Daily Growth Increments Research Articles

Daily and annual shell growth in a long-lived freshwater bivalve as a proxy for winter snowpack

To assess the potential of long-lived freshwater bivalve shells as a proxy for river environments, we examined daily and annual growth increments and analysed trace elements in the shells of Margaritifera laevis collected alive from the Shiribetsu River, located in the central western region of Hokkaido, Japan. The sum of the daily growth lines within a single annual increment corresponded to the days when the average daily water temperature was greater than 9 °C, suggesting that shell deposition occurred from spring to autumn. The growing degree days (GDD) are correlated with the maximum snow depth in winter and the average river discharge in spring. We conducted trace element microanalysis across daily and annual growth increments by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and compared the results with in situ environmental data. Sharp peaks in the barium/calcium ratio (Ba/Ca) observed during spring river discharge also suggest that the growth of Margaritifera shells is influenced by winter snow volume and meltwater. A 67-year profile of the standard growth index (SGI) estimated from twelve individual annual growth histories correlates to the annual snowfall accumulation. Distinct decadal variability is observed in the SGI and synchronized with the North Pacific Index and Pacific Decadal Oscillation. Sclerochronological approaches using Margaritifera shells could be valuable for reconstructing river and atmospheric conditions during the winter season, which is influenced by the Asian winter monsoon in the central Hokkaido region.

The World Vegetable Center Okra (Abelmoschus esculentus) Core Collection as a Source for Flooding Stress Tolerance Traits for Breeding

Okra (Abelmoschus esculentus) is a heat tolerant vegetable crop with high economic and nutritional importance in parts of Asia, Africa, and America. The okra biodiversity held in gene bank collections could be mined for traits for breeding more stress tolerant and nutritional cultivars. An okra core collection of 166 accessions comprising A. esculentus, A. moschatus, A. caillei, and A. manihot has been assembled from the World Vegetable Center germplasm collection (840 accessions) based on diversity analysis with 20 microsatellite markers. A selection of A. esculentus accessions of the core collection (75 accessions) and 20 breeder-selected genotypes have been screened for variation of their response to flooding stress under field conditions using a high throughput phenotyping system. Growth increment per day and changes of physiological indices were measured before, during, and after application of 9 days of flooding stress. Several accessions showed only a small reduction in daily growth increment during flooding. Across the germplasm panel, maintained growth was correlated with maintained normalized differential vegetation index and was negatively correlated with plant senescence index. Accessions with maintained growth and health under flooding were selected for future further analysis and use in breeding.

An inconstant biorhythm: The changing pace of Retzius periodicity in human permanent teeth

Human tooth enamel retains evidence of growth in the form of Retzius lines. The number of daily growth increments between the regularly occurring lines defines their repeat interval, or periodicity. Retzius periodicity is often incorporated into enamel formation times, age-at-death reconstructions, or used to provide a basis from which to explore an underlying biorhythm. Biological anthropologists typically assume that RP remains constant within an individual and does not vary along the tooth-row. Here, we test that assumption. RP was calculated from n = 223 thin sections of human permanent teeth from individuals of British and southern African origin. Forty individuals provided multiple teeth (n = 102 teeth) and a further 121 individuals each provided a single tooth. We report first evidence that RP of permanent teeth does not always remain constant within an individual. Of those individuals that provided multiple teeth, 42% (n = 17/40) demonstrated a decrease in RP along the tooth row, with most shifting by two or more days (n = 11). Across the entire sample, mean RP of anterior teeth was significantly higher than molars. Mean premolar RP tended to be intermediate between anterior teeth and molars. Our data do not support the assumption that RP invariably remains constant within the permanent teeth of an individual. Transferring RP from molars to incisors within an individual can result in a miscalculation of formation time and age-at-death by up to 1 year. Implications for biological anthropologists and the source of the underlying long period biorhythm are discussed.

