However, this approach has fallen short of identifying migrations between regions as it is difficult to determine variations in isotopic shifts without temporal sampling of prey items. Here, we apply analysis of δ15N values of amino acids to five baleen plates belonging to three species, revealing novel insights on trophic position, metabolic state and migration between regions. Humpback and minke whales had higher reconstructed trophic levels than fin whales  as expected due to different feeding specialization. Isotopic niche areas between baleen minima and maxima were well separated, indicating regional resource use for individuals during migration that aligned with isotopic gradients in Atlantic Ocean particulate organic matter. Phenylanine δ15N values confirmed regional separation between the niche areas for two fin whales as migrations occurred and elevated glycine and threonine δ15N values suggested physiological changes due to fasting. Simultaneous resolution of trophic level and physiological changes allow for identification of regional migrations in mysticetes.Baleen from mysticete whales is a well-preserved proteinaceous material that can be used to identify migrations and feeding habits for species whose migration pathways are unknown. Analysis of δ DespiFeeding strategy and trophic ecology of mysticetes have primarily been identified through visual confirmation during feeding or stomach content analysis from strandings and historic catch data ,12. Howe13C and δ15N values, respectively) provides insights into the diet composition and habitat [13C and δ15N values by 0.5–2‰ and 0.5–5‰, respectively, compared with their diet [13C and δ15N values of resources supporting primary production shift substantially both spatially and temporally depending on the balance of biogeochemical processes affecting available inorganic and organic C and N sources within an ecosystem [15N values are observed for particulate organic matter (POM) in the higher latitudes  compared with lower mid-Atlantic areas . In the regions ,36.15N values from amino acids contained in baleen. Isotopic differences that arise due to metabolic pathway differences between amino acid types can help to address regional shifts caused by the underlying δ15N baseline by providing simultaneous temporal information on trophic level and baseline δ15N values supporting the consumer [β, the difference between trophic and source amino acids in underlying primary producers, trophic level estimates for individuals can be calculated [The issue depicted above may be resolved by the application of compound-specific analysis of δconsumer . Baselinconsumer ,39). By consumer  and β, tlculated . Trophiclculated ) and reslculated ,42.15N in amino acids from baleen sourced from stranded or bow-caught fin whales , a stranded humpback whale (Megaptera novaeangliae) and a minke whale , all opportunistically sampled in The Netherlands. We applied δ15N values of amino acids to baleen to: (i) resolve trophic levels for these species, (ii) identify changes from regional biogeochemical source δ15N values being used during migrations, and (iii) characterize any potential metabolic effects from fasting and episodic feeding during migration. This work serves as a proof of concept that amino acids from baleen can be used as a continuous ecological indicator in mysticete whales. Application and further development of this novel method to materials derived from marine mammal strandings have the potential to inform ecological knowledge about marine mammals, particularly in mysticete whales. Once established, this method will help to reconstruct life histories and further identify the ecological overlap between species. We also provide evidence of feeding in different regions due to migration between mid-Atlantic breeding and high-latitude feeding areas through application of isotopic niche areas from trophic level-corrected bulk isotope data from baleen.In this study, we are among the first to combine bulk stable isotope with analysis of δ. 2. 2.1M. novaeangliae) was acquired from Naturalis Biodiversity Center, Leiden, and the minke whale (B. acutorostrata) baleen was acquired from a private collection.Baleen plates were cut from below the gumline of dead mysticetes that stranded on the Dutch coast or were brought into Dutch harbours caught on a ship's bow in 2012 and 2013. Details about sex and estimates of animal's maturity are located in . 2.215N of amino acids was selected based on the variation within the bulk δ15N values observed for each individual and was used to target minimum and maximum values observed across the lengths of plate.One baleen plate per individual whale was air-dried (greater than 2 days), cleaned with bidistilled water, then dichloromethane, and dried at 40°C for approximately 10 h. Powdered keratin was collected using a hand drill (3 mm bit) along the leg  of the plate at either 0.5 cm or 1 cm intervals  depending on the relative size of the plate, from the gingiva along the full length of the main plate. Powdered baleen was collected and stored at −20°C until further processing. Since baleen grows continuously throughout a whale's life the material closest to the gingiva reflects the most recently produced layer with the material farther away from the gums reflecting increasingly older periods of the whale's foraging history. Subsampled material (3–5 mg) for analysis of δ. 2.313C) and nitrogen (δ15N) isotopic ratios for bulk material, as well as carbon and nitrogen content (%) of bulk biomass. Samples were analysed on a Flash 200 elemental analyser coupled to a Delta V Advantage isotope ratio mass spectrometry .Approximately 0.5–0.8 mg of dry, homogenized keratin powder was weighed into tin cups in duplicates for determination of carbon (δ13C and atmospheric N2 (air) for δ15N. A laboratory acetanilide standard with δ13C and δ15N values calibrated against NBS-22 and IAEA-N1, respectively, and known %TOC and %TN contents, was used for calibration. Analytical precision for the standards  for δ13C and δ15N analyses were 0.18‰ and 0.20‰, respectively.Stable isotope ratios are expressed using the δ notation in units per mil:. 2.4et al. [et al. [et al. [15N values for 12 amino acids including alanine (Ala), aspartic acid (Asp), Glu, Gly, isoleucine, leucine, lysine, Phe, serine, Thr, tyrosine and valine. The precision for samples and standards was less than 0.5‰ for all amino acids in standards and samples across the 13 sequences that comprise this dataset .The method is a modified version of the amino acid analysis method by Chikaraishi et al.  as descr [et al. . In shor [et al. . We repo. 2.515N values for bulk material along the length of the main plate, we fitted a generalized additive model (GAM) for each individual. GAM models were produced using the geom_smooth function in the ggplot2 package with model = ‘gam’ to apply smoothing parameters selected by data-driven methods using Akaike information criteria to time series in R (v. 4.0.0) with R Studio (v. 1.1.463) [15N values of baleen are assumed to reflect residence times in mid-Atlantic breeding grounds (minima) and high-latitude feeding grounds (maxima) with substantial differences in the underlying δ15N values for POM in these regions [13C values for individuals were less distinct, having a smaller range than those for δ15N values often due to closer similarity in prey δ13C values and are known to be further confounded due to coastal foraging in areas with gradients in δ13C [15N values were used to estimate baleen growth rates for each individual . Growth estimates were determined as the distance between sequential minimum δ15N values and this interval was used to estimate a weekly growth rate as in Busquets-Vass et al. [15N values we plotted a linear regression across δ15N values for each baleen and binned regions of each baleen into minimum (below midpoint) and maximum (above midpoint) values depending on relative position to the conditional mean to allow for further analysis of regional differences for δ15N values. Using the conditional mean to demarcate periods provided a robust and independent indicator of minimum and maximum regions, especially in baleen with less well-defined oscillations. It is more conservative than using narrowly binned regions selected in an arbitrary manner.To examine the relative rates of change for the oscillations in the δ1.1.463) ,46. The  regions . Oscilla in δ13C . Therefodividual  by assums et al. . To furt. 2.6et al. [β values appropriate to the trophic-source AA pairings or weighted averages.15NTrophic and δ15NSource are either the δ15N values for Glu and Phe or the weighted mean values for grouped trophic  and source (lysine and Phe) amino acids. Values for β, the ‰ difference between Glu and Phe or the grouped trophic and source amino acids in the underlying phytoplankton, and TDF are presented in et al. [et al. [et al. [et al. [Trophic level (TL) estimated from baleen amino acids is presented using either the individual amino acids Glu and Phe  or the wet al.  using TDn et al.  and McCa [et al. . The TDF [et al.  by rearry et al.  to allow15N using phenylalanine was calculated aset al. [15NPhe–Base values.Correction for trophic enrichment to establish baseline estimates for δtabolism  and trop [et al. . Error p13C and δ15N values for bulk measurements using the equations:13CBulk and δ15NBulk represent the C and N isotopic composition of bulk material, 2.3 ± 0.3‰ and 2.8 ± 0.2‰ represent the offset between diet and baleen for carbon and nitrogen [trophic–source for each individual  for their sample size were constructed containing 70% of the variation in each group for the binned minimum and maximum values for δ13CBase versus δ15NBase for each individual using the SIBER package [c, followed by a residual permutation and Hotelling t2-test to evaluate statistical differences [α = 0.05) using the package ‘Hotelling’.To analyse differences in isotopic niches within each individual baleen, standard ellipse areas corrected  than for the humpback whale (−19.6‰), but lower than for the minke whale . Within-individual variation in δ15N values was larger than seen for δ13C values . Oscillations in δ15N values also showed greater amplitude from 0.5 to approximately 3‰, with median values for the humpback and minke plates (12.8‰ and 12.2‰) higher than those for all three fin whale plates . δ15N values were higher for both the minke and humpback whale  than for the fin whales (9.2–10‰).The δ15N values that imply baleen growth rates of 2–3.5 mm week−1, calculated based on GAM modelling  and below (grey) the conditional mean ) with means ranging from 3 to 3.2 