Tanabe, K., Shigeta, Y., Sasaki, T. & Hirano, H. (eds.) 2010. - Present and Past Tokai University Press, Tokyo, p. 67-75. Reconstructing the life history of modern and fossil based on the nitrogen isotopic composition of shell organic matter and amino acids

YUICHIRO KASHIYAMA1, 2*, NAOKO O. OGAWA1, YOSHITO CHIKARAISHI1, NAPUSSAKORN KASHIYAMA1, SABURO SAKAI1, KAZUSHIGE TANABE 3, AND NAOHIKO OHKOUCHI1

1 Japan Agency for Marine-Earth Science and Technology 2-15 Natsushima-cho, Yokosuka 237-0061, Japan 2 Present Address: Department of Chemistry, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8571 Ibaraki, Japan (*correspond- ing author, e-mail: [email protected]) 3 Department of Earth and Planetary Science, University of Tokyo 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan

Received April 30, 2008; Revised manuscript accepted September 22, 2008

Abstract. We determined the nitrogen isotopic compositions of organic matter in the shell material of mod- ern nautilids ( pompilius) and a mid- cymatoceratid ( sakalavus from the of Madagascar) to reconstruct the trophic ecology in natural habitats and its change over the ’ lifetimes. The profiles of δ15N values along the growth direction exhibited a common pattern in modern Nautilus specimens, suggesting three ontogenetic stages in the trophic ecology of the . The Embry- onic Stage, represented by the innermost shell formed during embryogenesis, is characterized by relatively elevated δ15N values (12–14‰). The δ15N values show a sharp decrease (by approximately 3‰) shortly after hatching of the shell, recorded between the 7th and 12th septa (the Post-hatching Stage). In the following Juvenile-mature Stage, δ15N values exhibit a relatively stable pattern in the range 10–12‰. The decrease in δ15N values during the transition from the Embryonic Stage to the Juvenile-mature Stage probably reflects a change in nutrient source from yolk to a diet. Analysis of shell material formed in an aquarium following capture from the ocean revealed a sudden drop in δ15N values, probably reflecting the change from natural to artificial diet. The compound-specific nitrogen isotopic compositions of amino acids from the shell of N. pompilius suggest that the above isotopic patterns obtained from the bulk material do indeed reflect changes in the apparent trophic level throughout its life. In contrast to modern Nautilus, δ15N values obtained for C. sakalavus show a gradual increase from 1.2 to 3.8‰ with increasing septum number, suggesting an increase of approximately one trophic step in the food web throughout its life.

Key words: Nautilus pompilius; fossil ; nitrogen isotopes; trophic level; amino acids trophic level; shell organic matter.

