Introduction
Sea water salinity varies over time and space as a function of continental
ice volume, evaporation, precipitation and river runoff. Salinity
reconstructions could provide important constraints on past ocean
circulation, the hydrological cycle and glacial–interglacial sea level
changes. Currently, most reconstructions of salinity are indirect and based
on the correlation between salinity and δ18Owater,
assuming this relationship to be constant over space and time (Rohling and
Bigg, 1998). An independent
salinity proxy may reduce the uncertainties inherently associated with such
approaches (Rohling and Hilgen, 2007) and should preferably be based on one
of the main components of sea water salinity, for instance sodium (Na).
Results from a culture study showed that the sodium content of foraminiferal
calcite (Na / Cacc) correlates positively and linearly with
salinity for the benthic low-Mg, symbiont-barren species Ammonia tepida, with a sensitivity of 0.22 mmol mol-1 for every change of 1
salinity unit between salinities 30 and 38.6 (Wit et al., 2013). Various
culture studies earlier showed that also Mg / Ca is affected by salinity
but responds more strongly to changes in temperature (Lea et al., 1999;
Dissard et al., 2010b; Nürnberg et al., 1996; Hönisch et al., 2013).
Although an effect of salinity on foraminiferal Sr / Cacc has
been reported in some studies (Kısakürek et al., 2008; Dissard et al.,
2010b; Wit et al., 2013), other studies did not find a relation between
salinity and foraminiferal Sr / Ca (Dueñas-Bohórquez et al.,
2009; Diz et al., 2012; Allen et al., 2016), which led to the hypothesis
that foraminiferal Sr / Ca mainly reflects sea water inorganic carbon
chemistry (Keul et al., 2017) in addition to its response to temperature (Lea
et al., 1999; Raja et al., 2007). Hence, an independent salinity proxy would
not only be useful for constraining past (changes in) salinity but would also
improve temperature reconstructions based on Mg / Cacc and
reconstructions of past sea water carbonate chemistry based on Sr / Ca.
Following the culture-based Na / Cacc–salinity calibration for
A. tepida (Wit et al., 2013), a culture study with planktonic
symbiont-bearing species also showed a significant linear relationship for
Globigerinoides ruber (Allen et al., 2016). Although no significant
relationship was observed in this study for G. sacculifer (Allen et
al., 2016), a recent field calibration observed positive linear relationships
for both these species (Mezger et al., 2016). Still, the Na / Ca–salinity
sensitivities observed between the different species and studies differed
considerably (ranging from a change in 0.074 to 0.66 mmol mol-1 in
Na / Cacc for a change in 1 salinity unit). Whereas Wit et
al. (2013) suggested an incorporation mechanism similar to that observed in
inorganic calcite, field and culture studies also show that different species
of foraminifera have varying calcite chemistries, thereby resulting in the
need of species-specific calibrations similar to many other foraminiferal
trace-metal-based proxies (e.g. Elderfield and Ganssen, 2000; Rosenthal et
al., 2000; Anand et al., 2003; Bemis et al., 1998; Toyofuku et al., 2011).
For example, Mg / Cacc values are different between groups of
low-Mg, high-Mg hyaline and porcelaneous foraminifera (Toyofuku et al.,
2000; Segev and Erez, 2006; Raja et al., 2007), which also seems to be
reflected in other co-precipitated cations (De Nooijer et al., 2017). Hence,
calibration of Na / Cacc as a function of salinity for other
species is not only necessary to test the applicability of this novel proxy
for other groups of foraminifera but also allows testing whether monovalent
cations follow the inter-species trends described for divalent cations
(Terakado et al., 2010).
Here we calibrated Na, Mg and Sr incorporation in the intermediate-Mg
calcite benthic foraminiferal species Amphistegina lessonii and the
low-Mg calcite species Ammonia tepida over a salinity range of 20
units (from 25 to 45). We thus compare the El / Ca versus salinity trends
in a tropical, symbiont-bearing species (A. lessonii) to a temperate
intertidal symbiont-barren species (A. tepida) and both of them to
existing calibrations. The chemical composition of newly formed calcite was
determined by laser ablation inductively coupled plasma mass spectrometry
(LA-Q-ICP-MS), providing insights into concentrations and variability therein
between specimens and between single chambers. To investigate intra-specimen
variability at the scale of the chamber wall we also performed electron probe
micro-analysis (EPMA), mapping the Ca, Na and Mg distribution throughout the
chamber wall for specimens of both species cultured.
Methods
Culture media preparation and chemistry
In total, 50 L of sea water with a salinity of 50 was prepared by
sub-boiling 0.2 µm filtered North Atlantic sea water for 48 h at
45 ∘C. Subsequently, culture media were obtained by diluting this
high-salinity sea water with double de-ionized sea water (∼ 18 MΩ) in batches of approximately 10 L with salinity increasing from 25 to 45
in steps of 5 units, resulting in five unique salinity conditions. Using a
single batch of concentrated sea water to subsequently dilute to the desired
salinities ensures constant element to Ca ratios. Salinity of the media was
measured with a salinometer (VWR CO310), based on conductivity. Culture media
were stored in Nalgene containers and kept in the dark at 10 ∘C. Sea
water pH was determined with a pH meter (pH110, VWR). Subsamples were taken
prior to and at the end of the experiment and analysed for DIC and element
concentrations to monitor the effect of sub-boiling on the sea water's
inorganic carbon chemistry and element composition (Table 1). Subsamples for
DIC were collected in headspace-free vials and conserved with a saturated
HgCl2 solution (10 µL HgCl2/10 mL sample). DIC
measurements were performed on an autoanalyser spectrometric system TRAACS
800; Stoll et al. (2001). This analysis requires only a small amount of
sample, while yielding high accuracy (±2 µmol kg-1) and
precision (±1.5 µmol kg-1). The minor and major elemental
composition of the culture media was measured using a sector field ICP-MS
(Element2, Thermo Scientific) by sampling 1 mL from the culture media and
dilution by a factor 300 with 0.14 M HNO3 (Table 1).
