Pteropods are excellent recorders of surface temperature and carbonate ion concentration

Pteropods are among the first responders to ocean acidification and warming, but have not yet been widely explored as carriers of marine paleoenvironmental signals. In order to characterize the stable isotopic composition of aragonitic pteropod shells and their variation in response to climate change parameters, such as seawater temperature, pteropod shells (Heliconoides inflatus) were collected along a latitudinal transect in the Atlantic Ocean (31° N to 38° S). Comparison of shell oxygen isotopic composition to depth changes in the calculated aragonite equilibrium oxygen isotope values implies shallow calcification depths for H. inflatus (75 m). This species is therefore a good potential proxy carrier for past variations in surface ocean properties. Furthermore, we identified pteropod shells to be excellent recorders of climate change, as carbonate ion concentration and temperature in the upper water column have dominant influences on pteropod shell carbon and oxygen isotopic composition. These results, in combination with a broad distribution and high abundance, make the pteropod species studied here, H. inflatus, a promising new proxy carrier in paleoceanography.

δ 13 C in calcium carbonate shells is assumed to be in equilibrium with, or offset by a constant amount from δ 13 C DIC , the carbon isotopic composition of dissolved inorganic carbon (DIC; the sum of CO 2(aq) , H 2 CO 3 , HCO 3 − and CO 3 2− ). However, several studies found discrepancies for other groups of calcifiers, e.g. foraminifera 10 , as physiological processes, such as the respiration of symbionts in foraminifera, influence shell δ 13 C 11 . Juranek and colleagues 12 found a correlation between pteropod δ 13 C ptero and carbonate ion concentration and hypothesized that this was caused either by a carbonate ion dependence, also demonstrated for foraminifera 13 , or by the influence of temperature on metabolic CO 2 incorporation.
The life cycle of the pteropod species Heliconoides inflatus (d'Orbigny, 1834 14 ), formerly and more commonly known as Limacina inflata, has been estimated to be approximately 7-9 months, with a maximum life span of about one year 15,16 . Reproduction occurs continuously throughout the year, with females retaining developing embryos in the mantle cavity up to a size of about 70 μm until release of the veliger larvae 15 . Sediment trap studies revealed high interannual variability in the abundance of H. inflatus as well as a pronounced seasonal cycle in abundance, e.g. in the Sargasso Sea 16 . Here, the highest flux of shells was found during summer, whereas the flux throughout the rest of the year was very low 16,17 . While H. inflatus has been found as deep as 1000 m, this species primarily occurs in the upper water column 12 . Seasonal shifts in pteropod depth habitat were also reported for the Sargasso Sea, with H. inflatus preferring shallower waters in the fall (100 to 250 m) than in the spring/ early summer (200-400 m) 9 . Diel vertical migration, common in pteropods, has been described for H. inflatus, with most of the population below 200 m during the day and highest night-time abundance in the upper 75m 9 . The species also undergoes ontogenetic migration: juveniles tend to stay in surface waters, whereas adults are mainly found in deeper waters 17 .
Here we present δ 18 O ptero and δ 13 C ptero measurements of Heliconoides inflatus shells to assess the potential of pteropods to serve as proxies of seawater temperature (via δ 18 O) and carbonate ion concentration (via δ 13 C). We particularly focus on the calcification depth of the species and characterize environmental controls on shell composition. Our material was collected along a meridional transect in the Atlantic Ocean, ranging from 31° N to 38° S. This provides an unique opportunity to assess calcification depths across the entire Atlantic basin, in contrast to previous sediment trap studies that were conducted within a single oceanographic context 12,16,18,19 . Furthermore, the broad spatial scope of the study allows us to assess different environmental parameters as controls on the stable isotopic composition in pteropod shells, as oceanographic conditions change significantly over such a large latitudinal range. The calibrations established here will be of use to the ocean acidification and paleo-oceanographic community, as the studied species, H. inflatus, occurs in high abundance in sediments worldwide 20 , for instance, in the Central and South Atlantic 21 or the Caribbean Sea 22 .