Introduction compositions of different stages of marginal-growing shell carbonate, for example, the life histories of both The ecology of extinct animals has primarily been extant and extinct cephalopods can be reconstructed estimated based on fossil morphology and analogy in terms of their living habitat (e.g., Oba et al., 1992; to modern relatives. Even for extant species, the Landman et al., 1994; Moriya et al., 2003). natural lives of organisms are poorly understood due to Analyses of the stable nitrogen isotopes of organic difficulties in undertaking direct observations of their matter within calcified skeletal remains are potentially life habits in nature. The composition of the skeletal useful in estimating the nutritional sources during remains of such organisms, however, potentially records an animal’s lifetime. The stable nitrogen isotopic their ecology as well as their living environment, composition of animal organic tissue is known to reflect the animal’s diet, with a systematically increase in thereby providing a geochemical approach to the 15 indirect observation of remote and past lives. One δ N values toward the upper part of the hierarchy of of the most successful applications in this regard is the food web (e.g., Hobson and Welch, 1992; Keough et al., 1996; Yoshii et al., 1999; Ogawa et al., 2001; stable oxygen isotopic analyses of carbonate mollusk 15 shells, from which the ambient water temperature Post, 2002). The mean enrichment factor of δ N in can be inferred. By determining the oxygen isotopic body tissue is ~3.4‰ per trophic step (e.g., DeNiro and Epstein, 1981; Minagawa and Wada, 1984). Calcified 68 Yuichiro Kashiyama et al. skeletal tissue encapsulates organic material that is analyzed a specimen of the fossil nautiloid Cymatoceras biosynthesized at the time of formation, resulting in the sakalavus () recovered from the Albian potential for preservation for much longer periods than of Mahajanga Province, Madagascar, which preserves body tissue, even in fossil specimens of the geologic alagonitic shell materials and its original nacreous past. Therefore, the nitrogen isotopic composition of structure (Figure 2). skeletal tissue potentially preserves the pristine isotopic signature of organisms, directly reflecting their trophic Isotopic analyses of bulk shell material ecology (e.g., Ostrom et al., 1993). Aragonitic shell material corresponding to various In this context, we have been exploring a new growth stages was milled from septa and the nacreous methodology to reconstruct the trophic ecology layer of the ventral shell wall along its growth direction. of modern and fossil nautiloids based on nitrogen Shells of N. pompilius were cut parallel to the median isotopic analyses of shell material. As with other plane to obtain central slices of the shell (Figure 1). For mollusks, nautiloids deposit a marginal-growing shell specimens Tnb-53 and Rbt-1, each septum was carefully wall throughout their life, as well as periodically detached from the sliced specimens and pulverized after secreting aragonitic septa during the cycle of chamber the surface layer had been planed off. For Tnb-2, Tnb- formation. The organic matter of mollusk shells is 53, and Tnb-88, small amounts of powdered material largely conchiolin, a protein complex secreted from (4–6 mg) were sampled from the ventral shell wall of the mantle during shell growth, hence reflecting the the sliced specimens; here, only the core of the nacreous nitrogen isotopic composition of the animal’s diet layer was sampled by planing parallel to the shell during the period of the shell formation. Therefore, the surface after the removal of the outer surface. nautiloid shell material contains a continuous record of The fossil specimen was cut and polished along the nitrogen isotopic signature of its trophic state. Such the median plane. Small amounts of powdered shell data enable the reconstruction of dynamic changes in material (0.5–6 mg) were carefully sampled from the the trophic state of an individual throughout its life. polished cross-section of each septum wall using an We determined the nitrogen isotopic composition of automated micromill sampler (GEOMILL326; Sakai, the bulk shell material of three specimens of a modern 2007). The precision of the sampler (<10 µm) enabled nautiloid, Nautilus pompilius of the family Nautilidae selective sampling from only the core layer of each from the Philippines, sampled sequentially from the septum. Sample material obtained by micro-milling ventral shell wall along its growth direction, as well as of 2–3 adjacent septa were analyzed together because samples of septa. We also determined the compound- the amount of powder obtained from single septa was specific nitrogen isotopic composition of amino inadequate for accurate analyses. acids from selected samples, which is a novel tool in Nitrogen isotopic analyses were performed using estimating the trophic state of organisms. We also a ThermoFinnigan Delta plus XP isotope-ratio mass discuss the trophic history of N. pompilius and report spectrometer coupled to a Flash EA1112 automatic the results of nitrogen isotopic analyses of aragonitic elemental analyzer via a Conflo III interface (EA/IRMS; shell material from a fossil nautiloid, and consider the Ohkouchi et al., 2005). Nitrogen isotopic compositions possible implications of these data for the animals’ are expressed as conventional δ-notation against trophic ecology. atmospheric N2 (AIR):

15 3 15 14 15 14 Materials and methods δ N (‰) ≡ 10 [( N/ N)sample/( N/ N)standard - 1]

Materials In practice, isotopic compositions were normalized We analyzed one male (Tnb-53) and two female (Tnb- using cross-calibrated in-house standards (Proline [δ15N 2, Tnb-88) specimens of Nautilus pompilius captured = 13.31 ± 0.06 ‰]; and Tyrosine [δ15N = 8.44 ± 0.05 live in Balayan Bay, Batangas, Luzon, the Philippines, ‰]). The powdered shell material of N. pompilius by K. Tanabe and T. Ubukata in 1993 (Figure 1). The was wrapped in precleaned tin capsules for EA/IRMS male specimen was not kept alive, but the two female analysis. The powdered fossil shell was treated with 2 specimens were reared in an aquarium at the University N HCl in precleaned silver capsules to remove excess of Tokyo for approximately 1 year, fed mainly on carbonate and then wrapped in tin capsules (combustion commercially available cultivated prawns. Besides, an improver) after dehydration. The estimated analytical additional adult specimen of N. pompilius (Rbt-1; shell precisions (1σ) of standards (analytical range: 2-35 μg only; source unknown) was used for analysis. We also nitrogen) were <0.5‰. N isotope-based reconstruction of nautiloid life history 69

Figure 1. Shell of Nautilus pompilius (median dorsoventral section; specimen Tnb-53). Circles indicate sampling sites of powdered cal- cium carbonate from the nacreous layer of the ventral shell. The locations of sampling sites are expressed in terms of the spiral length from the center of coiling.

