Trophic niche separation of two non-spinose planktonic foraminifers Neogloboquadrina dutertrei and Pulleniatina obliquiloculata

Abstract

Based on laboratory observations, planktonic foraminifers are omnivorous, feeding zooplankton and phytoplankton. Spinose species tend toward greater dependence on zooplankton prey than on phytoplankton prey, while non-spinose species are more adapted to herbivorous diets. However, the trophic activity of planktonic foraminifers in the natural environment and their trophic positions in the marine food web have not yet been fully understood. The trophic position (TP) of two non-spinose species, Neogloboquadrina dutertrei and Pulleniatina obliquiloculata, was determined by differences in the nitrogen isotopic composition between two amino acids (glutamic acid and phenylalanine). Results show that TP values of N. dutertrei were ~ 2.4, indicating dependence on omnivorous (mixed herbivorous and carnivorous) diets, while those of P. obliquiloculata were ~ 2.1, indicating dependence on herbivorous diets. Together with previous laboratory observations, these TP values suggest that N. dutertrei is a detritivore or scavenger, while P. obliquiloculata is generally a herbivore. This trophic niche separation likely allows these two planktonic foraminiferal species to live within a similar depth zone in the open water column and provides a clue for understanding causes of spatial and temporal changes in their relative abundances in living and sediment assemblages.

1 Introduction

Planktonic foraminiferal species and assemblages are useful biological proxies for paleo-sea surface water properties (e.g., Berger 1969; Fischer and Wefer 1999; Kucera 2007) because the distribution and abundance of planktonic foraminiferal species are mainly controlled by environmental factors such as sea surface temperature, salinity, dissolved oxygen, light attenuation, and nutrient availability (e.g., Hemleben et al. 1989; Murray 1991; Watkins et al. 1998; Kuroyanagi and Kawahata 2004; Schiebel and Hemleben 2017). Biological factors such as the abundance of prey and predators may also control the distribution and abundance of planktonic foraminifers, especially on regional and seasonal scales dominated by upwelling (Thiede 1975; Ortiz et al. 1995; Schiebel et al. 2004). Laboratory observations of trophic activity have revealed that planktonic foraminifers are basically known as omnivorous, feeding zooplankton and phytoplankton. Spinose species tend toward greater dependence on zooplankton prey than on phytoplankton prey, while non-spinose species are more adapted to herbivorous diets (e.g., Anderson et al. 1979; Spindler et al. 1984). Diet preferences might also be diverse among different genera and species to avoid diet competition in the same water column. Such trophic niche separation might partly explain the diversity and distribution of planktonic foraminifers living in the surface to subsurface water column and their speciation among different species within a genus or among different genotypes within a morphospecies (Weiner et al. 2012). However, the trophic activity of planktonic foraminifers in the natural environment and their trophic positions in the marine food web have not yet been fully understood.

Trophic position can be estimated based on the nitrogen isotopic composition (δ¹⁵N) of two amino acids (glutamic acid and phenylalanine) in consumer organisms (Chikaraishi et al. 2009). Each amino acid experiences different isotopic fractionation during amino acid metabolism. Glutamic acid shows a large δ¹⁵N enrichment from one trophic level to the next, whereas phenylalanine shows little change in the δ¹⁵N value between trophic levels. The use of the δ¹⁵N differences between these two amino acids is a powerful tool for elucidating the trophic position of organisms in aquatic ecosystems with an error of 0.1–0.2 units (Chikaraishi et al. 2009). The traditional trophic position estimation techniques that rely on the δ¹⁵N values of bulk consumer tissues are sensitive to background isotopic variation between the basal resources of a food web and are hampered by spatial and temporal variations in the δ¹⁵N value of primary producers. In contrast, the trophic position based on δ¹⁵N values of glutamic acid and phenylalanine from a single consumer is independent of such factors and successfully applied in studying the trophic position of aquatic organisms (Chikaraishi et al. 2014; Ohkouchi et al. 2017). Another advantage of the amino acid δ¹⁵N approach is that it permits analyses of small specimens (2 nmol for each amino acid; Chikaraishi et al. 2009), which allows us to assess the trophic functions of innumerable meiofauna such as foraminifers (Tsuchiya et al. 2018).