for the fin whales, 3.7 for the humpback whale and 3.8 for the minke whale (a). The trophic level estimates did not vary significantly  between the minimum and maximum periods examined along the baleen. Therefore, single trophic level estimates (TLTrophic–Source) were used for each individual whale to establish baseline–corrected δ13C and δ15N values ; 15N values representing the base of the food web (δ15NPhe-Base) by accounting for fractionation due to trophic level increase (equation (2.3)). Changes in the δ15NPhe–Base values reflect the differences in underlying regional N source values supporting each individual during the period when the plate was formed. The δ15NPhe–Base values for time intervals with minimum and maximum bulk δ15N values were found to be significantly different between individuals and between minimum and maximum δ15N value periods . FW3 was found to have higher δ15NPhe–Base in the maximum periods for bulk δ15N values than in the minimum periods . While for FW2 the δ15NPhe–Base values were also higher in the maximum bulk 15N period than in the minimum periods , but not statistically significant using a threshold of α = 0.05 (figure 3b).Trophic levels were estimated using the weighted means of the δke whale a. The tra,b) whose δ15N values indicate the amount of metabolic reworking occurring during metabolism [15N values of Phe were subtracted from all amino acids δ15N values to adjust for changes in baseline values . δ15N values for Ala, Asp, Glu and Ser were not significantly different between minimum and maximum periods . δ15NThr values were higher for the fin whales  with a wider range (−29.3 to −11.2‰) than for the humpback whale (−28.4 to −25.9‰) and minke whale (−32.3 to −21.6‰). The ranges for δ15NThr values in the fin (11–18‰) and minke (10.7‰) whales were considerably larger than for any of the other trophic or metabolic amino acids examined (1.4–6.2‰). δ15NGly values were significantly different between minimum and maximum periods for individuals . For all individuals, the mean δ15NGly value for the bulk minimum periods was higher than for the bulk maximum, but was only statistically higher for FW3 , although FW1  was close to being significant at a threshold of α = 0.05 (figure 4c). δ15NAsp values were higher for both the humpback and minke than for the fin whales, and for δ15NSer the humpback had higher values than all the other whales . δ15NAla and δ15NGlu values for the trophic amino acids were generally higher for the humpback and minke whales , but post hoc Tukey's indicated different relationships between individuals  with FW3 being similar to the HB for Glu.Glu and Ala are trophic amino acids a,b whosetabolism . Thr andtabolism ,42. δ15Ne values b,c. δ15N. 3.3c) were calculated by applying a Bayesian statistical model (SIBER package) to the larger dataset of trophic level corrected δ13CBase and δ15NBase values (equations (2.4) and (2.5)). The overlap between the minimum and maximum baleen periods ranged from 0  to 56% (indicating some overlap) with Hotelling's t2-test, indicating significant differences between periods for all individuals  for all individuals besides HB, where separation occurred across the x-axis (δ13CBase).Isotopic niche areas  and high-latitude feeding grounds (maxima) with substantial differences in the underlying δ15N values for POM in these regions  combined with the impacts from metabolism and excretion of N on δ15N values depending on fasting status during migrations [15N values, but rather a distinct increase in δ15N value that progressed along the length of baleen, suggesting different migration behaviour than observed for the other whales in this study. The estimates for baleen growth rates for the four individuals with recurring minima in δ15Nbulk values  within these individuals. Other studies have examined considerably longer plates [The marked oscillations in δgrations ,59. The r−1; −1, ) and bowcm yr−1, ), all ofr plates ,62.. 4.215N values along the baleen are expected to reflect the largest contrasts between food resources throughout the multi-year periods recorded in the baleen. Trophic level estimates from amino acid δ15N values indicated no significant changes in trophic levels across the baleen records for these five individuals using any of the three approaches  for the fin whales than for the humpback and minke whales (a). This is fully consistent with the fact that fin whales preferentially consume krill in areas where these are abundant and only occasionally feeding opportunistically on schools of small fish when krill is scarce [Clupea harengus) and sprat (Sprattus sprattus) [15N values along the baleen are expected to reflect the largest contrasts between food resources throughout the multi-year periods recorded in the plate. Trophic level estimates for all of the whales (ranging from 3 to 3.8) were based on a relatively small TDF (3.6‰) compared with the typically applied value of 7.6‰ used for estimation of lower trophic levels (e.g. fish and invertebrates) [Balaena mysticetes TL of 1.9–3.0) as determined through amino acid analysis and stomach contents (TL 3) in Matthews et al. [Trophic level estimates from minimum and maximum regions of δ and Phe , TDF of  and Phe  or TDF oe whales a. This is scarce ,64. The prattus) –67. Tropebrates) ,40. A TDs et al. . Smallers et al.  and appls et al. . In futu. 4.315N values for each AA [15N values of glucogenic amino acids  are expected to increase as exogenous material becomes limited and endogenous protein starts to be metabolized [15NSer or δ15NAsp (a) but δ15NGly values were statistically higher in the minimum periods for bulk δ15N values than in the maximum periods for bulk δ15N in FW3 (c). However, all five individuals had higher averages for δ15NGly values in minimum periods ranging from 0.3 to 1.4‰ and the difference for FW2 was nearly statistically significant (p = 0.06). Furthermore, patterns of significant differences between individuals for Gly and Phe did not align, probably reflecting different processes affecting the metabolic versus source amino acids. These isotopic enrichments for δ15N are lower than the observed shift (2–6‰) in glucogenic amino acids for fasting southern elephant seals  across all individuals regardless of species (c) and suggests that there is a fasting effect that occurs as fat stores are accessed during migrations toward breeding grounds when feeding becomes opportunistic.The amino acids Gly and Thr have been found to respond to fasting and protein deficiency in mammals through variable enrichments in δ each AA ,42 as caabolized . Fastingabolized  and feedabolized ,62. We o δ15NAsp a but δ15N in FW3 c. Howeve effects . Althoug species c and sug15NThr values in mammals have been observed to coincide with reduced protein quality in their diet causing reduced reverse fractionation with higher δ15NThr values indicating periods of potential starvation [15NThr are typical of marine mammals from higher trophic levels [a) that are expected for adequate protein availability throughout the period covered by the baleen examined. However, the fin whales (FW1–3) had higher δ15NThr values (−22.1 to −20.6‰) and larger ranges (11–18‰) due to higher δ15N values (−15 to −11‰) for Thr occurring intermittently throughout the baleen records. The range in δ15N values observed for Thr were 2–13× those observed for trophic  or other metabolic AAs  and Thr and Glu had different patterns (b) indicating that Thr behaves differently to the ‘canonical’ trophic amino acid. These higher values observed for Thr are potentially the result of protein deficiency and may mark protein deficiency or starvation events across an individual's lifetime. This finding conflicts with decreased δ15NThr values observed in elephant seal whiskers during fasting [15N values in the baleen and differences in δ15NThr suggest more episodic onset than the more regularly occurring fasting effects observed for δ15NGly. Given these observations, the fin whales appear to have been more regularly under food stress than either the humpback or minke whale individuals examined in this study.Higher δarvation  althoughc levels . Both hupatterns b indicat fasting , suggest. 4.415N values had distinct values for both δ15NPhe–Base (b) and the isotope niches formed using the wider dataset of δ13CBase and δ15NBase (equations (2.4) and (2.5); 15N periods probably reflect underlying isotopic differences in resource values supporting the food web between mid-Atlantic breeding grounds and high-latitude feeding grounds. Although few individuals have been tracked, all three species have been observed to make southerly winter migrations away from high-latitude feeding grounds (greater than 70°N) with North Atlantic fin whales having been observed to migrate to the Azores [13C −20 to −24‰; δ15N −1 to 1‰) and high-latitude Arctic . The δ13C and δ15N values from these regions vary depending on local biogeochemical processes  and serve as variable end members for the source amino acids incorporated into baleen as it is produced from bloodstream metabolites derived from the animal's diet. The variations in those values are likely to be dampened and never reflect the end member values from the underlying isoscape depending on the relative feeding intensity (opportunistic feeding in transit), variations in seasonal values for underlying biogeochemical processes and the relative turnover of the internal source AA pool during migration and breeding. The ranges in δ15NPhe–Base (1.9–9.9‰), δ13CBase (−20.7 to −23‰) and δ15NBase (1.5–8.5‰) values all fall within the ranges expected for dietary intake of resources sourced from regions with distinct baseline isoscape values. Significant differences were observed between minimum and maximum regions for δ15NPhe–Base that did not align with those observed for δ15NGly–Phe or δ15NGly values, indicating that Phe fractionated differently to Gly in this dataset. Therefore, we have assumed that the fractionation associated with Phe is primarily due to changes in underlying biogeochemical values with minimal impacts from fasting, although other studies have observed considerable metabolic impacts [Minimum and maximum periods in baleen bulk δPhe–Base b and thee Azores , humpbace Azores  and minke Azores . These le Azores ,35, char impacts .15NPhe and δ15NBase  versus δ13CBase (2.3‰) are probably due to the large amounts of lipid stores that are primarily developed with time spent on feeding grounds [13C value that reflects the fractionation of C from food resources containing the regional δ15N values where they were consumed [13CBase values (hysteresis) of the baleen during fasting