Experiment culture media measurements per salinity condition.
Carbonate ion concentrations and saturation state with respect to calcite (at
25 ∘C) were calculated using CO2SYS (Van Heuven et al., 2011) and
the equilibrium constants K1 and K2 of Mehrbach et al. (1973), as
reformulated by Dickson and Millero (1987).
Experiment
Na / Casw
Mg / Casw
Sr / Casw
Salinity
DIC
pH
[CO32-]
Ωcalcite
(mol mol-1)
(mol mol-1)
(mmol mol-1)
(µmol kg-1)
(mmol kg SW-1)
S25
48.84
5.61
9.37
25.2
1087.3
8.32
164.90
4.28
S30
49.79
5.69
9.45
30.3
1305.3
8.28
205.98
5.15
S35
48.56
5.51
9.04
35.2
1512.0
8.22
258.84
6.22
S40
48.50
5.62
9.19
40.0
1734.4
8.17
267.23
6.16
S45
48.90
5.73
9.21
45.2
1947.4
8.10
284.67
6.23
Collection of foraminifera and culture setup
Surface sediment samples containing foraminifera (A. lessonii) were collected from the
Indo-Pacific Coral Reef aquarium in Burgers' Zoo (Arnhem, the
Netherlands; Ernst et al., 2011) and a tidal flat near Den Oever, the Wadden
Sea (A. tepida, genotype T6; Hayward et al., 2004). Sediment was stored in aerated
aquaria at 25 ∘C (A. lessonii) and 10 ∘C (A. tepida) with a day/night cycle
of 12 h/12 h, similar to conditions in the coral reef aquarium and Wadden
Sea, respectively. From both stocks, living specimens, recognized by chambers
that were filled with yellow cytoplasm and pseudopodial activity, were
isolated.
The culture protocol was the same for both species to facilitate comparison
of obtained element / Ca ratios between species. Since our specimens of A. tepida are
from a location with a much larger temperature range than where A. lessonii is derived
from (Ernst et al., 2011; Van Aken, 2008; De Nooijer et al., 2014a), both
species were incubated at 25 ∘C. Living specimens were placed in
groups of 25 individuals in Petri dishes with approximately 70 mL of North
Atlantic surface sea water (0.2 µm filtered) and fed with fresh cells
of the algae Dunaliella salina. After reproduction, which occurred in approximately two-thirds of
all incubated specimens in both species, 2–3 chambered juveniles were
isolated. The use of specimens from reproduction events guarantees that
virtually all chambers present at the end of the experiment were produced
under the culture conditions (De Nooijer et al., 2014a).
Strains of specimens of the reproduction events were divided over Petri
dishes (resulting in 2–10 individuals per dish) with approximately 10 mL of
culture medium and stored in a temperature-controlled incubator set at
25 ∘C with a day/night cycle of 12 h/12 h. The culture media in
the Petri dishes were replaced once every week, after which specimens were
fed with 1 mL of concentrated and freeze-dried Dunaliella salina diluted with the culture medium
for each salinity condition, to minimize changes in salinity when feeding
the foraminifers. The amount of food was adjusted so that it was not
depleted after a week, at the same time not resulting in an excess of debris
and hence reduce bacterial growth. Petri dishes were sealed with a lid to
minimize evaporation. After 6–8 weeks, specimens were harvested and
transferred to microvials to clean the specimens' carbonate shells from cell
material. Specimens were cleaned with an adapted version of the Barker
protocol (Barker et al., 2003), only applying the organic removal/oxidation
step, in which NaOH was replaced with NH4OH, in order to
avoid Na contamination of our samples. Organic matter was removed by adding
1 % H2O2 buffered with 0.1 M NH4OH at 90 ∘C and
gentle ultrasonication (80 kHz, 50 % power, in degas mode) for 1 min, which
is known not to affect obtained Mg / Ca and Sr / Ca (Barker et al., 2003).
Specimens were subsequently rinsed three times with double de-ionized water
and dried in a laminar flow cabinet, after which their size was determined (i.e.
the maximum diameter crossing the centre of the specimen). The specimens
were thereafter stored until geochemical analyses (LA-Q-ICP-MS; Sect. 2.2.2 and
EPMA; Sect. 2.4).
Accuracies (Ac) and precisions (Pr) for Na, Mg and Sr for the
various standards analysed.
Standard
n
Ac Na (%)
Pr Na (%)
Ac Mg (%)
Pr Mg (%)
Ac Sr (%)
Pr Sr (%)
JCp-1
51
99
6
96
6
96
4
NIST610
32
119
3
104
2
110
3
NIST612
29
119
3
104
2
110
2
Foraminiferal calcite chemistry
Specimens were fixed on a
laser ablation stub using double-sided tape, carefully positioning them to
allow ablation of the last chambers (Supplement S1). Element concentrations
of individual chambers were measured with LA-ICP-MS (Reichart et al., 2003).
The last 1–3 chambers of each specimen were ablated using a circular spot
with a diameter of 60 µm (A. tepida) and 80 µm
(A. lessonii) (NWR193UC, New Wave Research) in a helium environment
in a New Wave TV2 dual-volume cell (cup volume of ∼ 1 cm3) at a
repetition rate of 6 Hz and an energy density of approximately
1 J cm-2. The aerosol
was transported to a quadrupole ICP-MS (iCap, Thermo Scientific) on a helium
flow at a rate of 0.7 L min-1, with 0.4 L min-1 Argon make-up
gas being added before entering the torch. Nitrogen gas was added at a rate
of 5 mL min-1 to enhance sensitivity of the analysis. Before entering
the torch, the aerosol / Ar / He mixture passed through an in-house-made smoothing device to reduce temporal variations in signal strength.