Growth and development of the third permanent molar in Paranthropus robustus from Swartkrans, South Africa

Third permanent molars (M3s) are the last tooth to form but have not been used to estimate age at dental maturation in early fossil hominins because direct histological evidence for the timing of their growth has been lacking. We investigated an isolated maxillary M3 (SK 835) from the 1.5 to 1.8-million-year-old (Mya) site of Swartkrans, South Africa, attributed to Paranthropus robustus. Tissue proportions of this specimen were assessed using 3D X-ray micro-tomography. Thin ground sections were used to image daily growth increments in enamel and dentine. Transmitted light microscopy and synchrotron X-ray fluorescence imaging revealed fluctuations in Ca concentration that coincide with daily growth increments. We used regional daily secretion rates and Sr marker-lines to reconstruct tooth growth along the enamel/dentine and then cementum/dentine boundaries. Cumulative growth curves for increasing enamel thickness and tooth height and age-of-attainment estimates for fractional stages of tooth formation differed from those in modern humans. These now provide additional means for assessing late maturation in early hominins. M3 formation took ≥ 7 years in SK 835 and completion of the roots would have occurred between 11 and 14 years of age. Estimated age at dental maturation in this fossil hominin compares well with what is known for living great apes.

Can otolith microstructure and elemental fingerprints elucidate the early life history stages of the gadoid southern blue whiting (Micromesistius australis australis)?

Southern blue whiting (Micromesistius australis australis) once contributed to one of the largest finfish fisheries in the Southwest Atlantic. Intense exploitation began in the late 1970s with peak total annual catches attaining 258.000 tonnes in 1983. Continuing levels of exploitation hereafter resulted in a significant reduction in its abundance by the early 1990s and forced the implementation of a suite of conservation measures to rebuild these stocks. Improved recruitment levels in recent years suggests that these measures have been effective. However, more information on habitat use and behaviour during the early life history stages of this fish is required to be able to draw firm conclusions. Here, we use complimentary investigations into otolith microstructure and trace elemental composition (by LA-ICPMS) to study the early stages of ontogeny in M. a. australis. We provide evidentiary support for the formation of daily growth increments on the otoliths of juvenile M. a. australis and utilise these to estimate the age at onset and completion of metamorphosis (30.5 ± 0.3 and 60.7 ± 1.8 days after the first increment formation, respectively). The timing of a newly defined ‘juvenile transition-mark’ (corresponding to the transition from the epipelagic larval habitat to a deeper juvenile habitat) was estimated to form at 76.5 ± 2.0 days after first increment formation, suggesting that early juveniles remain in the epipelagic habitat for over two weeks post-metamorphosis. Significant shifts in element:42Ca ratios were found for elements 24Mg, 55Mn, 88Sr and 138Ba between otolith zones corresponding to different ontogenetic stages: (A) larval-, (B) metamorphosis- and (C) juvenile zone. These shifts could be correlated with changes in behaviour and habitat. Elemental ‘fingerprints’ for each of the three otolith zones were found to be distinct through high classification accuracies of the Random Forest classifier (81.1 ± 0.1 %), with 55Mn:42Ca contributing most to the classification. This holistic approach investigating both otolith microstructure and elemental composition provided evidence that ontogeny could be a confounding factor in natal (geographic) origin studies if elements predominantly controlled by physiology are included in the respective classification analyses.

Age determination for juvenile fourfinger threadfin (Eleutheronema rhadinum) by using otolith microstructure and length data obtained from commercial fisheries off northwestern Taiwan

The fourfinger threadfin (Eleutheronema rhadinum) is a commercially crucial species for coastal and inshore small-scale fisheries, but is rarely studied to understand its life history, including age and growth. The present study examined the daily growth increment in otoliths for the species during its early life stages by using otolith microstructure analysis, and corroborated the age estimates using alternative information and approaches. The daily growth increments were discovered to be formed and spaced regularly. The fish samples were successfully aged at between 174 and 652 days, with the growth rate fairly consistent among individuals. The mean length at age 1 year of E. rhadinum was estimated at 29.7 cm fork length using the fitted growth equation. Hatch dates, estimated using the daily increments and the date of capture, were mostly from April to August. We further corroborated the age estimates by using identifiable modes of younger fish in the monthly length–frequency samples. The lengths-at-age, predicted using the growth equation with best fit, were discovered to correspond favorably with the progress of the modes in the length–frequency distribution of sampled landed fish. A framework that incorporates information available from alternative approaches to determine and corroborate estimates of fish age can be developed, as demonstrated in this study.