conditions as metabolites from blood are incorporated into baleen. Use of C from lipid stores contributes to the dampening of variation in C values along the baleen, and although the metabolic N pool in whales is quite large, there is no comparable storage pool for N. Therefore, N from incidental feeding is expected to be more directly metabolized into animal tissues, while carbon from lipid-rich prey can be metabolically routed to either direct incorporation to tissue or to storage within large lipid stores depending on feeding status [15NBase values (range of minima from 1.5 to 5‰) never reach the expected low isoscape values for the mid-Atlantic of approximately −1 to 1‰ as the lower concentrations of N from opportunistic feeding may be insufficient to fully overcome the comparatively high δ15N fed on extensively at higher latitude feeding grounds (6–10‰). Alternatively, higher than expected δ15NBase values may reflect poorly constrained TDFs and should be considered in future work aiming to anchor δ15NBase values for marine mammals to biogeochemical isoscapes.The larger ranges observed for δ grounds . Under fconsumed . These lg status . δ15NBas15NBase axis . The combination of (i) no baseline shift for PheBase and (ii) no difference for δ15NGly values between minimum and maximum periods indicating fasting effects, support the hypothesis of higher trophic level predation within the same environment  across time. High δ15NGly values observed for the humpback  also mirror δ15NGly values found in small fish migrating from estuarine into coastal waters where elevated δ15NGly values have been previously observed, such as herring [Differentiation between periods of migration is supported in four individuals , with clear separation between niche areas for basal resources that predominantly separate along the δgrations , or a regrations . The gra herring .15NPhe–Base and δ15NGly, were males, and the remaining three individuals were female. Females are likely to display different isotopic patterns for both C and N as they reproduce, as the result of gestation and lactation altering the partitioning of resources and resultant isotopic values [In the above discussions, it should be noted that individuals FW1 and FW3 that have statistically relevant differences between δc values . These ec values ,79. Seasc values ,35, espe. 515N values further confirm distinct differences between maximum periods that reflect feeding in high-latitude feeding grounds and minimum periods that reflect resources used from mid-Atlantic breeding grounds for these individuals. δ15N values from Gly and Thr provided useful metabolic indicators across the baleen sequences. Gly had higher values that aligned with considerable time spent in breeding grounds where feeding is expected to become incidental and fasting is likely to occur. Differences in Thr occurred more episodically and indicate that food stress or starvation occurred more often in the fin whales in this study relative to the other whales examined. Analysis of δ15N values for amino acids provided further context into the movements and metabolic conditions for mysticete whales, information that is especially important in wide ranging, difficult to track, animals with threatened conservation status.This study used isotope analysis of bulk material and amino acids to provide metabolic and trophic context to the isotopic oscillations observed within whale baleen. Trophic level estimates from source and trophic amino acids were higher for the humpback and minke whales than for the fin whales, which corresponds to previous observations. Trophic level estimates using amino acids allow for the correction of bulk stable isotope values from consumers to the underlying baseline values of the primary producers supporting regional foodwebs. Isotopic niche areas constructed from these baseline values using periods of minimum and maximum bulk δClick here for additional data file.


Globally, mangroves are increasingly impacted by human and natural disturbances under climate warming, including pervasive pulsing tropical cyclones. However, there is limited information assessing cyclone’s functional role in regulating wetlands carbon cycling from annual to decadal scales. Here we show how cyclones with a wide range of integrated kinetic energy (IKE) impact C fluxes in the Everglades, a neotropical region with high cyclone landing frequency. Using long-term mangrove Net Primary Productivity  data (2001–2018), we estimated cyclone-induced litterfall particulate organic C (litter-POC) export from mangroves to estuarine waters. Our analysis revealed that this lateral litter-POC flux (71–205 g C m−2 year−1)—currently unaccounted in global C budgets—is similar to C burial rates (69–157 g C m−2 year−1) and dissolved inorganic carbon  export. We proposed a statistical model (PULITER) between IKE-based pulse index and NPPL to determine cyclone’s impact on mangrove role as C sink or source. Including the cyclone’s functional role in regulating mangrove C fluxes is critical to developing local and regional climate change mitigation plans.Mangroves are the most blue-carbon rich coastal wetlands contributing to the reduction of atmospheric CO This recognition is associated with the urgency to develop cost-effective mitigation measures to ameliorate climate change and variability, which are manifested by the increasing frequency of extreme events including excess precipitation and tropical cyclones6. Among blue carbon ecosystems, mangrove wetlands are the most productive8 and can significantly reduce carbon dioxide (CO2) in the atmosphere throughout photosynthesis  and the accumulation of organic matter in the soil 10. This accumulation is typically promoted by the pervasive anaerobic conditions—over thousands of years of geomorphological evolution—where mangroves are established due to regular and intermittent flooding occurring in a wide variety of coastal geomorphic settings across latitudinal gradients and climates16.Blue carbon—the organic carbon sequestered and stored in coastal and marine ecosystems—is the most recognized ecosystem service worldwide8 with a conspicuous carbon storage capacity, despite their limited extension and geographic distribution18 when compared to other blue carbon  and terrestrial  ecosystems10. It is estimated that mangrove wetlands globally store, in vegetation (above- and belowground) and soil compartments, approximately 4.19–6.17 Pg C19. During the last 10 years, this assessment has improved as more field data are acquired and through remote sensing tools at locations underrepresented in early attempts to produce a global mangrove carbon budget21. However, this value still has large uncertainties at the global scale, as unaccounted carbon budget can range from ~ 104–112 Tg C year−123. Further improvements in this budget estimate are needed to implement feasible local and regional blue carbon conservation programs within the context of regional carbon markets, especially in countries situated in the Neotropics, Indo-Malayan, and Australasian ecoregions where most of the mangrove area is located25. This assessment is even more critical when attempting to assign an economic value to blue carbon as an ecosystem service given the major differences in mangroves physiognomic types 26, climate differences , water availability, and spatial distribution within a wide range of geomorphic settings 13.Indeed, mangrove wetlands are among “the most carbon-rich” ecosystems28. One major challenge in estimating the net atmospheric carbon emission and capture in mangroves is the diversity of organic  and inorganic  carbon (C) compounds that enter or exit coastal systems29. In contrast to terrestrial forests, mangrove productivity is not only regulated by nutrient availability that controls CO2 exchange (photosynthesis and respiration) between the forest and the atmosphere above the canopy , but also by hydrology affecting local hydroperiod32.In contrast to current global mangrove carbon storage estimates, carbon sequestration by both photosynthesis and soil accretion has been more elusive, especially where mangroves are undergoing severe and recurrent natural  and human disturbances 33. Yet, this lateral carbon flux magnitude is uncertain. Previous studies proposed that > 50% of the carbon fixed by mangrove vegetation is unaccounted 23 after counting potential carbon sinks . This finding implies that in current mangrove carbon budgets, mineralization  is significantly underestimated and therefore hypothesized that most of the carbon exported from mangroves to adjacent waters occurs as DIC22. Although few studies have been performed globally across different geomorphic settings and mangrove ecotypes to test that hypothesis, recent studies measuring DIC40, in addition to CO2 and methane (CH4) fluxes49, have partially confirmed the role of DIC, thus advancing our current understanding of the relative significance of these fluxes in regional carbon fluxes and budgets.Hydroperiod—the frequency, duration, and depth of inundation—controls the lateral exchange of materials between mangrove wetlands and adjacent coastal waters, including the net exchange of both inorganic and organic carbon22 in global mangrove budgets since it represents a knowledge gap when accounting for the fate of the forest total Net Primary Productivity . This flux is apparent  since most cyclones are usually considered as major destructive, episodic events characterized by the Saffir-Simpson Hurricane Wind Scale 51. Hereafter, we used the term cyclone as equivalent to tropical cyclone that includes hurricanes , major hurricanes  and tropical storms (TS). These pulsing events can cause high tree mortality and defoliation60 triggering subsequent long-term post-cyclone forest recovery in periods lasting from 5 to 10 years65. Moreover, cyclones with less destructive power can also impact forest structural and functional properties by regulating litterfall fluxes affecting organic matter accumulation in the soil in the short- or even long-term67  Fig. , caused 71. We developed a robust statistical model (henceforth PULITER) directly relating cyclone strength to the magnitude of “pulsing” litter deposition  using the cyclone integrated kinetic energy (IKE) as an efficient metric of the destruction potential based on wind-field size and magnitude reported in energy units (from kilo- to terajoules)72. We hypothesized that the IKE is an efficient proxy to determine the magnitude of the “pulsing” excess litterfall net primary production  at different cyclone energies.The main objective of this study was to evaluate the functional