Results
Surface distribution of oceanographic parameters. The warmest surface temperatures in the study area of the Atlantic Ocean, up to ~30 °C, usually occurred in October/November just north of the equator (around 10° N; Fig. 1a). Temperature decreased gradually both north and south of this maximum to approximately 12 °C at 40° S, where our southernmost station (station 66) was located. The surface salinity distribution mimicked this general latitudinal zonation (Fig. 1b), however, at the latitude of the temperature maximum, low salinities of 35 to 36 prevailed. Highest surface salinities (37-37.5) occurred in two locations west of 30° W: one in the North Atlantic at around 25° N and one in the South Atlantic at around 20° S. The three southernmost stations (stations 60, 62, 66) lie in an area characterized by mean salinities between 35 and 36. Theoretical values for δ 18 O of aragonite (δ 18 O ara ), taking into account δ 18 O estimates of seawater and ambient temperatures, as well as values for δ 13 C DIC were calculated for surface waters (Fig. 1c, d; see Methods and Table 1 for notations). The strong influence of temperature on the surface distribution of δ 18 O ara is evident from the latitudinal zonation of this parameter (Fig. 1c). Lowest surface δ 18 O ara values (~−1.5‰) occurred just north of the equator at 10° N, in the same area where temperature was highest and salinity lowest. From this minimum, surface δ 18 O ara increased continuously to the north and to the south until highest values were reached in the southernmost part of the transect (stations 62 and 66: ~+2‰). The distribution of δ 13 C DIC in surface waters also exhibited latitudinal zonation, with highest values (~+2‰) occurring at the equator around 0° W and in the Western Atlantic south of 40° S (Fig. 1d). From the equator, surface values gradually decreased towards both the north and south with lowest surface δ 13 C DIC values in the North Eastern Atlantic. δ 18 O in the water column and in pteropod shells. Latitudinal variations in calculated δ 18 O ara for equilibrium aragonite values at different depths in the watercolumn reflect strong thermal stratification between 20° N and 20° S, with a maximum in stratification around the equator (4° N), where values ranged from about −1.1‰ at the surface to +2.6‰ at 300 m depth (Fig. 2a). In comparison, δ 18 O ara in the southern temperate region, e.g. at 34° S (station 62), was less variable across the water column (from +2.1‰ at the surface to +1.5‰ at 300 m depth).  Table 2, S2 and Fig. 2a): the lowest values (minimum: −0.44‰) occurred at the equator and increased towards higher latitudes (maximum: +2.11‰). Variability between specimens at each station was close to instrument precision, with an average standard deviation of 0.14 (range 0.05-0.28‰). The pteropod oxygen isotopic composition measured here was comparable to the study of Juranek and coworkers 12 on the same species (+0.15 to +2.04‰), in which specimens from a one year-sediment trap in the Sargasso Sea were analyzed. Another study, however, reported more depleted oxygen isotopic values (approximately by 0.8‰) 16 for the same species from the Sargasso Sea, which likely derived from low temperature ashing of the samples before isotopic measurement 12 .
Average pteropod δ 18 O ptero values were similar to those calculated near the 75m-depth isopleth of δ 18 O ara (Fig. 2a), with individual measurements corresponding to values typically found between the surface and a maximum depth  Table 3, top). Likewise, we found a strong linear relationship between temperature and pteropod δ 18 O ptero (p < 0.05; R 2 = 0.87 to 0.86) in the upper 75 m of the water column (Fig. 3, Table 3, top). δ 18 O ptero also showed a positive, but weaker relationship to carbonate ion concentration (p < 0.05, R 2 = 0.64) at the surface, while no statistically significant correlation with salinity was found. δ 13 C in the water column and in pteropod shells. Pteropod shell δ 13 C ptero showed little variation in samples that were collected between 32° N and 26° S (+0.77‰ ± 0.23‰), however, δ 13 C ptero increased sharply with mean values of +1.28, +1.60, and +1.97‰ at the southernmost stations 60, 62, and 66, respectively (Fig. 2b, Table 2 and S2). Calculated variation of δ 13 C DIC in the water column was highest at low latitudes, where the variability of δ 13 C ptero in pteropod shells was very low (Fig. 2b). Pteropod shell δ 13 C ptero at most stations was lower than calculated δ 13 C DIC in the water column. This result was previously observed in pteropod shells that calcified in shallow waters 12 , and was attributed to the carbonate ion effect, where higher carbonate ion concentrations caused lower δ 13 C ptero values than in equilibrium with δ 13 C DIC (see Discussion). We find the strongest linear relationship between pteropod δ 13 C ptero and carbonate ion concentration in the upper water column (Fig. 4a), with a negative regression of δ 13 C ptero = −0.02 * Carbonate (50 m) + 5.31 (p < 0.05, R 2 = 0.92; Table 3, middle). Furthermore, we observe linear relationships between pteropod δ 13 C ptero and temperature and salinity, but none with δ 13 C DIC (Table 3, middle). Pteropod shells from the southernmost stations have high δ 13 C ptero values, where phytoplankton standing stock was high in surface waters (see Discussion below and Fig. 4b). We observe a significant positive relationship between δ 13 C ptero and δ 18 O ptero in pteropod shells (p < 0.05, R 2 = 0.68; Fig. 5). Interestingly, this relationship is mostly influenced by measurements performed on shells from the three southernmost stations (60, 62 & 66; indicated by open circles in Fig. 5): omitting these stations renders the relationship statistically non-significant (p > 0.05). Comparison of our results to previous findings on the same species 16,18,19 revealed that the slope was similar to prior work (Fig. 5). However, the δ 13 C ptero values presented here cover a broader range   than observed in previous studies that were located in environments with less oceanographic variation (e.g., sediment trap studies, Sargasso Sea).

Discussion
Deriving past ocean temperature and chemistry from fossil pteropod shells provides a wealth of information about past climate change events. The present study shows that the species H. inflatus is well suited for paleo-reconstructions, as the stable isotopic composition of their shells can be used to track two climate change indicators: δ 18 O ptero records temperature (Fig. 3) and carbonate ion concentration is traced by δ 13 C ptero (Fig. 4a). Both proxies can be measured simultaneously on a single pteropod shell, making pteropods particularly promising new proxy carriers. Furthermore, we demonstrate that H. inflatus records latitudinal ranges of surface water parameters, as suggested previously from sediment trap studies at a local scale 12,16 . We confirm shallow water calcification of this species for a large area of the Atlantic Ocean (31° N to 38° S), which, in combination with its basin-wide distribution, renders H. inflatus an ideal candidate for proxy reconstructions.  Based on a comparison between the oxygen isotopic composition of pteropod shells and that of seawater, we observed no systematic latitudinal variation in calcification depth (Fig. 2a). Oxygen isotopic values for H. inflatus strongly correlated with δ 18 O ara equilibrium values in the upper 75 m of the water column (Fig. 2a, Table 3, top). These findings corroborate observations from other studies on the same species in the Sargasso Sea, suggesting calcification from 50-250 m depths 12,16 . Such shallow calcification depths are in contrast to reported preferential occurrences of H. inflatus in deeper waters (to 600 m) in the Sargasso Sea 17 . One possible explanation is that pteropods preferentially calcify near the surface, where calcification is energetically favored due to warmer temperatures, affecting calcium carbonate saturation. Furthermore, most chlorophyll a, and thus potential food resources, occurs in the upper water column, which may be another reason why H. inflatus calcifies in shallow waters.
Pteropod shells are produced over several months, therefore reflecting the sum of environmental conditions experienced throughout the animal's life. Thus, single-shell measurements, as presented here, are an average of these conditions, with a bias toward the more recently calcified material, as this makes up the largest part of the shell 16 . Pteropods alter their depth habitat daily, seasonally, and ontogenetically 9,17 . Consequently, the aragonite of a single pteropod shell could have been produced across a range of depths. Accordingly, the estimated calcification depth of about 75 m may be the average of varying isotopic signatures from different calcification depths.  However, several observations argue against this. First, we found no correlation of δ 18 O ptero and temperature below 100 m. Second, other studies on the same species also found that H. inflatus mostly calcified in the upper water column, rather than in deeper waters 12,16 . The material of a single pteropod shell is probably also the product of several seasons, as the average life span of several pteropod species is on the order of one year 15 . Sediment trap material is ideal to study the effect of seasonality on the stable isotopic composition of pteropods. Two other studies 12,16 found that δ 18 O ptero measurements on seasonal H. inflatus samples from sediment traps usually correlate strongly with seasonal temperature variations in the water column. The absence of an offset in time between δ 18 O ptero and δ 18 O ara in these studies indicates that the bulk of the shell must have been precipitated within the few months prior to collection, making pteropods reliable recorders of surface water masses.