Hatching Time and Early Growth of Diplodus sargus sargus Inhabiting a Coastal Area of Gökçeada Island, Turkey

Daily growth increments in sagittal otoliths were used to estimate the age and growth of young of the year (YOY) white seabream, Diplodus sargus sargus collected between June 2013 and June 2014 at 6 different locations in the shallow waters (<20 m) off Gokceada, Turkey. Estimated ages from otolith daily growth increments (DGI) were found 42–238 days old. The mean daily growth rate of YOY D. sargus sargus was estimated as 0.046 cm/day. ANCOVA indicated that there was significant difference in daily growth rates between the locations (F = 22.92, df = 5, P < 0.001). The maximum daily growth rate of 0.055 cm/day was found in the location 2, while the minimum value of 0.037 cm/day was observed in the location 4. Hatching time was determined by subtracting the daily age of the fish from the sampling date and then hatch date frequency distributions were determined to occur from January through June, and 2 main hatching peaks were observed from the different locations. Linear relationships between otolith morphology (otolith length; OL, width; OW and radius; OR) and fish length (TL) were obtained.

Characteristics of two populations of Thai river sprat Clupeichthys aesarnensis from man-made reservoirs in Thailand and Laos, with aspects of gonad development

Growth and reproduction of the Thai river sprat Clupeichthys aesarnensis (Teleostei: Clupeidae) in Sirindhorn Reservoir, Thailand, and Nam Ngum Reservoir, Laos, were investigated using age estimates obtained from daily growth increments in otoliths and gonad observations. This species breeds throughout the year in both reservoirs, although water temperature was higher in Sirindhorn than in Nam Ngum. The Sirindhorn population tended to mature at smaller size (length) and younger age than the Nam Ngum population; the Sirindhorn population also tended to grow faster before reaching sexual maturity, growing slowly afterwards, than did the Nam Ngum population. Additionally, the maximum size in the Sirindhorn population was observed to be smaller than that of the Nam Ngum population. These observations indicated that higher water temperature resulted in earlier maturation and smaller maximum size in C. aesarnensis. Meanwhile, the smaller maximum size observed in the Sirindhorn population was possibly caused by an evolutionary down-sizing resulting from the overexploitation of this population, because Sirindhorn reservoir is exposed to more intensive fishing pressure.

N-Isotopes in Feathers and Abundance of Eiders Respond to Nutrients in Seawater

Nitrification of the environment has resulted from tremendous increases in the use of fertilizers for crop plants. This has increased runoff to coastal marine areas with consequences for primary production, benthos and upper trophic-level consumers, including sea ducks such as eiders Somateria mollissima. This species primarily relies on filter-feeding bivalves, especially blue mussels Mytilus edulis. Stable isotopes of nitrogen (measured as δ15N) are incorporated in the feathers of eiders during molt at a constant rate reflecting the amount in food eaten. We examined if δ15N values in feathers can link eiders to the nitrogen loads in the marine waters surrounding Denmark, which is the main wintering grounds for the Baltic/Wadden Sea eider population. We also assessed how the abundance of eiders is related to nutrients and dead zones with oxygen deficit. During 2014–2018 we collected samples from 489 eiders, and of these 100 were analyzed for stable isotopes δ15N. Eider feather δ15N was positively related to nitrogen abundance in marine waters. Local aggregations of eiders in late summer (the molting period) and in winter increased with the amount of nitrogen in the marine environment. Large eiders took longer to molt, they molted later and the daily growth increments of feathers were larger. Finally, larger eiders and eiders in higher numbers occurred in areas with hypoxia. These findings show that anthropogenic inputs to the coastal marine environment positively influence the abundance and aggregations of eiders.

Age and growth of Myctophum asperum in the South China Sea based on otolith microstructure analysis

To estimate the age and growth of a dominant species of lanternfish, Myctophum asperum, in the South China Sea, 399 fish were collected by a midwater trawl in spring and autumn, 2014–2016. Age was estimated using counts of daily growth increments laid down across the sagittal section of otoliths (n = 52 fish). Three regions of deposition in the otolith microstructure were clearly evident: the larval, post-larval and post-metamorphic zones. Increments in the larval zone (average of 30) and the post-metamorphic zone appeared brighter under a light microscope than the increments in the post-larval zone. The age of the sampled fish ranged between 183 and 405 days. The von Bertalanffy growth model provided the best fit for the post-metamorphosis fish and was expressed as: SL = 85.3[1-exp{− 0.008(t − 28.1)}]. The maximum rate of growth occurred at 183 days after metamorphosis and 58 mm SL among the age determined samples, and the growth rate decreased with age. The length-weight relationship for the entire sample of fish was W = 0.000037234 SL2.7935, whereas for females and males it resulted Wf = 0.000050140 SL2.7255 and Wm = 0.000035459 SL2.8022, respectively. Fish reached the adult size by 74 mm SL and 5–8 g, and this size group was numerically the most and constituted the greatest portion of the biomass.