role of cyclones in influencing the seasonal and interannual variation of litterfall organic carbon fluxes between the mangrove forest and adjacent coastal waters in the Shark River Estuary (SRE), South Florida. This subtropical estuary is in the northern Gulf of Mexico where cyclone landing frequency is high and with a wide range in energy contentThe IKE differs from the SSHWS classification since the latter is based solely on the sustained wind speed on a scale of categories ranging from Cat-1 to Cat-5. In contrast, the IKE is a practical energy-based integrative measure of the destructive power at different spatiotemporal scales. The IKE can separate potential wind impacts based on cyclone trajectory while retaining the concise range of the SSHWS. To quantify the cyclone strength, we defined a new indicator—a pulse index ; we also evaluated the seasonal and interannual litterfall Net Primary Productivity (NPPL) variability during the presence and absence of cyclones of different strengths73. Using this information, we propose first-order estimates of the cyclone-induced EXS portion  that is exported to the adjacent estuary caused by the storm surge. Although NPPL is one of the most extensive and well-documented organic carbon fluxes from the canopy to the soil in mangrove forests78, it is unknown how much of this flux contributes to the POC lateral exchange between the mangrove wetland and adjacent coastal waters before and after a cyclone impact. Finally, we compare these litter-POC export rates to other published DIC and DOC exchange rates measured in the same study sites to evaluate the overall carbon lateral flux as a result of cyclone impacts in the SRE and their significance to develop global mangrove carbon budgets.Previously we have measured litterfall rates and their contribution to the NPP79.Historically, twenty-eight cyclones made landfall or followed a parallel trajectory to the Florida coastline while passing within 300-km distance from our study sites in the 1990–2018 period  after Wilma (2005) and in 2018 after Irma  impacts.The litterfall rate in the period 2001–2003, when no cyclones caused defoliation, was defined as the NPPar−1 see . Althougar−1 see , they molue Fig. , ranged −2 per cyclone (N = 8) in the period from 2004 (Charley) to 2017 (Irma) . The REXS values of other relatively low-energy cyclones ranged from 3.9 to 33.7%  values: one Cat-4 hurricane ; two Cat-3 hurricanes , four Cat-1 or -2 hurricanes  and one TS  Fig. . OverallT), the values ranged from low values  to 140.3 TJ (Irma) (Table T: 116.6–121.0 TJ) were the most powerful cyclones followed by Frances (81.4 TJ), Jeanne (63.1 TJ) and Dennis (53.1 TJ). Interestingly, TS Isaac IKET (41.1 TJ) was more than three times over the value estimated for Hurricanes Katrina and Charley (9.7–15.5 TJ). Overall, the sub-quadrant IKE values of two selected quadrants (IKEQ) exhibited similar patterns as the IKET but with lower magnitudes , defined as the cumulative energy of a cyclone during a timespan  to 28.33 (Irma) TJ km−1 (Table Q) was lower , moderate  and strong .The total Pulsing Index  and PIQ (Model 2)  was significant and high, with minor differences between Model 1  and Model 2   Fig. . The coeT (Model 1A) and PIQ (Model 2A) as independent variables  and 0.42 . This lower R2 is attributed to the highly variable REXS value registered among mangrove sites from Katrina where the impact was different among each study site due to the interaction between cyclone trajectory and speed  and Irma 140.3 TJ), which were strong cyclones , 131.1 ± 12.8 g m−2 (SRS-5), and 129.0 ± 15.0 g m−2 (SRS-4) .Using the cyclone-induced excess litter (EXS) values Table , we dete−2 year−1), followed by SRS-5 (84 ± 11 g C m−2 year−1), and SRS-4 (71 ± 5 g C m−2 year−1); these rates included the litterfall material exported through both tides and storm surge over the eighteen-year study period  help partition the potential energy of wind fields across the landscape for mangrove forests in a post-disturbance analysis. Thus, IKE is a core component to define the pulse index (PI) that reflects the cumulative energy during a given period and distance (TJ km−1) from the mangrove forests.Our analysis showed that the IKE is a valuable metric to operationally assess cyclone impacts with different energy content on mangrove forest structure, and to quantitatively evaluate the cumulative impacts on functional properties including NPPEXS, the temporal litterfall anomaly caused by a cyclone, see 23  for mangrove forests in the SRE driven by both tides and cyclone storm surges was comparable to current estimates of soil carbon burial (69–157 g C m−2 year−1)81 and dissolved inorganic carbon (DIC) export (61–229 g C m−2 year−1)83 , and found that these relationships have a weak association accounting for only a small portion of the total variance .Although several linear and non-linear models have been developed to assess mangrove forest structural damage  after cyclone impacts using one or two meteorological variables EXS Fig. . In cont86. The PI value used in the PULITER model defines this interaction directly and at a finer spatial scale by using either the total energy of the four quadrants (PIT) or the energy of two quadrants (PIQ) 89 despite having PIT ~ 3.5 × higher cumulative kinetic energy gy Table .Another example of the complex and wide range of cyclone impacts based on the interaction among cyclone trajectory, landing location, distance from study sites, and quadrant-energy content is the case of Cat-2 Hurricanes Frances and Jeanne in 2004 Table . Both cyEXS values are determined by the initial forest structure, particularly tree height and density, since taller trees are more susceptible to damage by variable wind speed94.The timespan  and SRS-4 (3.85 TJ km−1) and slightly higher at SRS-6 (4.36 TJ km−1); however, the REXS at SRS-5 (5.5%) was lower than values at SRS-6 (22.8%) and SRS-4 (33.7%). This comparative analysis among cyclones highlights how a cyclone trajectory and distance can have different impacts on forest canopy, especially considering the distinct forest structures  along the estuary65. Although Katrina-induced EXS at SRS-5 is not significantly different from zero   (Table EXS = 58.9%) across all mangrove sites. Based on these values, we estimated that > 80% of the litterfall was exported from the forest in the case of Wilma   Fig. . In that2) Table , indicat74. Currently, this wide range does not explicitly include the contribution of cyclones impact and it is partially explained by the strong interactions of a number of environmental process and variables including geomorphology, hydrology 100, the relative dominance of different ecotypes within a given geomorphic setting26, and local hydroperiod101. Since the actual tidal range in mangrove wetlands is also partially dependent on the wetland relative elevation and net freshwater discharge102, the tidal exchange between the wetland and the adjacent coastal water can vary even within the same geomorphic setting.Globally, the estimated percentage of litter material exported from mangrove forests varies widely 74. The litter-POC flux in those Florida mangrove sites ranged from 186–438 g C m−2 year−1 representing up to 75–93% of NPPL. In contrast, our estimated litter-POC export is a relatively conservative value as we assumed 20% or 40% of the litter is exported to coastal waters under normal tidal cycles and weak cyclone-induced storm surge 74 . As a result, the EXS export from the forest is generally > 40–80% of the observed EXS  under other low-energy cyclones (Table −2 year−1 over the period 2001–2018 (N = 18 years) or 18–67 g C m−2 per cyclone (N = 8 cyclones).The extreme condition when assessing EXS values is represented by a destructive cyclone impact that results in complete defoliation, tree snapping, and uprootingEXS Fig.  represenes Table ; in term−1; 3 × the average pre-2006 rate, 3 ± 2 mm year−1)104. It is expected that this rate will continue to increase because of climate change causing significant alterations in regional hydrology and local hydroperiod in our mangrove sites101. Due to the location of each sampling site along the estuary  and export must be considered in the long term Fig. , not onl23. This absence is partially due to the difficulty in evaluating cyclones’ impact at different spatiotemporal scales and the lack of long-term data before and after cyclone impact. Despite some limitations due to the number of cyclones with different IKE values, we were able to determine their impact on litterfall —one of the major contributors to ecosystem NPPT77. The availability of a long-term litterfall record since 200173 allowed us to capture the role of cyclone impact under different intensities. In addition to the lack of inclusion of this flux (EXS) in carbon budgets, there is a large uncertainty in other carbon flux estimates since most of the data used to construct carbon budgets is from different sites106. This approach is currently used since there are not enough mangrove sites with key information about carbon stocks and fluxes needed to implement a meta-analysis across different regions and climates23. Consequently, regardless of the accuracy and precision of each flux and stock data, current budgets are confounded by the intrinsic spatiotemporal variability given the differences in biogeochemical cycling among mangrove ecotypes with different spatial extent and distribution within a range of geomorphic settings107.Although several studies emphasize the importance of tropical cyclones on carbon cycling in mangrove forests, this carbon flux is not explicitly included in current mangrove carbon budgets108, early estimates using the eddy covariance method and a mass balance approach showed that in Shark River’s riverine mangrove (SRS-6), the Net Ecosystem Carbon Balance (NECB) was 1038 ± 88 g C m−2 year−1 while the Net Ecosystem Exchange of CO2 with the atmosphere (-NEE) was 1170 ± 127 g C m−2 year−1109. Specifically in that mass balance calculation109, the overall net carbon lateral flux (FTOT = -131 ± 155 g C m−2 year−1) was first estimated for the SRE by difference between those two fluxes . The FTOT value calculation a priori included different carbon forms  and volatile organic carbon) and then compared to available direct measurements of selected carbon forms  to constraint both the flux magnitude and uncertainty. However, partitioning the net carbon lateral flux  for validation purposes was difficult since in situ measurements for several carbon forms are not available for the SRE106.In the frequently cyclone-impacted Everglades karstic environment−2 year−1)110 