Carbon isotopic composition of pteropod shells was relatively invariant along the meridional transect in the Atlantic Ocean, with the exception of higher δ 13 C ptero values at stations south of 30°S (60, 62 and 66; Fig. 4a). Phytoplankton standing stock (chlorophyll a concentration) was very high in surface waters (upper 75 m of the water column) in the south subtropical convergence province (stations 62 and 66, see ref. 23 ). Photosynthesis actively removes the lighter carbon isotopes from the water column DIC reservoir, resulting in 13 C DIC enrichment. Since pteropods use ambient DIC to build their shells, δ 13 C ptero will also be higher in regions with high rates of photosynthesis, as was observed at these southernmost stations (Fig. 4b). The positive relationship between δ 13 C ptero and chlorophyll a at 25 and 50 m depth (Table 3, middle, p < 0.05, R 2 = 0.80 and 0.73, respectively) also reflects the effect of photosynthesis on δ 13 C ptero . Additionally, H. inflatus specimens from southern temperate waters (south of 34°S) along a similar transect (AMT24) were reported to be morphologically distinct, having coarser and thicker shells than specimens from the rest of the transect, and thus may represent a distinct population or (sub)species (referred to as H. inflatus S) 24 . In these respects, station 60 (30.20 °S) should be regarded as a transitional station: it is located in the oligotrophic south Atlantic gyral province with low chlorophyll a concentrations and H. inflatus specimens that were morphologically similar to specimens from tropical and subtropical waters, but average δ 13 C ptero in their shells was relatively high (Fig. 4b, station marked by asterisk). While the (calcium) isotopic signature of foraminiferal sub-species has been demonstrated to be the same 25 , small trace element compositional differences can still be caused by depth habitat preferences of different sub-species 26 . All pteropods analyzed here calcify in the same water depth (upper 75 m, Fig. 2a), allowing us to assume that the potentially different (sub) species have similar isotopic signatures under the same environmental conditions. Pteropod δ 13 C ptero did not show much variation between 32° N and 26° S (Fig. 2b), where chlorophyll a concentration was much lower (Fig. 4b). Apparently, photosynthesis by these low phytoplankton concentrations did not cause a 13 C enrichment of the DIC pool, explaining the relatively low δ 13 C ptero values of the pteropods across most of the transect (average + 0.70‰). However, as we observe no correlation between chlorophyll a fluorescence and δ 13 C ptero in the majority of the pteropod shells analyzed (Fig. 4b), other influences on δ 13 C ptero should be explored, such as the carbonate ion effect. This effect was reported in foraminifera 13,27 and in pteropods 12 , and describes the inverse relationship between carbonate ion concentration and δ 13 C ptero , which is also apparent in our results (Fig. 4a, R 2 = 0.94, p < 0.05). For the southernmost stations, it is impossible to disentangle the effects of 13 C enrichment via photosynthesis and the carbonate ion effect on pteropod δ 13 C ptero (Table 3). We therefore performed linear regressions on δ 13 C ptero and water column parameters while excluding stations 60, 62, and 66 (Table 3, bottom). Excluding these three stations clearly demonstrates the carbonate ion effect on δ 13 C ptero (Table 3, bottom, R 2 = 0.84, p < 0.05) in the upper water column of an area where no 13 C enrichment is occurring (no effect of chlorophyll a on δ 13 C ptero ; Table 3, bottom, p > 0.05).  19 , dotted (R 2 = 0.5, Cape Blanc) 18 . Small black filled circles denote our measurements (triplicates, station 50 in duplicates) from all stations, with measurements from the three southernmost stations (60, 62, 66) depicted in red. The cross in the lower right corner indicates standard deviation (for δ 13 C ptero and δ 18 O ptero ).