This amino acid δ¹⁵N approach differs from a recently advanced planktonic foraminiferal shell-bound δ¹⁵N approach, which measures δ¹⁵N in organic matter bound within the calcareous shells (e.g., Ren et al. 2009, 2012; Schiebel et al. 2018; Smart et al. 2018, 2020). Planktonic foraminifers are expected to track the δ¹⁵N of the organic matter produced in the surface ocean. The δ¹⁵N values of bulk tissue and shell-bound nitrogen are similar in absolute value and vary together, supporting the use of shell-bound nitrogen as a recorder of upper ocean δ¹⁵N changes (Smart et al. 2018). In oligotrophic subtropical sites, shell-bound δ¹⁵N values in modern surface sediments and net tows are strongly correlated with the δ¹⁵N values of thermocline (i.e., shallow subsurface water) nitrate (Ren et al. 2012; Smart et al. 2018, 2020). Thus, N₂ fixation and denitrification changes are recorded in foraminiferal shell-bound δ¹⁵N.

In this paper, we apply the amino acid δ¹⁵N approach to determine the trophic position of two non-spinose planktonic foraminiferal species, Neogloboquadrina dutertrei (d'Orbigny, 1839) and Pulleniatina obliquiloculata (Parker and Jones, 1865), both of which are common in tropical to subtropical, warm subsurface water around the deep chlorophyll maximum (DCM) and generally herbivorous (Hemleben et al. 1989; Schiebel and Hemleben 2017). Results of this study imply the trophic niche separation of coexisting planktonic foraminifers in the subsurface water column and a clue for understanding causes of spatial and temporal changes in their relative abundances in living and sediment assemblages.

2 Materials and methods

2.1 Hydrographic data and plankton tow samples

Hydrographic data and plankton tow samples were collected from 0 to 200 m in water depth during the R/V Tansei-Maru cruise (KT-11-25) in the afternoon on October 14, 2011 (full moon phase), at the mouth of Suruga Bay, Japan (34°38.989′N, 138˚33.045'E), where the Kuroshio Current (warm current) flowed off south of Kouzushima Island to the northeast (Fig. 1). Hydrographic data including temperature, salinity, density, dissolved oxygen, and chlorophyll a (Chl a) were measured near the plankton tow sampling site by a conductivity temperature depth sensor (CTD; SBE 9, Sea-Bird Scientific) and a fluorometer (Aquatracka MkIII, Chelsea Technologies Group Ltd.). The upper 200 m was sampled twice using a Vertical Multiple Plankton Sampler (VMPS, Tsurumi-Seiki Co. Ltd.). The tow sampler has a 0.25-m² opening and 100-μm mesh size (NXX13). Four depth intervals (0–20, 20–50, 50–100, and 100–200 m) were sampled during each tow. The samples were fixed with seawater-buffered 5% formalin in a 50-mL vial. Formalin fixation does not affect δ¹⁵N values of amino acids derived from an aquatic consumer (Ogawa et al. 2013).

Fig. 1

Location of a plankton tow site (white circle) in Suruga Bay, Japan. Orange line in the insert map indicates the Kuroshio current at the time of sampling. PO Pacific Ocean, PHS Philippine Sea

Samples were poured into petri dishes. Planktonic foraminifers alive at the time of sampling, which were identified by the presence of cytoplasm, were picked from a wet solution by pipetting using a Pasteur pipette, mounted on a microfossil slide, identified, and counted to the species level. Microphotographs of five common species (Neogloboquadrina dutertrei, Pulleniatina obliquiloculata, Globigerina bulloides d'Orbigny, 1826, Trilobatus sacculifer (Brady, 1877), Globigerinoides ruber white (d'Orbigny, 1839)) were taken using a digital camera attached to a binocular dissecting microscope. The maximum diameter was measured using Image J (NIH) to estimate the size-frequency distribution of each species at each depth interval (Additional file 1: Fig. S1). Since other zooplankton were more abundant than planktonic foraminifers, the remaining samples were divided into 2–5 mL subsamples by pipetting from stirred and homogenized samples according to methods described in Omori and Ikeda (1976). Zooplankton alive at the time of sampling, which were identified by the presence of organic matter, were picked from a wet solution by pipetting using a Pasteur pipette and stored in a glass vial filled with seawater-buffered 5% formalin. The zooplankton were identified and counted (Suidosha, Co., Ltd.) and finally classified to the order level. Counts of planktonic foraminifers and zooplankton were converted to the standing stock (SS; the number of individuals m⁻³ seawater) using the following equations:

$${\text{SS}}_{{\text{planktonic foraminifers}}} = n_{{{\text{sample}}}} \div v_{{{\text{seawater}}}}$$