and POC values obtained in other locations in South Florida (64–186 g C m−2 year−1)111. This POC flux, for instance, compares to our estimated litter-POC flux range . In the case of DIC, recent seasonal tracer release experiments have identified a range from 61–229 g C m−2 year−1 along the SRE83. The total sum of DOC + POC + DIC (Table −2 year−1) using these field-based estimates is higher than the mass balance based on the FTOT value  109) in previous studies. Interestingly, the range of field-based DIC measurements is much lower than the range used in other studies  when attempting to determine the fate of the NPPT using both the mass balance and eddy covariance methods44. Additionally, in situ soil CO2 efflux measurements at SRS-6 show that soil respiration over a range of flooding stages is 351 g C m−2 year−1  in current global carbon budgets. In the case of the SRE, we found that the DIC flux represented 7–34% of the total NPPT while POC accounted for 10–24%; these fluxes together represent 17–58% of the total NPPT (Table −2 year−1) is lower than that included in the global POC range (137–203 g C m−2 year−1)112 while our high estimate (205 ± 23 g C m−2 year−1) is closer to the maximum value in that range. This first-order calculation suggests that our POC flux, caused by the pulse cyclones with variable energy, can explain up to 24% of the missing carbon in this karstic coastal environment. This information can help constraint regional carbon budgets in neotropical mangrove wetlands impacted by increasing seasonal cyclone activity114.As mentioned, previous studies have hypothesized that DIC export might account for a large proportion of the “missing carbon” . Because of the inability to presently account for the “blown away” POC portion caused by extreme wind velocity, we consider our values as conservative estimates65 .When partitioning the NPP115—as is the case of South Florida. Presently, we do not know the magnitude of this accumulation and spatial distribution along the SRE and coastal waters  in Florida’s karstic environment also plays a major role in the export of dissolved inorganic/organic carbon and inorganic nutrient to the coastal zone requiring more field-based measurements in this region121.It is expected that in mangrove-dominated coastal areas impacted by cyclones, this large litter mass transported by strong winds and storm surge might end accumulating in different stages of decomposition at the bottom of shallow waters in creeks and embayment of deltaic or karstic coastsRhizophoramangle is more susceptible to wind damage than Avicenniagerminans63. Indeed, wind velocity has different effects on vegetation depending on the variable leaf angle, canopy geometry and canopy height spatial distribution124 that can directly influence the magnitude of EXS values. The difference in species composition along with species-specific resistance to wind impact from the same cyclone could explain litterfall differences among sites.Mangrove plant adaptations are manifested on the species-specific response to major disturbances including resistance to windstorm. It is reported that 127. For instance, Lagunculariaracemosa dominance in forest stands located close to the SRE mouth  highlights the recurrent impact of cyclones where this species growth rate and reproductive output are enhanced when light availability increase after defoliation or tree mortality causes large forest gaps128. This species dominance is further enhanced when soil P limitation is offset by the landscape-level inorganic P inputs by sediment deposition driven by storm surges89. As a result of this P deposition, mangrove forests in the SRE recovered quickly within five years after Wilma’s impact, which caused > 90% defoliation and low cumulative tree mortality (< 16%) post-disturbance62. Under this scenario, e.g., Wilma’s impact62, trees are expected to allocate more energy to foliage production for expedited refoliation132, hence potentially arresting stem growth  and development134 that can alter both carbon sequestration and storage rates in the short (1–3 years) and mid-term (decades) periods.Although few studies have directly measured mangrove canopy trait responses to wind impacts, modeling studies have explicitly simulated forest canopy geometry, demonstrating the importance of crown geometry when trees compete for resources  before and after cyclone impact135. As previously reported20, mangrove tree height and aboveground carbon stocks in cyclone-prone regions are globally lower than those in coastal areas not impacted by tropical cyclones. This difference in carbon allocation at the regional scale (> 10 km2) has major implications in the amount of carbon storage and net carbon export in the long term106. This geographic and latitudinal distinction is needed when determining—not only current and future blue carbon budgets per ecoregion and country— but also their economic value in climate change mitigation plans as carbon markets are developed and implemented within the next decade69.Finally, to assess the relative role of mangrove forests as carbon sinks or sources at the global scale, and comparatively to other blue-carbon wetlands, we recommend not only describing the site geographical features but also its ecogeomorphic setting, mangrove ecotype(s) spatial extension—and critically—the frequency of cyclone impacts along the coastline136, three mangrove study sites were established along Shark River Estuary (SRE) with different distance from the mouth of the estuary: 4.1 km (SRS-6), 9.9 km (SRS-5) and 18.2 km (SRS-4) , soil nutrient  and stressors 137. Tree height decreases (~ 18 to < 6 m) from downstream (SRS-6) to upstream (SRS-4)138; basal area ranges from ~ 40–20 m2 ha−1 and tree density increases (2838–7746 trees ha−1) from downstream to mid- (SRS-5) and upstream estuarine regions137. Two 20 m × 20 m permanent plots were established at each site and five wooden baskets (N = 10) were placed at random locations in each plot following standard sampling litterfall techniques106. The baskets are 0.5 m × 0.5 m and lined with 1- mm meshed screen and standing at ~ 1.5 m height above the forest floor. Monthly litterfall samples were collected in paper bags and transferred to the lab for further analysis. Litterfall samples were oven-dried at 60 °C for 72 h and then sorted into different components, including leaves, reproductive materials  per species, and twigs/wood. The dry weight was recorded within 0.01 g for each component73. A conversion factor of 0.44 was used to convert dry mass (g m−2) to carbon content (g C m−2)140.The Everglades mangrove wetlands are in the southern region of the Florida Peninsula, Florida (USA). This peninsula intersects the trajectory of highly frequent tropical cyclones in the Gulf of Mexico Fig. . As part71. In extreme cases, they can cross the Florida Peninsula or pass in parallel to the coastline within one to hundreds of kilometers141. In this study, we identify potential impact over our study sites, by first identifying cyclones registered from 2001 through 2018 (https://coast.noaa.gov/hurricanes), and then selecting events that could impact our mangrove study sites within the period when Litterfall Net Primary Productivity (NPPL) data was available. Thus, we delimited a circular area with the center reference point established at the midstream study site  with a 300-km radius ; V is the volume of the storm domain. For each cyclone, we calculated the IKE for each quadrant. The sum of IKE for these four quadrants becomes the total IKE (IKET). We also identified two quadrants that the study sites were inside or close to, and then defined the sum of IKE for these two quadrants as sub-quadrant IKE value (IKEQ).Tropical cyclone structure is complex due to the compound effect of variable components including wind speed, circulation size, trajectory, and translation motion. Integrated kinetic energy (IKE)https://www.aoml.noaa.gov/hrd/ike/Calculator_AllQuad.php). Because the historical dataset was reported in six-hour intervals, we performed a linear interpolation to estimate IKE values for short intervals (1–3 h) to be consistent with the track records (https://www.nhc.noaa.gov/data/hurdat/hurdat2-1851-2019-052520.txt).We identified times when the cyclones moved near to the study sites within 300-km along the track and calculated the quadrant and total IKE using an on-line tool  using IKET and sub-quadrant PI (PIQ) using IKEQ, respectively.The sum of L anomaly over the study period (2001–2018) to identify years with frequent cyclone impacts. Given the minimum cyclone impacts on SRE mangrove sites in the period 2001–2003, the average NPPL over these three years was considered as baseline value to estimate the anomaly . The magnitude of this anomaly can be above  or below  the baseline; the concept is equivalent to the air temperature anomaly used in climate change studies142.We assessed the annual NPPy Table. . The annEXS as the ratio of the EXS value to the basket-based annual baseline . The REXS value normalizes the differences for each cyclone and facilitates its comparison across all sites . The value was calculated using the difference between the amount of the first samples collected after cyclone impact and its corresponding monthly baseline per basket. We defined the term RL estimates and the degree of canopy defoliation at each study site after Wilma’s impact in 2005, Danielson et al.62 used the SRS-4 excess litterfall value as a baseline to estimate how much litterfall in the baskets was removed due to the storm surge and high-speed wind impact on the other two sites (SRS-5 and SRS-6) where defoliation was > 50–90%. We followed Danielson et al.’s approach to calculate the EXS and REXS values for cyclone Wilma (2005) in all study sites  indicating that most of the canopy material was exported. Although canopy defoliation at SRS-5 and SRS-4 was less evident compared to SRS-6, still excess litter produced by high winds was deposited in the litter baskets. Because only at SRS-6 partial litterfall in baskets were removed, we used a linear regression between observed REXS values at SRS-5 and SRS-4 and the distances from each study site to the SRE mouth to determine the potential REXS value at SRS-6  before export and removal by Irma’s storm surge.Using annual NPPes Table . This ex-6 Table . We then143. Due to the lack of direct measurements assessing the lateral flux at the boundary between the forest and adjacent coastal waters along the SRE during a tidal cycle, we used previously estimated export rates based on a soil organic matter and nutrient accumulation modeling study performed