Monitored masses included 7Li, 11B, 23Na, 24Mg,
25Mg, 27Al, 43Ca, 44Ca, 60Ni, 66Zn, 88Sr,
137Ba and 238U, with one full cycle through the different masses
taking 120 ms. Calibration was performed against a MACS-3 (synthetic calcium
carbonate) pressed powder carbonate standard with 43Ca as an internal
standard. Count rates for the different masses were directly translated into
element / Cacc (El / Cacc) ratios. Internal
precision based on MACS-3 is 4 % for Na, 3 % for Mg and 4 % for
Sr. Accuracy and relative analytical errors, based on measuring international
standards JCp-1 coral (Porites sp.) powder and the NIST (National
Institute of Standards and Technology) SRM 610 and SRM 612 (glass), are listed
in Table 2. The relatively large offset between the glass standards and the
pressed powders (both MACS-3 and JCp-1) is known not to influence obtained
El / Cacc ratios when either one is used as a calibration
standard (Hathorne et al., 2008), but due to the similar matrix, MACS-3 was
chosen as the calibration standard.
In total, 675 chambers were measured (336 for Amphistegina and 339 for Ammonia), resulting in
between 52 and 125 single-chamber measurements per salinity condition per
species. These measurements were done on the last three (final or F,
penultimate or F-1 and pre-penultimate or F-2) chambers of these specimens
(see Table 3 for number of specimens and average number of spots per
specimen). Element concentrations were calculated from the time-resolved (i.e.
ablation depth) profiles using an adapted version of the SILLS
(Signal Integration for Laboratory Laser Systems; Guillong et al.,
2008) package for MATLAB (for details see Van Dijk et al., 2017a), while
taking care to exclude contaminations potentially present on chamber walls
(examples of profile selection: Dueñas-Bohórquez et al., 2011;
Wit et al., 2013; Mewes et al., 2014; Mezger et al., 2016; Van Dijk et al.,
2017b). Measurements with ablation yields or integrations times < 5 s
were excluded from further analysis.
Foraminiferal Na / Cacc, Mg / Cacc and
Sr / Cacc versus salinity. Light blue dots represent the
average per specimen (n=359 for A. lessonii, n=339 for
A. tepida, with 2–3 measured chambers per individual) and dark blue
dots indicate the mean, with inner error bars indicating the standard error
and outer error bars the standard deviation for each treatment. The linear
regression model (red line) is based on the mean of individuals, with the
95 % confidence interval of the regression in dashed lines.
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The LA measurements were also used to investigate the co-occurrence of
elements within specimens. Since there is variability in Ca counts between
the laser ablation measurements, single-spot-based
element / Cacc ratios may cause spurious correlation due to
coupled differences in Ca counts. To test whether observed correlations
between Na / Cacc, Sr / Cacc and
Mg / Cacc, based on single spots, are due to the use of a
common denominator (Ca), we performed a Monte Carlo simulation. In short, the
correlation coefficients between randomly drawn single-spot Mg concentration,
divided by measured Ca, and measured Na / Cacc concentrations
were compared to the correlation coefficient of measured
Na / Cacc and Mg / Cacc concentration ratios in
our dataset. By using a kernel fit of the measured data set to draw the
random data set and using the measured Ca as a common denominator we
effectively simulate the spurious correlation. The Monte Carlo results show
that inter-element correlations are not spurious, since the measured
correlation coefficient is significantly higher then the distribution of the
correlation coefficients between 10 000 randomly drawn El1
concentrations/measured Ca concentration and measured El2 / Ca
concentrations (Supplement S2).
Furthermore, to test whether Sr / Cacc and
Na / Cacc variability in A. lessonii is not caused by
variability in Mg content due to a potential closed-sum effect (since high
amounts of incorporated Mg cations could reduce the Ca content of the shell
and hence result in apparently elevated Sr / Cacc and
Na / Cacc), we calculated maximum variability due to the sole
effect of Mg substitution. For A. lessonii, variability (standard
deviation) of ±0.09 mmol mol-1 in Na / Cacc and
±0.016 mmol mol-1 in Sr / Cacc around the mean
could be caused by variability in Mg / Cacc (assuming Mg
substitutes for Ca in the calcite lattice, and Mg plus Ca approximates 1 mol
per mol calcite). This may have influenced the Sr / Cacc and
Na / Cacc regression slopes over salinity and also the calculated
inter-element correlation coefficients, but only by a maximum of ±1 %
for both elements, which is considerably lower than the total observed
variability within the dataset of 16 and 9 %, respectively.
Electron microprobe mapping
To investigate variation of element distribution across the chamber wall, a
number of cultured specimens were prepared for electron microprobe analysis
(EPMA). From each of the five salinity conditions, six specimens from both
species were selected and embedded in resin (Araldite 2020) in an aluminium
ring (diameter 1 cm) in a vacuum chamber. Samples were polished with a final
polishing step using a diamond emulsion with grains of 0.04 µm.
This procedure resulted in exposure of a cross section of the foraminiferal
chamber wall from which areas for EPMA mapping were selected (Supplement S1).
These areas were selected for being perpendicular to the shell outer surface,
resulting in pores completely crossing the exposed chamber wall. Elemental
distributions were mapped in chambers prior to F-3 to study the element
distribution across the various layers of calcite (lamella) produced with the
addition of each new chamber in rotaliid foraminifera (Reiss, 1957, 1960).
Elemental distribution in the shell wall was measured using a field emission
electron probe micro-analyser (JEOL JXA-8530F HyperProbe) at 7.0 kV with a
dwell time of 350 ms, using a spot diameter of 80 nm and a step size
between 0.1538 µm and 0.4072 µm
(130 × 130 pixels).