Scientific RepoRts | 7: 12645 | DOI:10.1038/s41598-017-11708-w Our study shows that the pteropod species H. inflatus calcifies across a number of oceanographic provinces in the Atlantic at the same, shallow depth (upper 75 m of the water column, Fig. 2a, Table 3), making these pteropod shells good recorders of surface water masses. Correlations between stable isotopic composition of shells and parameters of the water column indicate that H. inflatus shells are good proxy carriers for temperature and carbonate ion reconstructions with the following regressions (for values at 50 m depth): 18 ptero with p < 0.05, R 2 = 0.86 (Table 3, 13 ptero with p < 0.05, R 2 = 0.92 (Table 3, bottom), only valid for δ 13 C ptero < 1‰ (see Discussion below). The uncertainty in the estimations for temperature (equation 1) and carbonate ion concentration (equation 2) based on an error propagation calculation sums to an error of 17% and 25%, respectively, assuming a measurement precision of 0.09‰ and 0.05‰ for the measurements of δ 18 O ptero and δ 13 C ptero , respectively. Please note that the regressions reported above (equations 1 and 2) have been derived from different datasets. While the δ 18 O ptero -temperature regression (equation 1) includes all stations, the δ 13 C ptero -carbonate ion regression (equation 2) is not valid for high productivity waters (here 31° S to 38° S), as the δ 13 C ptero in these regions may be influenced by 13 C enrichment (see Discussion above). Consequently, the calibration (equation 2) should only be used on δ 13 C ptero values < 1‰ limiting the resolvable carbonate ion concentration to values of 200 μmol/kg-sw or higher.
Heliconoides inflatus is a pteropod species that not only occurs in the Atlantic, but has a circumglobal distribution in tropical and subtropical waters (including the Caribbean, Mediterranean and Indo-Pacific). Therefore, it is a good proxy carrier to assess surface water variations over paleo-timescales worldwide. Heliconoides is the oldest known pteropod genus in the fossil record (72-79 million years ago (mya) 28 ), and the species H. inflatus has been described to occur at least since the early Miocene (Aquitanian) from the Aquitaine and North Sea basins (23.03-20.44 mya 29 and pers. comm. Janssen 2017). One limitation on the application of this new proxy is the occurrence of well-preserved pteropod shells in sediments, confined to waters above the lysocline of aragonite. However, there are a number of sediment cores available in which H. inflatus is abundant and where the calibrations reported here can be applied. The CAR-MON2 core 22 would be an ideal candidate from the Caribbean Sea, as it contains H. inflatus in great abundance. The core spans the last 250,000 years, and the associated changes in the ocean's temperature and carbonate ion concentration during glacial/ interglacial cycles are well resolvable by the proxy calibrations reported here. This holds true even under the restriction of the δ 13 C ptero calibration, as surface carbonate ion concentration in the Caribbean Sea has been >250 μmol/kg-sw for the last 100, 000 years 30 .

Methods
Pteropod collection. Bulk zooplankton was collected on the Atlantic Meridional Transect Cruise 22 (AMT22) between 10/19/2012 and 11/16/2012. Oblique tows were conducted with bongo nets (200 µm, 333 µm), towed between on average 361 m depth and the sea surface. Pteropods were collected from a total of 11 stations, between 31° N to 38° S latitude, in the pre-dawn hours (Table S1). After collection, pteropods were immediately fixed in pure ethanol (96-99%), which was renewed within 12-24 hours of collection. Specimens were stored at −20 °C until analysis.

Measurements of stable isotopes (δ 18 O and δ 13 C) of pteropod shells. Pteropods (H. inflatus) within
a narrow size range (800-1200 μm shell width) were removed from ethanol and dried at room temperature. All individual shells were weighed on a microbalance to ensure sufficient material for isotopic analysis (sample mass 120 ± 60 (1 SD) μg on average). Shells were broken to allow removal of the soft-tissue. All shell pieces were collected, triple rinsed with ultrapure water, dried at room temperature and weighed. The isotopic composition was analyzed at the Leibniz Laboratory for Radiometric Dating and Stable Isotope Research (Kiel University, Germany) using a Kiel IV carbonate preparation device connected to a ThermoScientific MAT 253 mass spectrometer. The aragonitic shells were reacted with 100% phosphoric acid (H 3 PO 4 ) under vacuum at 75 °C, and the evolved carbon dioxide gas was analyzed eight times for each individual sample. All values are reported in the Vienna Pee Dee Belemnite notation (VPDB) relative to NBS19. Precision of all different laboratory internal and international standards (NBS19) is ≤±0.05‰ for δ 13 C and ≤±0.09‰ for δ 18 O values. For notations related to shell chemistry, see Table 1.