for the same study sites. This study considered the site-specific influence of hydroperiod, topography, and consumers activity144. These export rates assumed a site-specific percentage removal of soil litter accumulated on the soil surface by tidal exchange . We also used these rates to estimate the litter exported by the storm surge caused by weak and moderate cyclones. In the case of strong cyclones (Wilma and Irma), we observed litter export based on the material collected in the baskets at SRS-5 and SRS-6 during Wilma and only at SRS-6 in the case of Irma. Thus, litter export rates in those cases were estimated by the difference between the litter collected in the basket and the “reconstructed” total litter before being exported or removed by the storm surge (see above). Given the complex dynamics in assessing these export rates, these values are considered first-order estimations where 20% of the litterfall is assumed to be exported from the forest ground at SRS-4 during Wilma; this percentage was also used for the sites SRS-4 and SRS-5 in the case of Irma’s impact. Overall, we consider these export values as underestimates since the rates do not explicitly include the canopy materials “blown away” by high winds during the cyclone approach . Before the regression analyses, the normality of residuals was tested by the Shapiro–Wilk test (α = 0.05). We also performed linear regressions (α = 0.05) between REXS and other cyclone properties . All statistical analysis and graphs were performed using JMP Pro 15.2.1 .Since the EXS value represents excess litter caused by cyclones, we conducted one-sample upper tail Student’s t-test to determine which cyclone deposited excess litter  that was significantly greater than zero . Regression analyses (α = 0.05) were performed to establish the relationship between RSupplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Tables.


This study aimed to (a) assess if the combination of techniques yields a more precise assessment of past dietary and subsistence practices, and (b) contribute to our understanding of the dietary patterns of the prehistoric Hungarian populations. Overall, no correlations between microwear and δ13C and δ15N isotope variables were observed, except for a relationship between nitrogen and the vertical and horizontal index. However, we found that diachronic differences are influenced by the variation within the period. Particularly, we found differences in microwear and isotope variables between Middle Neolithic sites, indicating that there were different dietary practices among those populations. Additionally, microwear results suggest no changes in the abrasiveness of the diet, neither food processing methods, despite higher C4 plant resource consumption shown by carbon isotopic signal. Thus, we demonstrate that the integration of dental microwear and carbon and nitrogen stable isotope methodologies can provide complementary information for making inferences about paleodietary habits.Dietary reconstruction is used to make inferences about the subsistence strategies of ancient human populations, but it may also serve as a proxy to characterise their diverse cultural and technological manifestations. Dental microwear and stable isotope analyses have been shown to be successful techniques for paleodietary reconstruction of ancient populations but, despite yielding complementary dietary information, these techniques have rarely been combined within the same study. Here we present for the first time a comprehensive approach to interpreting ancient lifeways through the results of buccal and occlusal microwear, and δ The adoption of an agriculture and/or a pastoral lifestyle is one of the most important events in human history, resulting in significant biological, environmental, cultural and health changes6. By reconstructing the diet of these transitional populations, we can better understand the changes these societies experienced at different levels, including subsistence strategies, landscape use, health, and/or social stratification12.The transitions between the Neolithic, Copper, and Bronze Ages constitute key periods of important social changes in the configuration of European societies that included the creation of hierarchies and the establishment of agriculture14 , a series of changes in settlement patterns and subsistence strategies occurred from the Neolithic through the Copper Age and into the Bronze Age35, but these studies are now most often being complemented by a variety of bioarchaeological and molecular approaches41. Dental microwear and stable isotope analyses have been largely used to infer dietary practices in Holocene populations47. Dental microwear studies on buccal and occlusal surfaces of teeth provide insight into the physical properties of the food ingested by humans and other animals by yielding information about the abrasiveness of the food coupled with how these foods were processed prior to consumption53. During chewing, pits of different sizes and striations of different lengths and orientations are formed across enamel surfaces caused by particles harder than enamel, such as plant phytoliths, grit, or quartz dust 57. As such, distinct dietary habits and food processing methods result in different microwear patterns, allowing for the distinction, for example, between individuals who ingested foodstuffs obtained through different subsistence practices, such as foraging and agriculture, or between farming and pastoralism59.Dietary patterns and subsistence practices of prehistoric populations have long been inferred from cultural and archaeological remains62. The isotopic signature in archaeological human tissues differs from resources consumed and consumers in a predictable isotopic fractionation, with a generally accepted stepwise increase of 0–2‰ for carbon63 and 3–5‰ for nitrogen isotopic signatures66. Carbon (δ13C) and nitrogen (δ15N) isotope ratios measured from bone collagen of archaeological samples are indicative of the protein consumed by the individual about 10–15 years prior to death67. In brief, δ13C is primarily used to identify the consumption of plants that use different metabolic pathways for fixing carbon during photosynthesis and the animals raised on them. These δ13C differences can then be used to identify the consumption of C3 versus C4 terrestrial resources in past diets70. Values of carbon ratios may also inform about the consumption of marine foods74 and freshwater resources77.Carbon and nitrogen stable isotope analyses, in turn, are based on the principle that the biochemical composition of the food consumed by animals and humans is preserved in their body tissues15N in bone collagen are primarily used to analyse the relative presence of plant versus animal protein in the diet78. It is based on the observed increase of δ15N in tissues with the increase of trophic level79. Nitrogen isotope signals are also used to distinguish between the consumption of terrestrial foods and marine and freshwater resources75. Other environmental factors can also influence δ15N in tissues, such as the consumption of fertilized crops81, the health of the individual82, nursing practices83, and/or other environmental factors86.In addition to carbon isotope signals, values of δ90. Still, some research has been done in this line90, showing interesting results of the combination of buccal dental microwear and stable isotopes and enhancing the importance of integrating both methods. Within dental microwear, analyses combining buccal and occlusal surfaces are also becoming more popular92 due to differences in indentation enamel processes, which can provide dietary information over both short and long time scales96. The central aim of this study is to assess whether the combined use of dental microwear and isotope data provide a better understanding of the dietary patterns and the types of food consumed by past Hungarian populations. Remarkably, this study takes a step further, not only by including information from the occlusal surface too, but also and unlike previous studies89, by employing the same individuals for all the data obtained. The integration of the data obtained from these three proxies contributes for a dietary approximation with a more fine-grained approach.Both approaches offer different insights into dietary reconstruction, and although they are complementary, their combined use for paleodietary reconstruction is rareHere, we contribute to a better understanding of the dietary practices of people in the GHP from the Middle Neolithic to the Late Bronze Age by studying their diets at different levels: (a) at the individual level, by comparing both techniques in the same individual; (b) at the diachronic level, by observing potential differences between chronologies; and (c) at the regional level, by comparing contemporaneous sites.Archaeological human skeletal samples of both sexes from a wide range of ages at death, spanning from the Middle Neolithic to the Late Bronze Age, along with faunal material, were sampled from 17 sites across the GHP Fig. . Postcra98, there was a great amount of teeth in which dental microwear could not be observed for different reasons (e.g. enamel preservation). This fact made that small sample size be a typical unavoidable reality. From a total number of 65 individuals, buccal and occlusal well preserved microwear were obtained in 49 human posterior teeth from different individuals of both sexes and various ages. Teeth present different post-depositional modification, from no detectable to severe modification. Although, experimental studies have shown that post-mortem damage could be discerned from ante-mortem microwear100, teeth with severe evidence of post-mortem damage were excluded from the analysis. In the case of occlusal surface, enamel wear related with abrasive diets or the effect of age, affect directly to this surface and obscure dental microwear as wear progresses93. Lower molars were preferred as sample size was higher. Nevertheless, when dental microwear was not possible to be observed in the lower molars, upper molars from the same individual were chosen instead. Carbon and nitrogen stable isotope analysis was carried out on collagen from bone samples of 89 human individuals from both sexes and various ages, along with 29 faunal bone samples  from different adult individuals, while 34 molars from different adult individuals were obtained from occlusal microwear Table  and S2. es Table  and S10.15N, which was correlated with the vertical (NV/BTN) and horizontal index (NH/BTN) in buccal microwear.Within individuals, results from buccal and occlusal microwear analysis were compared to stable isotope data. Spearman correlation results indicated that the majority of microwear variables and stable isotope ratios were not statistically correlated Table  and S2. 