Spatial resolution of the EPMA mapping was determined using the software
package CASINO (monte CArlo SImulation of electroN trajectory in SOlids,
v 2.48). With the input parameters identical as used in our analysis (80 nm
spot size, beam current 7 keV, etc.), the simulated surface radius of the
backscattered electrons (i.e. the spatial resolution) equals 590 nm.
Semi-quantitative El / Cacc profiles were calculated by
averaging the El / Cacc intensities parallel to the banding
direction and applying a constant calibration factor obtained from LA-ICP-MS
measurements on the same specimen, similar to the procedure of Eggins et
al. (2004). We did not use the depth-resolved laser ablation profiles for
this purpose but used the average value from the profiles for correlation
with the EPMA-derived intensities.
Individual chamber LA-ICP-MS analyses showing correlations between
foraminiferal Mg / Cacc, Sr / Cacc and
Na / Cacc for A. tepida (a) and A. lessonii (b) per salinity condition. Significant orthogonal linear
regressions are indicated with a line, colour coded for salinity (see
legend). Correlation coefficients, slope and intercepts of these regressions
can be found in Supplement S3. In short, within salinity conditions, element
ratios are strongly correlated with each other in A. lessonii,
whereas in A. tepida, element ratios do not (strongly) correlate
with each other. When combining all single-spot data in A. tepida,
element ratios correlate amongst each other because the incorporation of all
three elements increases with salinity, shifting the distributions to higher
values. In A. lessonii, only the Na / Cacc
distributions shift towards higher values with increasing salinity, whereas
Mg / Cacc and Sr / Cacc distributions are
relatively similar between salinity conditions.
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Results
Foraminiferal calcite element ratios and partitioning coefficients
as a function of salinity
Per treatment, from lowest to highest salinity, average
Na / Cacc of the newly formed calcite varied between 9.3 and
10.8 mmol mol-1 for A. lessonii and between 4.7 and
6.4 mmol mol-1 (highest salinity) for A. tepida (Fig. 1),
with a corresponding partition coefficient (note that partition coefficients
are “apparent”, not taking into account speciation/activity of Na) ranging
from 1.90 × 10-4 to 2.20 × 10-4 and from
0.97 × 10-4 to 1.30 × 10-4 for
Amphistegina and Ammonia, respectively (Table 3). For both
species, sets of single-specimen Na / Cacc show slightly skewed
distributions towards higher Na / Cacc for all salinities
(Kolmogorov–Smirnov test, at the 95 % confidence level). Combining all
specimens (based on the average of single-spot measurements per specimen),
Na / Cacc shows a positive linear relationship with salinity
for both A. lessonii and A. tepida (Na / Cacc=0.077±0.017×S+7.13±0.60, F1,186 =20.9, p<0.001 for A. lessonii and Na / Cacc=0.064±0.013×S+3.29±0.44, F1,172=25.9, p<0.001
for A. tepida; Fig. 1). The observed average relative standard
deviation between specimens in Na / Cacc at each of the five
salinities is 15 % for A. lessonii and 20 % for A. tepida. The variance in Na / Cacc between individual specimens
explained by salinity is η2=0.08 for A. lessonii and
η2=0.14 for A. tepida.
Specimen averages of Mg / Cacc and Sr / Cacc
correlate positively with salinity in A. tepida
(Mg / Cacc=0.060±0.011×S+0.51±0.38F1,172=29.9p<0.001 and Sr / Cacc = 0.014±12×10-4×S+1.00±0.04, F1,337=254, p<0.001), whereas neither ratio correlates with salinity in A. lessonii. Average relative standard deviations for the five salinity conditions
per element are 27 % for Mg / Cacc and 9 % for
Sr / Cacc in A. lessonii and 32 % in
Mg / Cacc and 7 % for Sr / Cacc for
A. tepida. In A. lessonii, the proportion of variance in
Sr / Cacc explained by salinity is η2=0.04 (p<0.01) (Mg / Cacc not significant) and for A. tepida,
the proportion of variance in Sr / Cacc explained by salinity
is η2=0.44 and in Mg / Caccη2=0.19 (p<0.001).
Single-spot analyses on Ammonia tepida show that
Na / Cacc and Mg / Cacc are significantly
correlated within the salinity treatments, except for condition S=30
(Fig. 2). For the individual salinity treatments, single-spot
Sr / Cacc and Mg / Cacc, as well as
Na / Cacc and Sr / Cacc are not correlated
significantly with each other, except for S=25. Between salinity
treatments, distributions in this species shift towards higher
Na / Cacc, Sr / Cacc and
Mg / Cacc values with increasing salinity, although for the
range between 30 and 40 Na / Cacc distributions remain rather
similar (Fig. 2). For Amphistegina lessonii, distributions of
Sr / Cacc and Mg / Cacc ratios overlap largely
between salinities, and only Na / Cacc distributions shift
towards higher values (Fig. 2). Within each salinity condition, however,
single-spot Na / Cacc, Mg / Cacc and
Sr / Cacc in this species are positively correlated amongst
each other, whereby the Na / Cacc intercept of these
relationships increases with increasing salinity (Fig. 2 and Supplement S3).
Average El / Cacc ratios of the foraminiferal calcite
(based on average of average specimens value per salinity (Sal) condition
(S25–S45)) ± standard error and corresponding apparent partitioning
coefficients, defined as
DEl = (El / Cacc) / (El / CaSeawater) for A. lessonii
(A.l.) and A. tepida (A.t.). “n/spots” stands
for number of specimens and average number of spots per specimen.
Sal
n/spots
Na / Cacc
DNa
Mg / Cacc
DMg
Sr / Cacc
DSr
mmol mol-1
mmol mol-1
mmol mol-1
A.l.