(1)

$${\text{SS}}_{{{\text{zooplankton}}}} = n_{{{\text{subsample}}}} \div v_{{{\text{subsample}}}} \times v_{{{\text{sample}}}} \div v_{{{\text{seawater}}}}$$

(2)

where n is the count in a sample or subsample (individuals), v_subsample is the volume of a subsample (2–5 mL), v_sample is the volume of a sample (50 mL), and v_seawater is the volume of seawater. Since the volume of seawater filtered by a plankton net was not obtained using a flow meter, v_seawater is calculated by the opening area of a plankton sampler (0.25 m²) multiplied by the water depth interval (20–100 m).

In order to estimate the abundance of particulate organic matter (POM), the remaining residue excluding planktonic foraminifers and zooplankton (referred to as residue POM) and another 5-mL subsample (referred to as total POM) were filtered, wet weighed, and converted to the wet weight of POM (mg m⁻³) using the following equation:

$${\text{POM}} = \, w_{{{\text{subsample}}}} \div v_{{{\text{subsample}}}} \times v_{{{\text{sample}}}} \div v_{{{\text{seawater}}}}$$

(3)

where w_subsample is the mass of POM in a subsample.

In order to correlate the standing stock data with environmental variables, the median of environmental variables such as temperature, salinity, density, dissolved oxygen, and Chl a, was calculated for each depth interval. Standing stock data of planktonic foraminifers (N. dutertrei, P. obliquiloculata) and zooplankton (Calanoida, Cyclopoida, Poecilostomatoida, Harpacticoida, all copepods, other zooplankton, all zooplankton) and the abundance of POM (residue and total) were square-root transformed. Transformed standing stock data of planktonic foraminifers were correlated with other variables (environmental, zooplankton, POM) using Pearson’s correlations. In addition, the standing stock data were analyzed with a linear model using Chl a and total POM as explanatory variables to examine the effects of biological variables. These statistical analyses were conducted in R 4.1.1 (R Core Team 2021). We also calculated correlations between the standing stock data and the mean of environmental variables. The correlation results using the mean were similar to those using the median.

2.2 Amino acid nitrogen isotope analysis

In this study, we measured δ¹⁵N values of the bulk cells (i.e., the sum of the cell cytoplasm, organic membranes, and intracrystalline protein in a shell) of fixed specimens of N. dutertrei and P. obliquiloculata. Since the amount of intracrystalline proteins is fewer than the cell cytoplasm, δ¹⁵N values in the bulk cells would be expected to indicate the short-term value of their metabolism. Due to the low number of specimens, fixed specimens from two replicate samples for each water depth interval were pooled for the bulk cell analysis (i.e., one pooled sample of each water depth interval was measured for each species). The number of N. dutertrei and P. obliquiloculata specimens measured for each water depth interval ranged from 18 to 353 and 14 to 137, respectively. Measured test size ranged from 150 to 650 μm for all depth intervals of both species.

The δ¹⁵N value of amino acids was determined according to Chikaraishi et al. (2009). Briefly, each specimen was hydrolyzed in 12 M HCl at 110 °C, and then, the hydrolyzate was washed with n-hexane/dichloromethane (3:2, v/v) to remove any hydrophobic constituents. After derivatization with thionyl chloride/2-propanol (1:4, v/v) and subsequently with pivaloyl chloride/dichloromethane (1:4, v/v), the derivatives of the amino acids were extracted with n-hexane/dichloromethane (3:2, v/v). The δ¹⁵N value of individual amino acids was determined by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) using a Delta^plusXP isotope ratio mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled with a 6890 N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) via combustion and reduction furnaces. δ¹⁵N value is defined as δ¹⁵N = ([¹⁵N/¹⁴N]_sample/[¹⁵N/¹⁴N]_Air − 1) × 1000 and expressed in conventional (‰) against that of air (Air).