2 = 13.81, p = 0.007) and their length   Fig. .Table 4S2 = 6.44, p = 0.039). We also found differences in the horizontal  and vertical indexes  between the Bükkábrány-Bánya XI/A and Bükkábrány-Bánya XII/B sites  could be analysed, as only those sites with n ≥ 3 ], and a range of δ15N of 5.5–11.0‰ (mean = 7.9‰ ± 1.4‰ (1σ)), similar to those previously reported in the area103. Significant δ15N values are found among domesticates , and particularly pigs show higher enriched δ15N values than sheep/goats . High δ15N values are also seen in the GHP site of Polgár-Ferenci-hát104, suggesting they might eat a fair amount of meat and most likely being fed human food scraps.The local terrestrial fauna analysed ]. The human sample HUNG896 had an unusually low δ15N value , as seen in different physiological conditions  in soft tissues (i.e. hair)82. Although it still remains uncertain the detection of physiological stress in archaeological skeletal remains82, presumably this individual might have suffer some kind of physiological stress occurred during a long term (as it is reflected in bone collagen), or at least episodes of nutritionally deficient diets during early years as suggested by the presence of linear enamel hypoplasia in the anterior teeth107. That is why we decided to remove this individual from statistical analyses. Some infant individuals showed high δ15N values (HUNG892 and HUNG967), most probably due to breastfeeding effect108. Therefore, Infants I (1–6 years109) were also excluded from statistical analyses . When comparing human samples between different periods (N = 74), significant differences appeared between the isotope ratios . Individuals from the Middle Neolithic to the Late Copper Age had similar δ13C values consistent with a diet based on terrestrial C3 resources  , but no differences in δ13C values . Differences were significant between Bükkábrány-Bánya XII/B and the other sites  . Notably, markedly higher δ13C values from the Vatta-Dobogó site indicate a signature indicative of C4 plant consumption . These were not statistically different when comparing females and males by period  were not statistically significant for either δ110. By contrast, isotope data indicate the types of resources consumed by individuals78. Each approach describes dietary patterns at different levels. Therefore, they can be combined in paleodietary research to explore dietary preferences in past populations at short  and long  timescales, within individuals and across groups90.In Middle Neolithic to Late Bronze Age populations that lived in the GHP, dental microwear and stable isotope analyses have been shown to provide complementary information rather than correlated variables. Microwear data from both occlusal and buccal surfaces show the abrasiveness of food, which might be related to cultural and technological changes such as food processing or dietary preferences15N and the buccal variables of vertical (NV/BTN) and horizontal (NH/BTN) index, respectively. This means that individuals with high δ15N values (presumably with high meat consumption) are characterized as having a relatively more vertical and less horizontals striations. These results are in accordance with studies suggesting meat-eaters usually present high frequencies of vertical striations on the enamel surface due to the movements of the mandible while chewing meat111. In contrast, chewing hard foods, such as cereals, requires more horizontal movements111, which increases the horizontal index.There were significant positive and negative correlations between δ112. This contrast with a slower turnover of the buccal surface, which stabilizes at maturity54, and provides information of the diet over time. Nevertheless, we found interesting trends among the samples on the occlusal surface, which we discuss below.Generally, we did not find significant differences between periods, sites or sex in occlusal dental microwear. The lack of differences in the occlusal surfaces might be due to the so-called “last supper effect”, which consists of a fast renewal of occlusal enamel, thus recording the diet of the most recent days or weeks111. The high nitrogen isotopic values [mean = 11.0 ± 0.8‰ (1σ)] were similar to those found at other Middle Neolithic sites104. Freshwater resources consumption (e.g. fish) can potentially explain high δ15N values, due to longer and complex foodwebs113, and to its proximity to freshwater resources access for some of the sites. However, although not completely discarded, the consumption of freshwater fish would not necessary lead to significant nitrogen isotope enrichment. Even if the freshwater fish represented 20% of human’s diet, the isotopic effect is quite small (+ 0.7‰)66. Additionally, freshwater fish consumption would have resulted in a more 13C-depleted values, as documented in other Mesolithic and Early Neolithic individuals from the same geographical area114. The carbon isotopic signal from bone collagen of Middle Neolithic sites are indicative of a C3 terrestrial protein source.We observed significant diachronic differences between the Middle Neolithic and the rest of the periods in the buccal microwear analysis, with the exception of Late Copper Age individuals. The low number of striations (BTN) observed from the Middle Neolithic might indicate that foods were highly processed, and thus less abrasive, and/or that a high proportion of consumed foods were soft, such as meat, which left little wear on the enamel surface103 (ca 0–1‰ for δ13C and ca 2–3‰ for δ15N), high δ15N could be explained by high reliance on meat consumption, particularly pig meat with significant high enriched 15N ratios among domesticates. However that would have required that 100% of the protein in diet had contributed essentially from terrestrial mammalian fauna66, ignoring that other dietary macronutrients, such as carbohydrates and lipids, are required to meet body’s energy requirements116. Another possibility is the ingestion of young animals meat, which shows higher δ15N due to, most probably, nursing signal117. Different patterns on bone assemblages were found on Middle Neolithic sites of the northern part of the GHP. Findings in Nagykálló-Harangod yielded several young animals (mostly sheep)118, while in Kompolt-Kíser high amounts of elderly individuals were found, probably due to their complex exploitation 119. Unfortunately, the zooarchaeological small sample size makes difficult assess the extent to which livestock exploitation prevailed in the sites presented in this study. High δ15N values may be the result of a combined effect of fertilized crops and meat consumption, as suggested elsewhere103. Long-term experimental studies have shown an increase in δ15N values in modern manured crops120. This has led some researchers to suggest that there has been an overestimation of animal protein intake in Neolithic paleodietary reconstructions121. Thus, these researchers propose that the combined effects of fertilized crops and animal protein consumption in Early Neolithic European farmers should result in bone collagen δ15N values of approximately 9–11‰121. For Middle Neolithic samples represented here, manured crops consumption, together with animal protein intake, in our opinion, is the hypothesis more feasible to us to explain high δ15N values.Assuming a range for trophic level enrichment generally accepted in the literature and observed in the archaeological record of the GHP55. Bükkábrány-Bánya XII/B samples instead presented the lowest BTN, indicating that the foods in their diets were less abrasive. Although not statistically significant, we observed the same trend for the total number of occlusal striations in both Middle Neolithic sites. This is also in accordance with δ15N values, where in generally high isotopic values were observed for all Middle Neolithic sites; in particular, we further found significantly more consumption of meat, a minimally abrasive resource, by the individuals at Bükkábrány-Bánya XII/B. Although we found significant differences between Bükkábrány-Bánya XI/A and Bükkábrány-Bánya XII/B sites in the horizontal and vertical indexes, the values of both indexes within Bükkábrány-Bánya XII/B were similar (around 0.2%), indicating they ate a mixed diet. Differences between the Middle Neolithic sites might have been due to cultural differences, particularly the type of food intake and/or how food was processed  or cooked , as has been observed in other prehistoric populations122. It is important to keep in mind that Bükkábrány-Bánya XI/A and Bükkábrány-Bánya XII/B are closer to each other than either of them is to Bükkábrány-Bánya VII123  when compared to other Middle Neolithic sites, which were significantly different from those found at Bükkábrány-Bánya XI/A. This might indicate that the diet at Bükkábrány-Bánya VII was more abrasive due to the consumption of hard foods and/or to the inclusion of hard extrinsic particles in food15N values suggest that there was a change in dietary preferences among Middle Copper Age individuals. Significantly higher BTN values than those found in Middle Neolithic individuals indicate they were consuming a more abrasive diet such as cereals, or to the presence of exogenous particles, such as grit, as a result of processing or cooking methods58. In keeping with this finding, lower δ15N values also indicate lower animal protein intake, in accordance with a lower reliance on softer resources, and/or on manured crops. Isotopic faunal offsets suggest that potentially they could have relied on cattle, pig and sheep/goats as an animal protein source, although only ovicaprid remains were recovered from this period in this study. In contrast, Late Copper Age individuals presented significant δ15N-rich nitrates compared to other periods, suggesting a higher meat intake , and/or consumption of fertilized crops or animals that consumed them103. Late Copper Age individuals had lower BTN values, which is related to less abrasive foods, such as meat110. Additionally, despite not being significant, there was a tendency for the values of the vertical indexes on the buccal surfaces to be high, which also might indicate that there was more consumption of meat49. The nitrogen isotope signal in these Late Copper Age individuals was noticeably higher (mean = 12.0 ± 0.3‰ (1σ)) than those from other Late Copper Age sites21. These results are in accordance with findings from other Late Copper Age