeze-dried prior to transport and further treatments.

Total Hg concentration (THg) was determined in a 20 mg aliquot of each sample using a Direct Mercury Analyzer (DMA-80, Milestone, USA), by combustion, gold trapping and atomic absorption spectrophotometry detection (Observatoire Midi-Pyrénées, Toulouse, France). THg analytical detection limit was 0.005 μg·g−1 dw (dry weight). Repeated measurements of a tuna flesh homogenate (BCR-464, Institute for Reference Materials and Measurements, 5.24 ± 0.10 μg·g−1 dw) tested the analysis's reproducibility and accuracy. BCR-464 measurements (n=9) were reproduced within the confidence limits: 5.30 ± 0.44 μg·g−1 dw.

Another 20 mg muscle sample was then diluted into 3 mL of pure bi-distilled nitric acid (HNO3), left overnight at room temperature and digested at 100°C for 6 hours in pyrolyzed glass vessels closed by Teflon caps on a hotplate. We added 1 mL of hydrogen peroxide (H2O2), left the digestion to continue for another 6 hours and completed the extraction with 100 µL of bromine monochloride (BrCl) to convert solubilized MeHg into inorganic Hg. Samples were diluted to THg concentration of 1 ng·mL−1 using an inverse aqua regia (3:1 HNO3:HCl with 20 vol.% of MilliQ water). Hg isotope composition was measured at the Observatoire Midi-Pyrénées (Toulouse, France) by inductively coupled plasma mass spectrometry (MC−ICP−MS, Thermo Finnigan Neptune) with continuous-flow cold vapor (CV) generation using Sn(II) reduction (CETAC HGX-200). Mass-dependent fractionation is expressed in δ notation (‰). Sample values are expressed relative to their deviation from the National Institute of Standards and Technology (NIST) SRM-3133 standard and calculated by sample-standard bracketing. Along the procedure, THg in the solution was monitored by the 202Hg signal provided by MC-ICP-MS. We measured blanks and BCR-464-certified materials following the same procedure as for shark samples. We recovered 84 ± 19% of THg in shark samples and 90 ± 2% in BCR-464 replicates (n=10). Isotopic measurement reproducibility was assessed by analyzing UM-Almadén (n=8), ETH-Fluka (n=8) and the biological tissue procedural standards BCR-464 (n=10).

1. 洄游类群的管理依赖于其移动规律的认知。其中，个体发育栖息地转移（ontogenetic habitat shift）——即从育幼场向成体栖息地的迁移——是海洋类群共有的行为特征，可实现不同生活史阶段的资源分化并降低捕食风险。由于此类移动具有时间一致性，明确其发生时序对于制定高效管理方案至关重要，尤其在近岸海域，可借此减轻渔业对幼鱼种群的冲击。

2. 在墨西哥太平洋海域，路氏双髻鲨（Sphyrna zygaena）的栖息地利用情况尚缺乏详细描述，而该物种正遭受高强度捕捞。目前，关于其在性成熟前从近岸育幼场向远海水域迁出的时间节点存在较大不确定性，因此亟需对路氏双髻鲨的移动规律开展更精准的评估。

3. 汞的光化学降解会产生质量独立同位素分馏（mass-independent isotope fractionation，Δ¹⁹⁹Hg），该信号可用于区分新生幼体的近岸浅海栖息地与晚期幼体的远海深层觅食生境。本研究基于初始栖息环境与抵达栖息环境的同位素组成，以及肌肉组织的同位素周转速率，将肌肉Δ¹⁹⁹Hg作为"分子钟"，用于预测路氏双髻鲨的个体发育栖息地转移时序。

4. 研究发现，Δ¹⁹⁹Hg值随鲨鱼体长增长而降低，这反映出随着年龄增加，鲨鱼对远海中层大洋带猎物的依赖程度不断提升。近岸居留时长估算结果显示，路氏双髻鲨在向外海洄游前会利用近岸资源长达两年，表明该物种会在这些近岸生态系统中经历较长的居留阶段。

政策启示：本研究证明，汞稳定同位素与同位素"分子钟"可作为种群管理的辅助工具，通过预测动物洄游时序——这是海洋类群保护的关键环节——来支撑管理决策。在墨西哥太平洋海域，针对鲨鱼的捕捞活动多发生在近岸栖息地，导致幼鱼种群被过度消耗。因此，亟需开发管理决策支持工具，以长期有效维持早期生活史阶段的种群规模。本次研究发现，路氏双髻鲨在产仔后会有两年时间高度依赖受高强度捕捞的近岸生境，这一结果佐证了在近岸渔业中设置体长限制的必要性，同时也表明现行的鲨鱼临时禁渔制度对该物种而言可能缺乏有效性。

方法
鲨鱼样本采集于墨西哥下加利福尼亚半岛西海岸海域。2009年及2014年至2018年间，科研人员通过刺网与延绳钓捕获幼年路氏双髻鲨，共在4处小型手工渔业作业点（托尔图加斯湾、拉斯巴兰卡斯、圣拉萨罗以及蓬塔洛博斯）采集了102尾鲨鱼样本。每尾个体采集约1克背侧白肌组织。样本置于冰上运输，实验室中保存于-20℃环境，最终经冷冻干燥后进行后续运输与实验处理。

总汞浓度（THg）测定：称取每份样本的20 mg分装试样，采用直接测汞仪（DMA-80，迈尔斯通（Milestone）公司，美国），通过燃烧、金捕集与原子吸收分光光度法检测（法国图卢兹米迪-比利牛斯观测站）。总汞的分析检出限为0.005 μg·g⁻¹ dw（干重）。以金枪鱼肉匀浆标准物质（BCR-464，欧盟参考物质与测量研究所，5.24 ± 0.10 μg·g⁻¹ dw）验证分析的重现性与准确性，9次重复测定结果为5.30 ± 0.44 μg·g⁻¹ dw，落在置信区间内。

另取20 mg肌肉样本，稀释于3 mL纯双蒸硝酸（HNO₃）中，室温静置过夜后，在聚四氟乙烯密封的热解玻璃容器中于100℃消解6小时。随后加入1 mL过氧化氢（H₂O₂），继续消解6小时，再加入100 μL一溴化氯（BrCl）将溶解的甲基汞转化为无机汞。采用逆王水（3:1硝酸:盐酸，含20 vol.% 超纯水）将样本稀释至总汞浓度为1 ng·mL⁻¹。汞同位素组成在法国图卢兹米迪-比利牛斯观测站通过多接收电感耦合等离子体质谱仪（MC−ICP−MS，赛默飞世尔 Neptune）测定，采用锡(II)还原法（CETAC HGX-200）实现连续流动冷蒸气（CV）进样。质量依赖分馏以δ符号（‰）表示，样本值以相对于美国国家标准与技术研究院（NIST）SRM-3133标准的偏差进行计算，并采用样本-标准交叉校准法进行校正。实验过程中，通过MC−ICP−MS提供的²⁰²Hg信号监测溶液中的总汞浓度。按照与鲨鱼样本相同的流程进行空白实验与BCR-464标准物质测定。鲨鱼样本的总汞回收率为84 ± 19%，BCR-464重复样本的回收率为90 ± 2%（n=10）。通过分析UM-Almadén（n=8）、ETH-Fluka（n=8）以及生物组织流程标准物质BCR-464（n=10）评估同位素测量的重现性。