Age-based life-history parameters of the mesopelagic fish Notoscopelus resplendens (Richardson, 1845) in the Central Eastern Atlantic

The mesopelagic fish Notoscopelus resplendens is distributed mainly between 500 and 1000 m of depth during the day, with diel migration to surface waters at night (<90 m). Fish move during their diel migrations across different water masses, which show strong changes in temperature with depth that can reach 7 °C in the first 300 m during the warm season. N. resplendens show a type of diel migration pattern of the “entire migrant”, showing a clear day-night habitat separation, with peak abundance above 200 m at night. The difference between sexes in maximum size was 1.97 mm SL (84.36 mm, males; 82.39 mm females). The spawning season can be determined from December to March by back calculating the hatching date from daily growth increments. Age at first maturity was 1.7 year for males and 2.05 years for females. The sex ratio showed a predominance of males (1:0.67). Males were significantly more abundant than females in the 1-year age class. In the rest of the age classes, no significant differences were observed. A sexually dimorphic nature in relation to the position of the accessory luminous glands was observed. Three growth regions were observed in the otolith corresponding to different rates of deposition during fish ontogeny. Daily growth increments were calculated as validation of the annuli pattern. The pattern of the increment formation showed that each annulus has a unimodal distribution. The increment width decreases with age from 1 to 4 years. A strong relationship was found between both diameters of the otolith and length and between otolith weight and age. Different growth models have been analysed to understand the growth of this species. Gomperzt and von Bertalanffy were the best models obtained and revealed differences in growth between sexes. The maximum age recorded was 4 years. The SL-TW relationship showed allometric positive growth. The natural mortality coefficient for the overall population derived from the age-frequency distribution was 0.579 year−1. M obtained from the length converted catch curve was very similar at M = 0.549 year−1.

Unravelling growth trajectories from complicated otoliths-the case of Brazilian codling Urophycis brasiliensis.

Uncertainty regarding the age determination of the Brazilian codling Urophycis brasiliensis has hampered its stock assessment. Transverse sections of otoliths displayed up to seven (in males) and 12 (in females) alternate opaque and translucent bands that could not be conclusively validated as annuli, resulting in unrealistically high ages of first maturity (A50 ) (A50male = 4·5years and A50female = 6years). Therefore, growth was described by the von Bertalanffy (VB) model using an alternative approach that combined microstructure data (daily growth increments) and a fixed asymptotic total length (L∞ ). This approach was supported by applying it to two other co-occurring species, the whitemouth croaker Micropogonias furnieri and the king weakfish Macrodon atricauda, for which daily and annual ring formation has previously been validated. The sensitivity to realistic errors associated with the choice of the L∞ and the daily increment readings was shown to be low. The results show that U. brasiliensis has a fast growth rate (Kmale = 1·19 year-1 , Kfemale = 0·71 year-1 ) and early maturation (A50male = 1·1-1·5years, A50female =1·6-1·8years); typical life-history traits for a sub-tropical coastal gadiform. This novel study offers an alternative approach for age and growth reconstruction for species with complex patterns of opaque and translucent bands provided that daily growth increments in the yearlings can be counted and L∞ reliably estimated.

Relationships between the daily growth rate of Japanese anchovy Engraulis japonicus larvae and environmental factors in Osaka Bay, Seto Inland Sea, Japan

Seasonal changes in the growth rate of larval Japanese anchovy Engraulis japonicus collected by shirasu seine net boats in Osaka Bay in 2008 were estimated using otolith daily growth increments to examine the effects of environmental factors. Larval anchovy were caught from May to November. The range in standard length (SL) of the larval anchovy was 10–35 mm, and larvae of SL 20–25 mm dominated numerically, accounting for more than 40% of all anchovy larvae. Daily growth rates for the last five days before catch increased from May to July, decreased or were stagnant in August and September, increased again in October, and decreased in November. Seawater temperature and density of copepod nauplii were correlated with the daily growth rate. In this study, the relationship between growth rate and temperature showed a skewed dome shape, with an optimum temperature for growth of around 23 °C. These results in summer of 2008 suggest that the growth rate of Japanese anchovy increased with increasing temperature for temperatures < 23 °C, but growth was limited by prey availability and/or other factors, such as low dissolved oxygen, at higher temperatures. Recent environmental improvements in nutrient inputs to Osaka Bay are discussed in relation to larval growth rate.