The trophic position (TP_Glu/Phe) of the sample was calculated based on the following equation proposed by Chikaraishi et al. (2009):

$${\text{TP}}_{{{\text{Glu}}/{\text{Phe}}}} = \, \left( {\left( {\delta^{{{15}}} {\text{N}}_{{{\text{Glu}}}} - \, \delta^{{{15}}} {\text{N}}_{{{\text{Phe}}}} - { 3}.{4}} \right)/{7}.{6}} \right) \, + { 1}$$

(4)

where δ¹⁵N_Glu and δ¹⁵N_Phe represent the δ¹⁵N values of glutamic acid and phenylalanine, respectively, 3.4 is the isotopic difference between glutamic acid and phenylalanine in primary producers, and 7.6 is the offset of the trophic discrimination factor of these two amino acids per trophic position increase. Although this equation may need modification (particularly for the trophic discrimination factor) for some specific organisms, such modification is not required for organisms in the lower-trophic-level hierarchy of food webs (McMahon and McCarthy 2016). The trophic position is expected to be 1.0 for a “pure” primary producer and 2.0 for a “pure” primary consumer.

Propagation error (potential uncertainty) in TP_Glu∕Phe value has been calculated by taking into account the propagation of 1σ for δ¹⁵N_Glu, δ¹⁵N_Phe, the isotopic difference between glutamic acid and phenylalanine in primary producers, and the offset of the trophic discrimination factor (TDF) of these two amino acids per trophic position increase in Eq. (4) (Chikaraishi et al. 2009, 2014), and is expressed in the following equation (Kruse et al. 2015):

$${1}\sigma_{{{\text{TL}}}} = \, \left[ {\left( {y^{{2}} {/7}.{6}x} \right) \, + \, \left( {{1}\sigma_{{{\text{TDF}}}} {\text{/TDF}}} \right)^{{2}} } \right]^{{{1}/{2}}}$$

(5)

where x = δ¹⁵N_Glu − δ¹⁵N_Phe − 3.4, y = [2(1σ_m)² + (1σ_β)²]^1/2, 1σ_m = 0.5‰, 1σ_β = 0.9‰, and 1σ_TDF = 1.2‰, after Chikaraishi et al. (2009). Previous studies have indicated that the potential uncertainty in the TP_Glu/Phe value calculated via propagation of error is 0.2–0.4 for each trophic level based on an assumed standard deviation of 0.5‰ (1σ) for the observed δ¹⁵N values of glutamic acid and phenylalanine (Chikaraishi et al. 2009).

3 Results

3.1 Environmental variables

Water temperature was relatively stable (approx. 22.7 °C) at 0–40 m depth, and the thermocline was located at 40–60 m depth (Fig. 2, Additional file 2: Table S1). Salinity was stable (approx. 34.0) at 0–40 m depth, and the pycnocline was located at 40–60 m depth (Fig. 2). Chl a concentrations were 0.3 μM on average at 0–49 m depth, showed the highest peak (1.1 μM) at 50 m depth, with the deep chlorophyll maximum (DCM) being located in the center of the pycnocline, and then gradually decreased down to 0.04 μM at a deeper depth. Dissolved oxygen was stable (4.7 ml L⁻¹ on average) at 0–40 m depth and then decreased at 40–60 m across the pycnocline.

Fig. 2

Hydrographic conditions at the plankton tow site in Suruga Bay, Japan. A gray zone in a indicates the thermocline/halocline/pycnocline, while a dashed line indicates the deep chlorophyll maximum (DCM)

3.2 Planktonic foraminifers

A total of 11 planktonic foraminiferal species were identified (Fig. 3, Additional file 2: Table S2). The assemblage was mainly composed of N. dutertrei, P. obliquiloculata, G. bulloides, T. sacculifer, and G. ruber white. Of these five species, N. dutertrei, P. obliquiloculata, and G. bulloides were common at all the water depths sampled and account for > 90% of the total foraminiferal standing stock. The standing stock of planktonic foraminifers was high in the upper 50 m. The highest value (~ 50 individuals m⁻³) was recovered in the upper 20 m. N. dutertrei and P. obliquiloculata were more common in the upper 50 m but decreased at water depth below 50 m. The standing stock of G. bulloides was reasonably uniform (2.8 individuals m⁻³) throughout the water depths sampled. T. sacculifer and G. ruber white were low in abundances (~ 1 individual m⁻³) throughout the water depths sampled.