Isotope values are reported in standard δ notation where: Seawater parameters: temperature, salinity, carbonate chemistry and chlorophyll a. Seawater temperature, salinity, and chlorophyll a concentrations in the upper 500 m of the water column were obtained by conductivity-temperature-depth (CTD) casts (Sea-Bird Electronics, models: ocean logger, SBE45, 9plus) and Chelsea MKIII Aquatracka Fluorometer, respectively. Sensors were calibrated and data archived by the British Oceanographic Data Centre (BODC). Discrete seawater samples taken from Niskin bottles were used to measure pH, TA (total alkalinity) and DIC. In order to calibrate the CTD chlorophyll fluorometer, discrete Chlorophyll a samples were analyzed fluorometrically following standard acetone extraction 31 . Briefly, discrete chlorophyll a samples were filtered through GF/F filters (0.7 μm) and placed in acetone for 18-36 hours before fluorescence was measured on a Turner Designs AU10 fluorometer. pH was measured (~11 samples per station) spectrophotometrically according to Clayton and Byrne 32 . TA was measured at selected depths, including ~3 samples per station (e.g. at approximately 300, 100 and 2 m depth for station 15), and analyzed by open-cell-titration 33 . TA measurements were related to salinity and temperature 34 according to the polynomial described by Lee and colleagues 35 , and were subsequently used to estimate TA at all depths. TA and pH were used to calculate the complete C-system (DIC, bicarbonate, carbonate, Omega and Revelle Factor at all depths) using the CO2SYS software 36 . These calculations were consistent with measured DIC (at selected depths, ~3 depths per station, same depths as TA measurements) and surface pCO 2 (CO 2 partial pressure) measured continuously every 20 minutes 34 .
Seawater composition in the sampling area. In order to characterize environmental controls on pteropod stable isotopic composition (δ 18 O ptero and δ 13 C ptero ), seawater δ 18 O ara and δ 13 C DIC isopleths were calculated (surface to 300 m). The salinity-δ 18 O SW (seawater: sw) calibrations from Le Grande and Schmidt 37 for Atlantic provinces were used, with CTD-derived salinity (S) as an input parameter: North Atlantic (δ 18  , using temperature (T) from CTD measurements. There was no suitable calibration for the correlation of δ 13 C DIC and DIC, therefore we used the GLODAP data set (http://cdiac.ornl.gov/oceans/ GLODAPv238) to calculate linear regressions between δ 13 C DIC and DIC. We defined six oceanic provinces according to latitude (45°N-30°N, 30°N-15°N, 15°N -0°N, 0°S-23°S, 23°S-30°S, 30°S-45°S) and used all available data in the upper 105 m between 10° E and 60° E, yielding six regressions for the relationship between δ 13 C DIC and DIC (Table S3). The uncertainty in these calculations based on an error propagation calculation assuming a DIC concentration of 2200 (±10) μmol/kg sums to an average error of 22% for the δ 13 C DIC estimation and to an error of 5% in the δ 18 O SW estimation, when assuming a salinity of 35 (±0.1) and temperature of 24 (±0.1) ° C. These values have been calculated using the standard errors listed in Table S3 and the respective publications 7,37 . Data from the World Ocean Database (WOD) 38 and GLODAP 39 were used to generate surface distribution maps of the Atlantic for temperature, salinity and seawater δ 18 O ara and δ 13 C DIC . Plots present average values from October through November in order to obtain a representation of the typical surface distribution of these parameters during the period of the cruise (10/13/2012 to 11/19/2012). The WOD 38 data collection contained all surface data available from 1986 to 2011, and the GLODAP 39 data collection contained all data from 1972 to 2011.

Statistical analyses.
To test the effect of temperature, salinity, carbonate ion concentration, chlorophyll a concentration, δ 18 O ara and δ 13 C DIC on pteropod shell isotopic composition (δ 18 O ptero and δ 13 C ptero ), linear regressions were calculated for specific depths (2,25,50,75,100,200, 250, 300 m). Temperature, salinity and chlorophyll a were taken from the CTD casts. Carbonate ion concentration was interpolated to these depths, while δ 18 O ara and δ 13 C DIC were calculated at these depths (see above).
Data availability. All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).