S25
65/2.6
9.29 ± 0.27
1.90 × 10-4
33.35 ± 1.20
5.94 × 10-3
1.80 ± 0.026
0.199
S30
74/1.9
9.47 ± 0.21
1.90 × 10-4
32.10 ± 1.20
5.64 × 10-3
1.74 ± 0.020
0.189
S35
103/1.9
9.63 ± 0.18
1.98 × 10-4
32.71 ± 1.07
5.94 × 10-3
1.76 ± 0.018
0.191
S40
50/2
10.25 ± 0.31
2.11 × 10-4
35.22 ± 2.60
6.27 × 10-3
1.74 ± 0.034
0.184
S45
44/1.4
10.78 ± 0.30
2.20 × 10-4
33.80 ± 1.68
5.90 × 10-3
1.82 ± 0.036
0.189
A.t.
S25
109/2.5
4.75 ± 0.11
0.97 × 10-4
1.90 ± 0.06
3.40 × 10-4
1.34 ± 0.016
0.156
S30
58/1.8
5.63 ± 0.22
1.13 × 10-4
2.41 ± 0.09
4.24 × 10-4
1.44 ± 0.013
0.156
S35
59/1.8
5.58 ± 0.19
1.15 × 10-4
2.85 ± 0.24
5.17 × 10-4
1.50 ± 0.012
0.163
S40
93/1.8
5.70 ± 0.16
1.17 × 10-4
2.73 ± 0.15
4.86 × 10-4
1.55 ± 0.017
0.164
S45
201.3
6.39 ± 0.37
1.31 × 10-4
3.27 ± 0.27
5.70 × 10-4
1.61 ± 0.038
0.168
Size and chamber effect on Na <inline-formula><mml:math id="M380" display="inline"><mml:mo>/</mml:mo></mml:math></inline-formula> Ca<inline-formula><mml:math id="M381" display="inline"><mml:msub><mml:mi/><mml:mtext>cc </mml:mtext></mml:msub></mml:math></inline-formula>and inter-specimen
variance
Specimens of A. lessonii produced most new chambers at salinities of
25, 30 and 35, closest to the salinity in their “natural” habitat (Burgers'
Zoo aquarium, salinity 33.9–34.3; Ernst et al., 2011). Size averages are
not significantly different between these salinity treatments, based on a
Kruskal–Wallis test, whereas specimens grown at salinities of 40 and 45 were
significantly smaller than those from lower salinities, reflecting lower
chamber addition rates over the course of the culturing experiment at higher
salinity (Fig. 3). With all specimens combined, Na / Cacc is not
significantly related to size in A. lessonii. Specimens of
A. tepida produced less chambers at salinity 45, possibly because
such a high salinity is probably close to its tolerance levels (Murray,
2014), even though this species is adapted to relatively large salinity
shifts in their tidal flat habitat. Specimens in the lower-salinity groups
(25, 30, 35) grew larger compared to specimens grown in two of the highest-salinity groups (Fig. 3). Combining all specimens, Na / Cacc is
significantly related to size in A. tepida, yet with a small slope
(-0.003) and just within the 95 % confidence interval (p=0.04).
Box plots (a, b) showing the size distributions (median,
first
and third quartiles, minimum and maximum values) for each salinity condition
– n=24, 40, 60, 27, 33 for A. lessonii and n=38, 24, 28, 41, 15
for A. tepida. Letters a, b, c indicate significant different
population means, based on ANOVA (p<0.001). Panels (c, d) show
the Na / Ca values against size measurements per individual, colour coded
per salinity condition (see legend), for A. lessonii and A. tepida. Significant linear regression lines are plotted for A. lessonii.
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Within each salinity tested, single-chambered Na / Cacc
is slightly positively related to size for the specimens of A. lessonii cultured at salinities 25 (slope = 0.008, R2=0.32, p<0.01), 30 (slope = 0.002, R2=0.11, p<0.05)
and 35 (slope = 0.005, R2=0.18, p<0.001). For the
same species, Mg / Cacc is positively correlated with size at
salinities 25, 30 and 35, with a similar slope of 0.03 (p<0.05).
Sr / Cacc also shows a positive relationship to size within
salinities 25, 30 and 35 with slopes of 0.0007, 0.0003, and 0.0005 (p<0.001) respectively. For A. tepida, there is only a slight negative
correlation between size and Sr / Cacc for specimens cultured
at salinity 25 (slope = 9.9×10-4, p<0.001) and no
significant correlation for the other conditions, or between size and
Na / Cacc and Mg / Cacc in any of the
salinities.
Foraminiferal Mg / Cacc (panels a) and
Na / Cacc (panels b) intensity ratio maps, obtained
with EPMA, for two specimens of A. lessonii grown at a salinity of
30 (row 1) and 25 (row 2) and one specimen of A. tepida (row 3).
Panels (d) show profiles for Mg / Ca (blue) and Na / Ca
(red), based on averaged EPMA ratios scaled to LA-ICP-MS measurements of the
same specimen, of an averaged lateral profile area through the chamber wall
perpendicular to the lamella separated by organic linings (purple
rectangles in c). The transect area is indicated with a purple
rectangle, on top of a backscatter SEM image (c), showing that the
high El / Ca bands overlap with the primary organic sheet (POS, marked
with dashed red line) and subsequent organic linings. See Supplement S4 for the
results for three more specimens.
![]()
At the lowest salinity, Na / Cacc in the F chamber (newest
chamber) shows slight (0.9 mmol mol-1 Na / Ca higher median) but
significantly
higher values than the F-2 chambers for A. lessonii (multicomparison
test based on Kruskal–Wallis test, p < 0.05). For specimens of
A. lessonii cultured at other salinities and for A. tepida
at any of the salinities tested, no significant correlations between
Na / Cacc and chamber position were observed (note that only
chamber positions F to F-2 were taken into account, as for the lower chamber
position sample numbers were insufficient). Furthermore, chamber position
shows no significant effect on Mg / Cacc and
Sr / Cacc.