Fig. 3

Standing stocks of living planktonic foraminifers at four depth intervals (0–20, 20–50, 50–100, and 100–200 m; two replicates) in Suruga Bay, Japan

3.3 Zooplankton and particulate organic matters

Zooplankton, excluding planktonic foraminifers, were abundant in the upper 50 m, gradually decreasing at deeper depth (Fig. 4, Additional file 2: Table S3). Copepods were the most common zooplankton, accounting for more than 80% of the total zooplankton standing stock. Of the copepod assemblage, Calanoida, Cyclopoida, and Poecilostomatoida were more common than other taxa, comprising more than 95% of the assemblage. Decreasing zooplankton abundance with depth was mostly explained by the standing stocks of Calanoida and Poecilostomatoida, both of which showed the maximum abundance of 382 and 294 individuals m⁻³ in the upper 50 m, respectively. Total POMs were also abundant in the upper 50 m (~ 213 mg m⁻³), decreasing at deeper depth (Additional file 2: Table S3).

Fig. 4

Standing stocks of living zooplankton at four depth intervals (0–20, 20–50, 50–100, and 100–200 m; two replicates) in Suruga Bay, Japan

3.4 Correlations and regression with biological variables

The standing stock of N. dutertrei was highly correlated with most environmental and biological variables (> 0.9; Additional file 2: Table S4). The standing stock was highly correlated with temperature (0.98), salinity (− 0.91), and Chl a (0.97), while it was weakly correlated with standing stocks of Cyclopoida (0.57) and Harpacticoida (0.46). The standing stock of P. obliquiloculata was highly correlated with most environmental and biological variables (> 0.8). In particular, the standing stock was highly correlated with that of Poecilostomatoida (0.93), temperature (0.88), salinity (− 0.81), and Chl a (0.86), while it was weakly correlated with residue POM (0.57), standing stocks of Cyclopoida (0.46) and Harpacticoida (0.43).

Linear models indicate that standing stocks of both species were significantly affected by Chl a (N. dutertrei, p < 0.0001; P. obliquiloculata p < 0.05), but not by total POM (ns, Table 1). Both species were highly correlated with Chl a concentration distribution over the upper water column (Fig. 5). Linear regression analysis indicates that the standing stock of N. dutertrei was explained as N_{N. dutertrei} = 13.65 Chl a + 0.53 (R² = 0.95, adjusted R² = 0.94, p < 0.00005), while that of P. obliquiloculata was explained as N_{P. obliquiloculata} = 6.75 Chl a + 0.64 (R² = 0.74, adjusted R² = 0.70, p < 0.006).

Table 1 Results of the linear model for standing stocks of Neogloboquadrina dutertrei and Pulleniatina obliquiloculata

Fig. 5

Correlations of standing stocks of two non-spinose planktonic foraminiferal species (Neogloboquadrina dutertrei and Pulleniatina obliquiloculata) with chlorophyll a concentration

3.5 Nitrogen isotopic composition of amino acids and estimated TP_Glu/Phe

Observed TP_Glu/Phe values are averages based on bulk specimens of each foraminiferal species and characterized by a low propagation error (potential uncertainly) (Table 2, Additional file 2: Table S5). However, since the propagation error was significant (0.67) for P. obliquiloculata at 100–200 m depth, the value was not used for further discussion. TP_Glu/Phe values for bulk specimens of both species were similar through the water column (Fig. 6). The TP_Glu/Phe values for bulk specimens of P. obliquiloculata (~ 2.1) were lower than the values for bulk specimens of N. dutertrei (~ 2.4). The exception was TP_Glu/Phe values at 20–50 m depth, similar for both species (~ 2.3).

Table 2 Nitrogen isotopic composition of glutamic acid (δ¹⁵N_Glu) and phenylalanine (δ¹⁵N_Phe), and estimated trophic position (TP_Glu/Phe) and the propagation error of two non-spinose planktonic foraminiferal species (Neogloboquadrina dutertrei and Pulleniatina obliquiloculata)

Fig. 6

Trophic position (TP_Glu/phe) of two non-spinose planktonic foraminiferal species (Neogloboquadrina dutertrei and Pulleniatina obliquiloculata) along four depth intervals in Suruga Bay, Japan. Error bars indicate the propagation error (potential uncertainly) in TP_Glu/Phe calculated in Eq. (5)

4 Discussion

The water column in the study area consisted of three layers including (1) the surface mixed layer, (2) the thermocline/halocline/pycnocline at the 40–60 m depth interval comprising the DCM, and (3) the subsurface layer below 60 m depth. These observations are consistent with previously reported water mass structure in Suruga Bay, where mixed coastal and outer surface water (0–50 m depth) covers outer Kuroshio water characterized by high salinity (100–200 m depth) (Nakamura and Muranaka 1979). The surface mixed layer and the thermocline/halocline/pycnocline are warmer and more productive than the subsurface water column and accommodate the highest standing stocks of planktonic foraminifers and zooplankton, as shown in high correlations with environmental variables. However, it is difficult to determine their primary limiting factors because many environmental variables covary with water depth.