Recent studies have developed a novel methodology Nitrogen isotopic analyses of individual amino acids in estimating the trophic level of organisms based on Powdered samples were obtained from selected parts the nitrogen isotopic composition of selected species of of the ventral shell wall that represent various ontogenic amino acids, such as glutamic acid and phenylalanine stages of the animal (Table 1). Amino acids were (McClelland and Montoya, 2002; Chikaraishi et al., prepared by HCl hydrolysis and derivatized (N-pivaloyl/ 2007). Chikaraishi et al. (submitted to Limnology and isopropyl derivatization) using a modified procedure Oceanography: Methods) suggested that glutamic acid described in Chikaraishi et al. (2007). The nitrogen is systematically enriched in 15N toward the upper levels isotopic composition of the individual amino acids of the food chain (8.0 ± 1.2 ‰ at each trophic step) as a was determined by gas chromatography/combustion/ result of metabolic processes; in contrast, phenylalanine isotope ratio mass spectrometry (GC/C/IRMS) using shows little enrichment in 15N because of the absence of a ThermoFinnigan Delta plus XP IRMS coupled to an nitrogen-involving reactions in its dominant metabolic Agilent Technologies 6890N GC via combustion and processes. Therefore, trophic level is estimated based reduction furnaces. The analytical conditions for GC/ on the δ15N values of glutamic acid and phenylalanine C/IRMS analysis are described in Chikaraishi et al. via the following equation, termed the “Amino acid (2007). In the present study, we focused on the δ15N Trophic Level (ATL):” values of glutamic acid and phenylalanine, from which 15 15 information regarding the trophic ecology of the animal ATL = (δ NGlu – δ NPhe + 4.2) / 7.6 can be evaluated, as described below. 70 Yuichiro Kashiyama et al.

Figure 2. a. Cross section of the fossil specimen (Cymatoceras sakalavus). The specimen is three dimensionally preserved with the matrix filled with spary calcite. The shell material was determined as alagonite by X-ray diffractometry. b. scanning electron micrograph of the fractured cross section of the septum (location indicated by arrow in Fig. 2a), indicating that primary nacreous structure is well preserved.

15 15 where δ NGlu and δ NPhe are the nitrogen isotopic pompilius (Tnb-2, Tnb-53, and Tnb-88) show similar compositions of glutamic acid and phenylalanine, relative isotopic variations along the growth direction respectively. Unlike conventional estimates of trophic (Figures 4 and 5). Elevated δ15N values are observed level based on isotopic analyses of bulk organic tissue, in the innermost shell where located less than 60 mm the ATL indicates the absolute trophic level without in spiral length from the center of coiling. The values requiring knowledge of the isotopic composition of the fall sharply (by 3–4 ‰) in the shell portion between primary producer at the base of the food web. approximately 60 and 100 mm in spiral length from the center of coiling, shifting from 13.4 to 9.7 ‰ for Tnb-2, Results 13.6 to 9.8 ‰ for Tnb-53, and 12.9 to 10.6 ‰ for Tnb- 88. The δ15N value varies only gradually, within a range The nitrogen isotopic compositions of septa in of ~2‰, in the shell portion located more than 100 mm the two specimens of N. pompilius (Rbt-1 and Tnb- in spiral length from the center of coiling; however, 15 53) demonstrated similar ontogenetic variations with Tnb-2 and Tnb-88 record a second sharp fall in δ N increasing septum number (Figure 3). From the 7th to values (~2%) in the apertural shell portion that formed 12th septa, the δ15N values fell sharply, from 15.0 to during the period in the aquarium. 11.1 ‰ in Rbt-1 and from 14.0 to 10.0 ‰ in Tnb-53. The nitrogen isotopic values of septa of the C. After the 13th septum, however, the δ15N values show sakalavus specimen are generally lower than those of only minor variations, increasing slightly (by about 1 N. pompilius, and gradually increase from 1.2 to 3.8 ‰ ‰) to the last septum. with increasing septum number (Figure 6). The nitrogen isotopic compositions of the ventral shell wall in the three live-caught specimens of N. N isotope-based reconstruction of nautiloid life history 71

Figure 3. Ontogenic changes in septal δ15N values in two specimens of Nautilus pompilius (Rbt-1 and Tnb-53). Both specimens show a sharp fall in δ15N values (by ~3 ‰) from the 7th to 12th septa.