To further investigate the variance between and within individuals, a
multiway ANOVA was performed to investigate the effect on
Na / Cacc per salinity condition. Inter-individual variance
is significant and larger than the variance between chamber groups and
intra-individual variance in all salinity groups, with the between-individual
variability accounting for η2=0.75±0.11/0.84±0.03 of the variance (p<0.001) for A. lessonii and
A. tepida, respectively. The variance due to chamber position
is not significant and the remaining intra-individual variance accounts for
η2=0.09±0.05/0.08±0.05 for A. lessonii and A. tepida, respectively.
Elemental distributions in the chamber wall
EPMA maps of cross-sectioned chamber walls of A. lessonii show that,
within the resolution limits of the technique, bands of elevated
Na / Cacc intensities overlap with zones of elevated
Mg / Cacc (Fig. 4 and Supplement S4). Mg bands show higher
amplitudes than Na bands but clearly coincide spatially. Comparing EPMA maps
with the backscatter SEM image of the exposed sections shows that the bands
with the highest Na / Cacc and Mg / Cacc occur in
the proximity of the organic linings, which are clearly visible in the
backscatter SEM image (Fig. 4), with a number of high Na- and Mg-rich bands
with slightly lower maximum intensities occurring towards the outer chamber
surface coinciding with subsequent organic linings. For A. tepida,
one band of elevated Mg / Cacc is visible, coinciding with
the POS with no clear Na / Cacc banding being detected.
Discussion
The effect of salinity and DIC on Na <inline-formula><mml:math id="M454" display="inline"><mml:mo>/</mml:mo></mml:math></inline-formula> Ca<inline-formula><mml:math id="M455" display="inline"><mml:msub><mml:mi/><mml:mtext>cc</mml:mtext></mml:msub></mml:math></inline-formula>,
Mg <inline-formula><mml:math id="M456" display="inline"><mml:mo>/</mml:mo></mml:math></inline-formula> Ca<inline-formula><mml:math id="M457" display="inline"><mml:msub><mml:mi/><mml:mtext>cc</mml:mtext></mml:msub></mml:math></inline-formula> and Sr <inline-formula><mml:math id="M458" display="inline"><mml:mo>/</mml:mo></mml:math></inline-formula> Ca<inline-formula><mml:math id="M459" display="inline"><mml:msub><mml:mi/><mml:mi mathvariant="normal">cc</mml:mi></mml:msub></mml:math></inline-formula>
The single-specimen Na / Cacc data of the cultured A. lessonii and A. tepida both correlate positively with salinity
(Table 3, Fig. 1). This is in line with previous calibrations (for
Ammonia tepida, Wit et al., 2013; for cultured
Globigerinoides ruber, Allen et al., 2016; for field-collected
G. ruber and G. sacculifer, Mezger et al., 2016). However,
our Na / Ca–salinity calibration for A. tepida is somewhat less
sensitive than that observed earlier for the same species (Wit et al., 2013).
An offset in Na / Cacc values between calibrations for a single
species has been reported previously for the planktonic G. ruber and
G. sacculifer (e.g. Mezger et al., 2016; Allen et al., 2016). Such
an apparent discrepancy between studies may be caused by differences between
cultures or in situ conditions in one of the conditions not focussed on (e.g.
carbon chemistry, light intensity). Alternatively, subtle analytical
differences (e.g. differences in cleaning procedures), statistical reasons
(for example differences in the number of analyses or sample size) or the
effect of genotypic variability on element incorporation (Sadekov et al.,
2016) may also play a role. Although the calibration presented here consists
of many more data points compared to those in Wit et al. (2013), we do not
want to dismiss the latter as several parameters (like cleaning procedures or
the source of the seawater used for the culture media) inherently vary
(marginally) between studies. As such the difference observed between studies
merely illustrates the potential range for this species.
Contrasts in sensitivities such as observed for Na / Cacc
between calibrations also apply to Mg / Cacc and
Sr / Cacc, both of which here show an increase with salinity
in A. tepida but not in A. lessonii (Fig. 1). Previous
culturing experiments with Ammonia tepida, however, showed a smaller
sensitivity of Mg / Cacc to salinity
(0.029–0.0044 mmol mol-1 change per salinity unit; Dissard et al.,
2010b) than that reported here (0.06). Still, all these sensitivities are
considerably lower than that reported in Kısakürek et al. (2008) for
the planktonic G. ruber (0.23 when Mg / Cacc is
assumed to increase linearly with salinity) but in the same range as that
reported by Nürnberg et al. (1996) for G. sacculifer (0.05). The
sensitivity of Sr / Cacc to salinity in A. tepida
(0.014; Table 3) is comparable to that for O. universa (0.008; Lea
et al., 2008) and G. ruber (0.02; Kisakürek et al., 2008) and
similar to the significant effect of salinity on Sr incorporation in the same
species (0.01–0.02, depending on temperature) found by Dissard et
al. (2010b).