N. dutertrei and P. obliquiloculata were dominant species in the planktonic foraminiferal assemblage of the upper 50 m. Both species generally show the maximum abundance around the DCM (Schiebel and Hemleben 2017). N. dutertrei is generally distributed in tropical to temperate open oceans and upwelling settings, showing the maximum standing stocks at the DCM (e.g., Ravelo et al. 1990; Schmuker and Schiebel 2002; Watkins et al. 1996, 1998). In water columns around the Japanese Islands, N. dutertrei is found below the pycnocline, and its abundance peaks just below the DCM (Kuroyanagi and Kawahata 2004). P. obliquiloculata is generally distributed in tropical to subtropical surface mixed layer around the thermocline and DCM (e.g., Watkins et al. 1996, 1998; Jentzen et al. 2018). These observations are consistent with high positive correlations of the standing stocks of both species with Chl a in this study and in previous studies (Watkins et al. 1998), although no correlation was observed for N. dutertrei in similar settings (Kuroyanagi and Kawahata 2004).

The TP_Glu/Phe value of a predator is one level higher than that of its prey. The TP_Glu/Phe values of N. dutertrei are ~ 2.4, indicating an omnivorous feeding strategy, while those of P. obliquiloculata are ~ 2.1, indicating dependence mostly on herbivorous diets. Similar TP_Glu/Phe values at 20–50 m depth indicate that P. obliquiloculata becomes more omnivorous near the DCM than above. Previous studies using a variety of marine and terrestrial organisms demonstrated that even if the δ¹⁵N value of amino acids has a variation between individuals of a species, their trophic position can remain unchanged during a given period, even though its food type and/or source has changed dramatically (Chikaraishi et al. 2014).

Laboratory studies demonstrated that diatoms are a significant part of many non-spinose species’ diets and are found in digestive vacuoles in these two species (Anderson et al. 1979; Spindler et al. 1984). Metazoan tissues are also found in the digestive vacuoles of non-spinose species collected in the open ocean, although they can only feebly catch and hold zooplankton prey when grown in the laboratory since the rhizopodial net of non-spinose species is not suited to capture living prey like copepods (Spindler et al. 1984; Hemleben et al. 1989). Therefore, the metazoan tissue is likely obtained from inactive (e.g., dead) organisms caught in the rhizopodia or by snaring fecal matter containing incompletely digested metazoan tissue (Hemleben et al. 1989). These previous laboratory observations and TP_Glu/Phe values in this study suggest that N. dutertrei is a detritivore or scavenger, while P. obliquiloculata generally depends on herbivorous diets. This assumption is supported by variable Ba/Ca signals in shells of N. dutertrei, which are likely due to calcification in a microenvironment enriched in Ba such as marine snow or other organic particulates (Fehrenbacher et al. 2018). Although the standing stock of N. dutertrei is more positively correlated with Chl a than that of P. obliquiloculata (Fig. 5), this might be explained by the increasing abundance of dead metazoan tissues and fecal matters in the upper water column. Slight trophic niche separation inferred from this study may allow these two planktonic foraminiferal species to live within a similar depth zone in the open water column.