stage (e.g., Arnold, 1987), and that primary constriction Discussion developed at the time of hatching (Uchiyama and Tanabe, 1999; Figure 4). Therefore, this trophic stage Modern nautiloid (Nautilus pompilius) corresponds to the period during which an embryo 15 develops solely on nutrition derived from its yolk. The δ N values of shell material from live-caught 15 specimens of N. pompilius suggest the occurrence of The δ N values of the embryonic stage are ~3‰ three ontogenetic stages in terms of trophic strategy: (1) higher than those of the juvenile-mature stage, corresponding to approximately one trophic level (3.4 the “embryonic stage,” when the yolk is the sole source 15 of nutrients; (2) the “post-hatching stage,” during the ‰; Minagawa and Wada, 1984). This difference in δ N early postembryonic stage; and (3) the “juvenile-mature value probably arises because the yolk, the nutritional stage” (Figure 4). An additional stage is identified for source of the animal during the embryonic stage, has a similar nitrogen isotopic composition to that of the those specimens reared and fed on artificial diets (see 15 below). mother (cf., Hobson, 1995). Therefore, the δ N value The embryonic stage is isotopically defined as an of the embryo should indicate one trophic level higher ontogenetic stage that occurs before the onset of a rapid than the adult animal in the juvenile-mature stage. drop in δ15N values within organic tissue. This stage is The post-hatching stage is isotopically defined as an ontogenetic stage that accompanies a rapid fall in the recorded in septa 1–7 and the early ventral shell portion 15 before the primary constriction (less than 60 mm in δ N value of organic tissue. This stage is recorded spiral length from the center of coiling) (Figures 3–5). in septa 7–12 and the ventral shell at 60 to 100 mm in Previous observations of early postembryonic Nautilus spiral length from the center of coiling (Figures 3–5). specimens newly hatched in aquaria have shown that The isotopic drop is probably related to a gradual change the first seven septa are secreted during the embryonic in nutrition source from yolk during the embryonic stage to its diet during the early postembryonic stage. 72 Yuichiro Kashiyama et al.

Figure 4. δ15N profiles derived from bulk shell material from the ventral shells of three specimens of Nautilus pompilius (Tnb-2, Tnb- 53, and Tnb-88). All three specimens show a sharp fall in δ15N values over the shell portion between ~60 and ~100 mm in spiral length from the center of coiling, suggesting the occurrence of three distinct trophic stages. Tnb-2 and Tnb-88 also show an additional fall in δ15N values in the most recently formed shell portions, reflecting the isotopically distinct nature of artificial diets given while in captivity.

In fact, newly hatched Nautilus are known to start Tanabe, 1985). A broad trend of ~2 ‰ increase in δ15N consuming diets while still possessing an external throughout the entire juvenile-mature stage of Tnb- yolk sac (Uchiyama and Tanabe, 1999). The observed 2 may reflect a change in the size of the diet, as larger gradual shift in isotopic values therefore appears to be diets tend to occur at higher trophic levels. partially attributed to the delayed response to the diet An additional trophic stage is recorded by shell change due to the gradual metabolic turnover of amino material formed after introduction to an aquarium acids in body tissue. environment (Figure 4), showing a rapid shift in The juvenile-mature stage is recorded in septum 13 nitrogen isotopic composition due to a change from and larger, as well as the ventral shell portion more natural to artificial diet. The observed drop in δ15N than 100 mm in spiral length from the center of coiling values in Tnb 2 and Tnb 88 probably reflects the (Figures 3–5). This stage is isotopically defined as the relatively low δ15N value of the artificial diet (e.g., stage after the end of the rapid drop in the δ15N value cultivated prawns) upon which they were fed while in of organic tissue, representing the life period from the the aquarium. early postembryonic stage to the fully developed adult The nitrogen isotopic compositions of specific stage, during which time nutrients are exclusively amino acids demonstrated that the isotopic stages supplied from the diet. The δ15N values of shell material identified based on analyses of bulk shell material do corresponding to this stage vary by only 2 ‰, less than indeed reflect changes in the apparent trophic level the expected change for a single trophic step. The rather than other factors such as environmental change above results are therefore concordant with an ontogeny (e.g., isotopic change of the primary producer) or of N. pompilius that develops directly and presumably physiological factors unique to the individual (Table feeds on a similar diet throughout its life (Saisho and 1). The ATL of Tnb-2 dropped from 4.5 during the N isotope-based reconstruction of nautiloid life history 73