Sea water carbonate chemistry is an additional factor potentially affecting
trace metal uptake (e.g. Lea et al., 1999; Keul et al., 2017; Russell et al.,
2004). Since salinity and dissolved inorganic carbon concentration in the
culture media co-varied in our experiments similar to the natural environment
(Table 1), Na / Cacc in our cultured specimens also correlates
positively to sea water [DIC]. However, sodium incorporation has been shown
to be independent from changes in carbonate chemistry in cultured
Amphistegina gibbosa and several other benthic hyaline and
porcelaneous species (Van Dijk et al., 2017a). Additionally, Allen et
al. (2016) also found no significant effect of carbonate chemistry (i.e.
varying [CO32-]) on Na incorporation in cultured G. ruber,
suggesting that the variability in Na / Cacc observed here in
A. lessonii can be attributed to changes in salinity rather than
[DIC]. However, future studies should disentangle the impacts of DIC and
salinity on Na / Ca, in order to increase proxy confidence in areas where
Na / Ca and DIC relationships differ from the global average. Previous
studies showed that Sr / Cacc correlates positively to [DIC]
in A. tepida (Keul et al., 2017), which may account for part of the
correlation between Sr / Cacc and salinity reported here for
this species. The published sensitivity of Sr / Cacc to [DIC]
is approximately 2 × 10-5 mmol mol-1 change in
Sr / Cacc for every 1 µmol kg-1 change in
[DIC], likely representing the maximum potential effect of DIC on Sr
partitioning given that others found no significant effect (Dissard et al.,
2010a). For a change in ∼ 850 µmol kg-1 (Table 1), this
would amount to an increase in Sr / Cacc of
0.019 mmol mol-1 (Keul et al., 2017) over the salinity range studied
here, thereby accounting for approximately 7 % of the total observed
change in Sr / Cacc (Table 3). Inorganic carbon chemistry is
known to affect growth rates and shell weights in benthic foraminifera
(Dissard et al., 2010a; Keul et al., 2013), which in turn may affect
incorporation of Sr and Mg, hence providing a mechanistic link between
inorganic carbon chemistry and element partitioning.
El / Ca ratios of specimens of both species grown within each salinity
condition are characterized by a relatively large variability. In the overall
data set, salinity only explains around 8 % of the variation in Na
incorporation for A. lessonii and 14, 19 and 44 % of Na, Mg and
Sr incorporation in A. tepida. Nevertheless, for A. lessonii, the Na / Ca mean values (which translates to the values
obtained from traditional solution-ICP-MS) fit the regression model
relatively well (Fig. 1). However, given the low sensitivity, many specimens
are required to reduce the uncertainty (Supplement S5). This is reflected by the
relatively wide prediction bounds for the Na / Ca–salinity regressions,
indicating an uncertainty associated with a single Na / Cacc
measurement. The relatively large inter-specimen variability in
element / Cacc ratios has been reported and discussed before
(e.g. Sadekov et al., 2008; De Nooijer et al., 2014a), but the cause for this
variability remains to be identified.
Inter-specimen, inter-species and intra-shell
El <inline-formula><mml:math id="M513" display="inline"><mml:mo>/</mml:mo></mml:math></inline-formula> Ca<inline-formula><mml:math id="M514" display="inline"><mml:msub><mml:mi/><mml:mtext>cc</mml:mtext></mml:msub></mml:math></inline-formula> variability
Single-chamber measurements show that Na / Cacc for both
species varies between chambers (i.e. specimens) with a RSD (relative
standard deviation) of 15–20 %, despite identical culture conditions
(Fig. 1). Since the analytical error on Na / Cacc accounts for
approximately 2 % (Table 2), a large portion of the observed variability
between specimens must be due to ontogeny and/or inter-specimen differences
in biomineralization controls (De Nooijer et al., 2014a).
Foraminiferal shell sizes at salinities 40 and 45 are significantly smaller
than those cultured at lower salinities. When combining data from all
salinities, however, there is no (A. lessonii) or only a very small
(A. tepida) negative correlation between Na / Cacc and
shell size, as opposed to a more substantial negative correlation as observed
by Wit et al. (2013). In fact, there appears to be a growth optimum around
salinity of 30–35, whereas growth at higher salinities might be hampered
(Fig. 3). This may indicate that the earlier observed negative correlation
between size and Na / Ca was the result of indirect co-variation with
salinity rather than a causal relationship resulting in lower Na / Ca
values in smaller specimens. This is corroborated by our observation that,
for individuals grown at a similar salinity, the relationship between
Na / Cacc and size is either slightly positive or absent.
Hence, size unlikely affects the observed inter-specimen variability in
Na / Cacc, which is supported by the absence of a correlation
between chamber position (and hence ontogenetic stage) and
Na / Cacc. This implies that measuring specimens of different
size fractions or measuring different or multiple chambers should not
significantly affect the application of the Na / Cacc salinity
proxy. However, sufficient specimens (n>30, for an error margin
< 5 % at the 95 % confidence level; Sadekov et al., 2008; De
Nooijer et al., 2014a) are required for measurements. As most variability is
between individuals rather than between chambers (Sect. 3.3), analysing more
chambers of the same specimen would increase the accuracy of the measurement
but not improve the precision of the salinity estimate, given the large
inter-specimen variability. Without a major effect of ontogeny, physiological
processes at the organismal level are more likely to cause observed large
inter-specimen variability in Na / Cacc; however, these
processes remain poorly understood.
In A. lessonii, single-spot Na / Cacc,
Sr / Cacc and Mg / Cacc are correlated amongst
each other within each salinity condition (Fig. 2). Correlation coefficients
between the three element ratios are similar for the different salinities,
with a superimposed increase in the Na / Cacc relative to that
of Mg / Cacc and Sr / Cacc with increasing
salinity (Supplement S3). In contrast, single-spot Sr / Cacc and
Mg / Cacc in A. tepida are not correlated, whereas
incorporation of all these elements increases significantly with salinity.
Within salinities Mg / Cacc and Na / Cacc are
significantly correlated in four out of the five salinities, but with much lower
correlation coefficients compared to A. lessonii (Fig. 2 and
Supplement S3). However, between the different salinities these elements are
correlated in A. tepida, implying that for A. tepida
salinity is one of the actual parameters controlling Na / Cacc,
Mg / Cacc and Sr / Cacc element uptake.