sites that suggest an increase in animal product use during this period16, but not from other Middle Copper Age sites103. As Giblin103 argued for Early Copper Age site differences, this could be due to regional site differences in meat consumption and nitrogen enrichment, rather than differences in subsistence practices. More Copper Age sites, particularly from the Middle and Late Copper Age, need to be analysed to better understand if the increase in animal products was a general trend or instead a local or regional process.Both buccal microwear and δ55 and/or less consumption of processed food43. Millet consumption starts to be noticeable in bone collagen as δ13C values suggest22, although it probably would have been identified in earlier and more individuals if bone or enamel apatite were also tested (see60). Higher consumption of this C4 plant resource was not probably the cause of the abrasiveness increase, since no significant positive correlations were found for BTN and XT with δ13C values. The nitrogen isotope signal, although high (MBA mean: 10.8 ± 0.9‰ (1σ); LBA mean: 10.9 ± 1.1‰ (1σ)), suggests a decreased reliance on animal products , and/or manured crops as compared to Late Copper Age individuals22.The few Middle and Late Bronze Age individuals analysed for buccal microwear presented high mean values of BTN and XT variables, which indicates the consumption of an abrasive diet15N values suggest they may have consumed more animal protein than females. This trend is supported by other studies in Holocene populations124. This issue needs more careful research, and additional studies using dental microwear and stable isotopes should be undertaken to explore potential sex-related differences and their dietary manifestations.There were no significant sex differences among any of the periods analyzed. Despite a limited sample size, there was an overarching trend in XT and NH/TN in the Middle Copper Age individuals for females and males: females presented shorter, more horizontally oriented buccal striations than males, which is consistent with higher cereal intake. Males’ higher δAs demonstrated here, the integration of dental microwear and stable isotope approaches employed within individuals can provide a more complete overview of the dietary preferences of past human populations. Our findings suggest these two proxies are complementary, confirming their significance in paleodietary research, and we encourage future studies its combined use.15N results for meat consumption. In addition, the results obtained here show that changes in C4 plant consumption in the prehistoric Hungarian populations did not necessarily entail changes in diet abrasiveness, either in the C4 resource itself (grain) or the way it is processed (extrinsic particles). It is the case of Middle Bronze Age sites where higher reliance on millet (C4) consumption did not imply differences in the abrasiveness of their diet.In general, the level of abrasiveness in diet, revealed by buccal dental microwear variables, is correlated with the isotopic signal at the individual/local level when interpreting δThis research demonstrates the importance of studying the variation between different sites from the same time period. We have shown the characteristics of each Middle Neolithic site, and, at the individual level, confirmed that both dietary proxies, dental microwear and stable isotopes, point toward the same interpretation. We also found that previous interpretations regarding an increase in the consumption of animal products by the Middle Copper Age could not be confirmed in the Middle Copper Age sites analysed here.We found no significant differences in dietary composition between females and males for each period. However, results for Middle Copper Age females and males suggest a differential trend in proportions of cereal and meat consumed. Further research involving bone apatite and/or tooth enamel, as well as the inclusion of more samples would contribute significantly to better understand these results.125. Polyvinylsiloxane dental impression material (Coltene President Ligh Body©) was used to mold the tooth crowns. High resolution casts were made using EPO-TEK 301. The replicas were sputter coated with gold, and aluminum tape was attached from the stub to the molar to improve the conductivity of electrons126. We analysed the buccal and occlusal surfaces of the molars under an FEI Quanta 600 Environmental Scanning Electron Microscope (ESEM) held in the Scientific and Technical Resources Service of the University Rovira i Virgili , using previously-published parameters43.Prior to microscopic analysis, molars were washed with cotton swabs soaked in 96% alcohol, then left to air dry completely125 with the free software GIMP (version 2.10.22), (https://gimp.es/). Then, digital images were cropped to cover an area of 0.56 mm2 following125. Afterwards, the total number of striations in the buccal surface (BTN), the average length , and their orientation  , vertical (V) (67.5°–112.5°), mesiodistal (MD) ; distomesial (DM) 50 were computed using open-source image processing software, Image J127 (version 1.52p), (https://imagej.nih.gov/ij/index.html). Two orientation indexes that track broad differences in dietary patterns across groups50 were calculated: total horizontal/total number (NH/BTN) and total vertical/total number (NV/BTN).Micrographs (1024 × 832 pixels) were taken with ESEM at 100× of the middle third of the surface. The images were enhanced to increase the contrast and facilitate the observation of striations129. Facet 9 is a crushing and grinding surface, which experiences both compression and shearing during chewing131. The images were also enhanced, and the contrast modified using GIMP. Each image was cropped to 700 × 500 pixels, which represents approximately 0.03 mm2 of the molar’s surface122. On this surface, four variables were considered: the number of striations (OTN), the number of pits, their area, and the percentage of pits (Pits/(TN + Pits) × 100)42. Striations were defined and measured as in buccal surface, whereas pits are defined as features with a length to width ratio ≤ 4:193. Both features (striations and pits) were computed and recorded with ImageJ.Micrographs (1024 × 832 pixels) were taken with ESEM at 500× of facet 9, which is located on the distobuccal cusp on the first and second molars132. For that reason, one author (RH) was responsible for the measurement of buccal and occlusal dental microwear.There is a potentially high level of inter-observer error of the dental microwear22  were also included in the analyses. Human postcranial samples were preferentially selected for collagen extraction except in those cases where only skull fragments were available  School of Archaeology lab facilities and UCD Conway Institute . Bone fragments were prepared by cutting a sample of approximately 0.2–1.0 g from the original sample material using a handheld rotary tool with a diamond-coated cutting wheel. The outer surfaces were then abraded using a diamond coated burr. Collagen extraction followed a modified Longin method13C and ± 0.06‰ (1σ) for δ15N. All δ13C results are expressed in standard delta notation relative to Vienna PeeDee Belemnite (V-PDB). All δ15N results are expressed in standard delta notation relative to air N2 (AIR).Stable isotope analyses were carried out following the routine procedures at Light Stable Isotope Mass Spectrometry Laboratory of the Department of Geological Science at University of Florida . A Thermo Electron DeltaV Advantage isotope ratio mass spectrometer was used coupled with a ConFlo II interface linked to a Carlo Erba NA 1500 CNS Elemental Analyzer. The accuracy and precision of the measurements, based on repeated measurements of two international laboratory standards USGS40 and USGS41, was ± 0.05‰ (1σ) for δ135. Powder was obtained from the isolated cochlea using a mixer mill (Retsch MM400), and aliquots of 50–75 mg were used for DNA extraction. The risk of modern DNA contamination was reduced by carrying out all steps in a lab dedicated to the preparation of ancient bone samples at the UCD Conway Institute of Biomolecular and Biomedical Research , as recommended by138. While handling ancient bone samples, researchers wore overall suits, double gloves, hair nets, and face masks; all surfaces were cleaned and decontaminated with DNA-ExitusPlus between sample preparation and UV irradiation was also used to ensure effective decontamination took place.Ancient DNA (aDNA) analyses were performed using human petrous bones following standard procedures to determine the sex of some of the individuals Table . The cocThe DNA extraction, library preparation, and sequencing steps for Neolithic and Copper Age samples were performed in a physically separated aDNA lab at UCD and at the University of Vienna, following standard stringent anti-contamination protocols. A detailed description of DNA extraction, library preparation, and sequencing steps is provided in the “139. Although non-parametric tests were done, with this kind of sample we also relied on overarching trend and data visualization when the groups were small141.Given the small sample size of some chronological groups, the use of non-parametric statistic was required13C and δ15N, for each individual. Biplot graphs were generated for graphical visualization, including Ordinary Least Squares (OLS) regressions with a 95% confidence interval , synchronic (between sites), and between-sex perspectives by employing non-parametric statistical analyses. Isotopic values from faunal samples were also statistically compared. Kruskal–Wallis tests were chosen when comparing more than two groups (e.g. between periods). Mann–Whitney tests were applied for pairwise comparisons (i.e. compare two groups).92. For isotope analyses, only data from adults and subadults 6 years and above of known age were considered, as they have been shown to have similar isotopic signals in the same geographical areas and time periods103. Boxplots were created to illustrate the general tendencies of the samples. Human isotope ratios were also compared with fauna isotope ratios. Faunal samples from all sites were grouped and compared with human samples for each time period separately. We assumed an isotopic fractionation range from consumed to consumers of a + 2–3‰ for δ15N and + 0–1‰ for δ13C, as it is the range that generally previous studies have found104. All statistical data was performed with R142, and graphs were generated using the package ggplot2143. All data generated are included in this article .Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.