Figure 5. δ15N profiles constructed based on septal 15δ N values of Nautilus pompilius (Tnb-53) compared with profiles derived from the phragmocone from the same specimen. Each septal δ15N value is plotted against the position of the aperture (i.e., the growing edge of the phrag- mocone) at the time of formation of the septum, as estimated by extrapolating the ratio of the positions of the last septum and the aperture in adult animals (ca. 10:13). The two profiles show identical 15δ N variations. embryonic stage to 3.8 at the start of the juvenile-mature stage, presumably reflecting the change of nutritional Fossil nautiloid (Cymatoceras sakalavus) source from yolk to diet. The ATL obtained at a later The δ15N values of shell samples of Cymatoceras period of the juvenile-mature stage (3.7) is similar to sakalavus did not exhibit the three trophic stages that of the earlier period (3.8). recorded by modern Nautilus (Figure 6); instead, the An ATL of 3.7–3.8 suggests that the trophic level values showed a gradual increase of 2.6 ‰ throughout of N. pompilius in its natural habitat in the Philippines its life, slightly less than the change thought to indicate was approximately 4, making it a tertiary consumer a single trophic step. This finding may reflect a gradual (a consumer placed three steps higher than a primary change in diet during the ontogeny of this species. producer in the food-chain hierarchy). In the natural The obtained δ15N values are generally low (1–4 ‰), food web, however, the trophic level of organisms is indicating a low trophic level and perhaps a herbivorous best expressed with decimal fractions because their diets diet during at least the animal’s early life stages. This could consist of a mixture of organisms from variable finding challenges the common idea that all modern trophic levels. For example, N. pompilius analyzed in (and perhaps fossil) nautiloids are predators. The the present study could have alternatively fed on a diet obtained δ15N values are unusually low, lower even than with a trophic level higher than 3 (e.g., crustaceans) those of common primary consumers in the modern and one with a trophic level lower than 3 (e.g., annelids marine environment (e.g., Chikaraishi et al., 2007). and fishes that feed on planktons and/or algae). This Moreover, the data do not indicate a distinct embryonic interpretation is strongly supported by a previous stage with elevated δ15N values, as observed in the analysis of the oesophagus and stomach contents of N. modern nautiloid. This result may suggest that the egg pompilius in Fiji (Saisho and Tanabe, 1985). of C. sakalavus hatched at a relatively early stage of 74 Yuichiro Kashiyama et al.

Figure 6. Septal δ15N values obtained for the fossil nautiloid Cymatoceras sakalavus. development, presumably prior to formation of the third consisting of only three stages, is consistent with septum. the predicted life history of a species that develops We sampled and subsequently treated the analyzed directly from an unusually large (ca. 3–4 cm in shell material with special care to minimize the maximum size) and yolk-rich egg, without a larval possibility of contamination in the laboratory; however, stage. This reconstruction of the trophic history was a potential pitfall of the current method is that we further reinforced by compound-specific nitrogen cannot exclude the possibility that organic matter was isotopic analyses of amino acids. Thus, it is suggested added to or formed within the samples during post- that the present method is applicable in gaining an mortem degradation. Nonetheless, the non-random understanding of the trophic ecology of other mollusks trends in the obtained profiles indicate that the isotopic and temporal changes in trophic ecology over the signature represents at least in part a primary signal, organism’s life, without the need for direct observations and is not entirely overprinted by post-mortem events. of feeding. Compound-specific isotopic analyses of amino acids of The method is also potentially applicable to studies of shell protein-derived peptides, may help to clarify the the trophic ecology of extinct mollusks. For example, absolute trophic level of this fossil nautiloid, as such an Ammonoidea, an extinct ectocochiliate analysis would be free of any post-mortem influences. group that flourished in –Cretaceous oceans, Such analyses were not possible in the present study are known to possess much smaller embryonic shells because of difficulties in analyzing the fossil specimen. (generally < 1 mm in diameter, excluding Early Devonian taxa) than nautiloids (Landman et al., 1996). Conclusion and implications This observation strongly suggests that newly hatched ammonoids fed on relatively small organisms (e.g., The nitrogen isotopic composition of shell material zooplankton) as a nutritional source, and that their of the modern nautiloid Nautilus pompilius records its trophic level at the early embryonic stage was lower trophic history through life. The apparently simple than that of modern and fossil nautiloids. trophic history suggested by the present results, An understanding of the ecological roles of ammonoids is particularly important for analyses of N isotope-based reconstruction of nautiloid life history 75

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