Within conditions, the correlations between both Sr / Cacc
and Na / Cacc with Mg / Cacc in A. lessonii differ from the correlation of Sr / Cacc with
Mg / Cacc (correlation absent) and Na / Cacc with
Sr / Cacc (weaker correlation) for A tepida. The
differences between the correlations likely reflects differences in their
calcification pathway (e.g. transport of ions to the site of calcification)
and/or might be explained by differences in lattice strain due to the higher
Mg content in A. lessonii, whereas this effect is expected to be
less prominent in low-Mg species such as A. tepida (Evans et al.,
2015). Differences in the calcification pathway may also explain why
Sr / Cacc and Mg / Cacc are correlated with
salinity in A. tepida, but not in A. lessonii
(Sect. 4.1).
In both species, Mg is found to be elevated in bands located close to the
primary organic sheet and to other organic layers (Fig. 4), present in
rotaliid species due to their lamellar calcification mode (Reiss, 1957,
1960). This is similar to reports of within-chamber wall banding in many
elements in other species (Branson et al., 2016; Eggins et al., 2004; Sadekov
et al., 2005; Paris et al., 2014; Spero et al., 2015; Fehrenbacher et al.,
2017; Kunioka et al., 2006; Steinhardt et al., 2015; Hathorne et al., 2009).
In planktonic species element banding has been related to diurnal light–dark
cycles rather than the addition of a new lamella with chamber addition (Spero
et al., 2015; Fehrenbacher et al., 2017). Whether, in the species studied
here, chamber addition (and hence element banding) is related to day–night
cycles remains to be investigated. As in other studies, the Na and Mg bands
are spatially correlated (Fig. 4). For Ammonia tepida, the banding
in both elements is less pronounced than for Amphistegina lessonii,
which is likely related to the (much) lower average Mg / Cacc
and Na / Cacc ratios in the former species. Alternatively, as
the observations are close to the spatial resolution of the method, the
observed pattern could also be due to the band's width being smaller in
A. tepida compared to A. lessonii.
Biomineralization controls on element uptake
How elements are transported to the site of calcification and what the
role of sea water vacuolization, leakage, trans-membrane transport of ions,
pH regulation and precipitation rate is, and how this differs between species and
specimens, remain to be discovered. The overall element composition of the
calcite precipitated by A. lessonii suggests that the calcification
process of this species may more closely resemble inorganic calcite
precipitation from sea water, compared to that in Ammonia tepida and
other low-Mg calcite precipitating species. As a result, more elements (like
Mg) are incorporated and crystal lattice strain in intermediate-Mg calcite
species is elevated, which may promote incorporation of other elements
through stress compensation (Mucci and Morse, 1983; Mewes et al., 2015). This
would explain the observed inter-element correlations within salinities.
Another difference between the species studied here may be caused by
differences in CaCO3 phase shifts during calcite precipitation (e.g.
Bots et al., 2012; De Yoreo et al., 2015). A metastable vaterite pre-cursor
phase recently found in two planktonic species may explain the low Mg
incorporation relative to inorganic calcite (Jacob et al., 2017). The higher
Mg contents of A. lessonii could be related to the (partial) absence
of a vaterite–calcite transformation in this species. An amorphous calcium
carbonate (ACC) pre-cursor phase has been observed in other marine
biomineralizing organisms (e.g. Weiner et al., 2003; Giuffre et al., 2015)
and often been hypothesized to play a role in foraminiferal calcification
(Erez, 2003; De Nooijer et al., 2014b), although it has not yet been directly
detected. A higher Mg concentration at the site of calcification could
hypothetically result in a phase shift from ACC
directly into to calcite, whereby Mg is stabilizing the ACC, as described by
Littlewood et al. (2017). In inorganic calcite, the absence of a vaterite
precursor phase also enhances the incorporation of other metals incompatible
with calcite, such as Sr (Littlewood et al., 2017), and a similar process could
hypothetically contribute to inter-species differences in element
partitioning similar to that observed here. Although the strong fractionation
against Mg in A. tepida could reflect double fractionation through a
vaterite–calcite transformation (Jacob et al., 2017) the low-Mg content might
as well reflect a more enclosed site of calcification, whereby ions are
mainly transported trans-membrane (Nehrke et al., 2013; De Nooijer et al.,
2014b). However, the experiments here do not allow distinguishing between
these (and other) potential mechanisms. Trans-membrane transport (TMT) of
Ca2+ and concomitant leakage of Mg2+ and Sr2+ might be more
sensitive to differences in ionic strength and element concentrations, hence
possibly explaining the salinity effect on the incorporation of these
elements in A. tepida, whereas it does not in A. lessonii,
assuming that TMT contributes relatively more to the supply of ions to the
site of calcification in this species compared to A. lessonii, which
might be relatively more dependent on sea water vacuolization. However, since
there are many, both biotic and abiotic, mechanisms that can (simultaneously)
influence (coupled) element partitioning, it is challenging to resolve the
exact mechanism responsible for inter-specimen and inter-species differences
in El / Ca.
The spatial correlation between the intra-shell distributions of Mg and Na,
associated with the organic linings, suggests a coupled control on these
elements during the calcification process, which is in line with the observed
inter-specimen correlations. This suggests that incorporation of these
cations is influenced by similar biomineralization mechanisms, related to sea
water vacuolization (Erez, 2003; Bentov and Erez, 2006), trans-membrane
transport of elements (Nehrke et al., 2013), the lattice-strain effect (Evans et
al., 2015) and/or metastable precursor phases (Jacob et al., 2017). The
relative contributions of these mechanisms might differ between species,
resulting in the observed differences in element incorporation and different
inter-element correlations between species. Differences in the efficiency of
such processes between specimens might cause the observed inter-specimen
variability, whereas changes in these processes during the calcification time
could be responsible for the observed correlation between elements within the
chamber wall.