Some non-spinose species, including N. dutertrei and P. obliquiloculata, appear to facultatively harbor algal endobionts, chrysophycophytes (chrysophytes), which are capable of photosynthesizing within the perialgal vacuoles (Gastrich 1987; Takagi et al. 2019). The algae are also frequently observed in stages of division within the host vacuoles, thus indicating they are in a healthy state (Hemleben et al. 1989). However, some specimens are entirely devoid of these algae, and some algae were observed in a state of being digested by the foraminifers (Hemleben et al. 1989). Predation on algal endobionts results in TP_Glu/Phe value around two due to a predator–prey interaction in terms of the amino acid metabolism between host and endobiont, while the use of photosynthate from algal endobionts as a nitrogen source results in TP_Glu/Phe value around one (Tsuchiya et al. 2018). Algal symbiont-bearing planktonic foraminifers likely obtain carbon and nitrogen from two distinct sources, the diet and the symbionts (Uhle et al. 1997). For example, the carbon and nitrogen isotopic data suggest that symbiont-bearing spinose species Orbulina universa is indicative of the transfer of isotopically heavy metabolic carbon and nitrogen from its symbionts and relatively lighter carbon and nitrogen from the diet. In contrast, diet is the sole source of metabolic carbon and nitrogen used for amino acid synthesis in symbiont-barren spinose species (Globigerina bulloides) (Uhle et al. 1997). Intermediate depth-dwelling and chrysophyte endobiont-bearing N. dutertrei and P. obliquiloculata exhibit intermediate ranges of tissue and shell-bound δ¹⁵N values between the low-δ¹⁵N surface-dwelling and dinoflagellate symbiont-bearing and high-δ¹⁵N subsurface-dwelling, symbiont-barren species, but more similar to the latter group (Smart et al. 2018, 2020). These previous studies and our results on TP_Glu/Phe values suggest that these two non-spinose species do not have a symbiotic relationship with algal endobionts, but hold algal endobionts to prey on them. Further studies are necessary to reveal a relative dependence between foods and algal endobionts for nitrogen sources of these non-spinose species.

Recently advanced analytical techniques of δ¹⁵N values of shell-bound organic matter of planktonic foraminifera (shell-bound δ¹⁵N) are potential tools for reconstructing past changes in global nitrogen cycling (e.g., Ren et al. 2009, 2012; Schiebel et al. 2018). Late Quaternary glacial-interglacial records indicate that shell-bound δ¹⁵N was higher during the glacial periods than the interglacial periods, suggesting that sea-level driven oscillations in the balance of N₂ fixation and denitrification (e.g., Ren et al. 2009, 2017). Future studies of shell-bound amino acid δ¹⁵N approaches combined with shell-bound organic matter δ¹⁵N analyses would provide information on temporal changes in paleo-trophic levels and food webs associated with glacial-interglacial changes in ocean productivity.

Trophic niche separation of N. dutertrei and P. obliquiloculata revealed in this study may also provide a clue for understanding the causes of spatial and temporal changes in their relative abundances in sediment assemblages. For example, in the East China Sea and the Ryukyu Arc regions, both species are common under the influence of Kuroshio Current, based on surface sediments (Ujiié et al. 2003) and sediment trap data (Xu et al. 2005). In contrast, temporal variations of the relative abundances of N. dutertrei and P. obliquiloculata from the last glacial to Holocene at the Okinawa Trough show a negative relationship (Li et al. 1997; Ujiié and Ujiié 1999; Ujiié et al. 2003). N. dutertrei was common in the last glacial period and the Holocene Pulleniatina Minimum Event (PME; ca. 4.5–3 ka), while P. obliquiloculata increased toward the interglacial but decreased during the PME (Li et al. 1997; Ujiié and Ujiié 1999; Ujiié et al. 2003; Lin et al. 2006). In lower latitudes of the West Pacific, the negative relationship of both species is less obvious (Lin et al. 2006). Temporal changes in ocean productivity associated with these paleoceanographic changes may have influenced food webs and these two species with slightly different trophic levels.

5 Conclusions

The trophic position (TP_Glu/Phe) of two non-spinose species, N. dutertrei and P. obliquiloculata, was determined by differences in the nitrogen isotopic composition between two amino acids (glutamic acid and phenylalanine). The TP_Glu/Phe values of N. dutertrei were ~ 2.4, indicating an omnivorous feeding strategy, while those of P. obliquiloculata were ~ 2.1, indicating dependence mostly on herbivorous diets. The TP_Glu/Phe values in this study, together with previous laboratory observations, suggest that N. dutertrei is a detritivore or scavenger, while P. obliquiloculata is generally a herbivore. The TP_Glu/Phe values also suggest that these two non-spinose species do not have a symbiotic relationship with algal endobionts, but hold those algal endobionts to prey on them. This trophic niche separation may allow these two planktonic foraminiferal species to live within a similar depth zone in the open water column and provide a clue for understanding causes of spatial and temporal changes in their relative abundances in living and sediment assemblages.