Validation of Growth Layer Group (GLG) depositional rate using daily incremental growth lines in the dentin of beluga (Delphinapterus leucas (Pallas, 1776)) teeth.

Counts of Growth Layer Groups (GLGs) in the dentin of marine mammal teeth are widely used as indicators of age. In most marine mammals, observations document that GLGs are deposited yearly, but in beluga whales, some studies have supported the view that two GLGs are deposited each year. Our understanding of beluga life-history differs substantially depending on assumptions regarding the timing of GLG deposition; therefore, resolving this issue has important considerations for population assessments. In this study, we used incremental lines that represent daily pulses of dentin mineralization to test the hypothesis that GLGs in beluga dentin are deposited on a yearly basis. Our estimate of the number of daily growth lines within one GLG is remarkably close to 365 days within error, supporting the hypothesis that GLGs are deposited annually in beluga. We show that measurement of daily growth increments can be used to validate the time represented by GLGs in beluga. Furthermore, we believe this methodology may have broader applications to age estimation in other taxa.

Digital Imaging Tool to Enhance Otolith Microstructure for Estimating Age in Days in Juvenile and Adult Fish

Age estimation based on fish otolith microstructure analysis is a very repetitive and time-consuming task. The lack of appropriate image analysis software, capable of both overlaying a number of images automatically and recording a high number of daily increments, has been a significant limitation in counting and measuring daily growth increments in large otoliths from juvenile and adult fish individuals. This paper presents a new software to assist marine biologists with faster, more efficient, and more reliable microstructure readings of fish otoliths. Open source code is preferred so that software packages can be updated with new image processing algorithms developed by the scientific community. The approach consists of three steps: 1) a single grayscale digital image combining images of different parts of the same fish otolith is obtained using the blind image registration technique fast normalized cross correlation; 2) the growth rings of the image are enhanced for age estimation purposes, using the adaptive histogram equalization technique; and 3) a semiautomatic interactive tool draws a simple polygonal chain along which the microstructures are easily identifiable and the points of interest can be marked, whose data will be saved automatically. This new tool opens up the opportunity of aging juvenile and adult fish individuals at regular intervals by counting growth rings in the otolith microstructure and facilitates working with other calcified pieces of marine species that exhibit a daily ring pattern, such as cephalopod beaks and mollusk shells.

Juveniles recruitment and daily growth of the southern stock of Mugil liza (Actinopterygii; Fam. Mugilidae): new evidence for the current life-history model

Mugil liza is distributed along the western Atlantic coast. It is a commercially exploited species in Argentina, supporting a small-scale fishery conducted by an artisanal fleet. Age determination of fishes constitutes an important key issue for fishery management. The age, growth and recruitment of M. liza juveniles in Mar Chiquita coastal lagoon and Las Brusquitas creek (Buenos Aires, Argentina), were estimated by means of the analysis of the sagittal otoliths of fish collected during January to December of 2014. Ages were estimated by counting and measuring daily growth increments in otoliths under a light microscope. A total of 735 specimens ranging from 19 to 71.5 mm SL and from 67 to 212 days age was analysed. Lengths at previous ages were determined by back-calculation, a linear growth model was fitted to the back-calculated data: SL = 0.2468 + 2.0516; R2 = 0.9945. Two peaks of recruiters were observed from February to March, and from October to November in 2014. Mean ages in days of Querimana and juveniles at the recruitment time were 84.07 ± 14.43 days and 87.56 ± 19.51 days, respectively. The hatching dates of specimens showed two spawning seasons. One was from December 2013 to January 2014, and the second one from July to August 2014. The assessment carried on this work generated age determination values that support previous findings, contributing to make a more accurate description of the life-history model currently used. In addition, valuable information has been generated to give better advice for improving the management of the fishery resource.

Age determination of Loligo vulgaris and Loligo forbesii using eye lens analysis

Eye lens of 196 Loligo vulgaris and 205 Loligo forbesii collected by trawl surveys from 2013 to 2015 in Sardinian waters (Mediterranean Sea) were analysed for age determination. In an alternative to the technique previously used in Sepia officinalis and Enteroctopus megalocyatus, in this study, we proposed the elimination of decalcification and dehydration steps to reduce the time of lens processing. In addition, lenses were included in epoxy resin, which was then subject to lapping, to ensure clear reading and counting of the increments. The number of increments counted in L. vulgaris (59–520) and in L. forbesii (51–543) was strongly related to mantle length and weight of the animals, with no statistical differences between the sexes. Assuming daily growth increments, L. vulgaris was estimated to have a life span of 17 months for males and 14 months for females, while L. forbesii males had an estimated life span of 18 and 16.5 months for females. The exponential model was the best fit for growth data for both species and sexes, showing higher growth for males in comparison with females. These results preliminarily indicate that the eye lens is a valid tool for age determination of the two Loliginidae species, but it would benefit from validation by additional studies.

Analysis of shape variability and life history strategies of Illex argentinus in the northern extreme of species distribution as a tool to differentiate spawning groups

ABSTRACTThis study aimed to differentiate local and migratory spawning groups of the shortfin squid Illex argentinus caught in Brazilian waters using geometric and traditional morphometric methods and size-selective processes. The back-calculated length distributions reconstructed from daily growth increments deposited in the gladius allowed the identification of size-selective processes that may be related to different life history strategies. Landmark analysis on body shape (geometric morphometric) revealed that spawning groups presented significant ontogenetic variations in terms of body outline. In addition, traditional morphometric methods, based on multivariate analysis, associated juveniles of the expected migratory group (large size) and differentiated them from the local group individuals (small size). The changes in form, probably linked to the environmental gradients experienced by individuals throughout ontogeny, were interpreted as adaptations to improve swimming capacity. Migrant individuals have being differentiated from the smaller sizes group by body characteristics (broad fins, elongated and thicker mantle), which may increase the ability to perform long migrations.

The trade-off between rapid feather growth and impaired feather quality increases risk of predation

Feathers are used for flight, and any damage to feathers impairs efficient escape from predators. Because individuals can either rapidly produce feathers of poor quality or slowly produce feathers of high quality, prey experience a trade-off between speed of molt, quality of feathers, and risk of predation. We analyzed feathers produced by Woodpigeon Columba palumbus prey captured by Goshawks Accipiter gentilis and compared those to feathers shed during molt in the same areas. Feathers that were produced rapidly as reflected by long daily growth increments suffered from a greater degree of feather wear than feathers that were produced slowly. Prey had longer daily growth increments and a shorter period of molt than non-prey. Woodpigeons with worn feathers were more likely to fall prey to Goshawks than those with little or no wear to the plumage. These effects were independent of age, year, and time during the breeding season. These findings are consistent with a trade-off between speed of molt and feather quality affecting predation risk.

A biomineralization study of the Indo-Pacific giant clam Tridacna gigas

The giant clam, Tridacna gigas, is an important faunal component of reef ecosystems of the Indo-Pacific region. In addition to its ecological role, shells of this bivalve species are useful bioarchives for past climate and environmental reconstructions. However, the biomineralization processes involved in shell aragonite deposition are insufficiently understood. Here, we present a study of the shell microstructure of modern specimens from Palm Island, Great Barrier Reef (GBR), Australia, and Huon Peninsula, Papua New Guinea (PNG), using a combination of petrography, scanning electron microscopy, electron backscatter diffraction, Raman spectroscopy and stable carbon isotope ratios. Daily growth increments were recognizable in all specimens through ontogeny, and counting these growth lines provides a robust specimen age estimate. For the internal layers, paired increments of organized aragonitic needles and compact, oblong crystals were recognized in a specimen from PNG, whereas specimens from GBR were composed of shield-like crystals that were not definable at the microscale. The combination of nutrient availability, rainfall and solar irradiance are likely to be the most significant factors controlling shell growth and may explain the observed differences in microstructure. The external layer, identical in all specimens, was composed of dendritic microstructure that is significantly enriched in 13C compared to the internal layer, suggesting different metabolic controls on layer deposition. We propose that the mineralization of the internal and external layers is independent from each other and associated with the activity of specific mantles. Future studies using T. gigas shells as bioarchives should consider the microstructure as it reflects the environment in which the individual lived and the differences in mineralization pathways of internal and external layers.