Isotopic evidence concerning the habitat of Nautilus macromphalus in New Caledonia.

Modern nautilids (Nautilus and Allonautilus) have often been studied by paleontologists to better understand the anatomy and ecology of fossil relatives. Because direct observations of these animals are difficult, the analysis of light stable isotopes (C, O) preserved in their shells has been employed to reveal their habitat and life history. We aim to (1) reconstruct the habitat depth of Nautilus macromphalus and (2) decipher the fraction of metabolic carbon in its shell by analyzing oxygen and carbon isotopes (δ18O, δ13C) in the septa of two specimens in combination with analyses of water samples from the area. Additionally, we investigate whether morphological changes during ontogeny are reflected in the isotopic values of the shells. Results reveal that the patterns of change of δ18O and δ13C in the septa of N. macromphalus pre- and post-hatching are consistent with previous studies. Values of δ18Owater range from 0.7 to 1.4‰ (VSMOW), with a maximum value coincident with a salinity maximum at ~150 m. We use the temperature and δ18Owater profiles to calculate equilibrium values of δ18Oaragonite with depth. Comparing these values with the measured δ18O of the septa shows that the habitat depth of N. macromphalus is ~140 m pre-hatching and ~370 m post-hatching. Using δ13C of shell carbonate and published data on metabolic carbon, the fraction of metabolic carbon is reconstructed as ~21% and 14% pre- and post-hatching, respectively. The reconstructed depth pre-hatching is slightly shallower than in N. pompilius from the Philippines and Fiji, but the post-hatching depth is similar. However, it is important to emphasize that these estimates represent average over time and space because nautilus is a mobile animal. Lastly, the changes in morphological parameters and the changes in δ13C and δ18O during ontogeny do not coincide except at hatching and at the onset of maturity.

Introduction

Nautilus is an iconic marine mollusk. It is the only externally shelled cephalopod alive today. It is a member of a vast group of shelled cephalopods, including ammonites and nautiloids, that once inhabited the planet. It comprises two genera Nautilus and Allonautilus. Based on morphological and molecular data, the two genera include at least eight species, which are restricted to isolated archipelagos across the Indo-Pacific [1].

Because nautilus live in deep fore-reef environments, direct observations of their habitat and life history have been difficult. Nevertheless, expeditionary research in the last 50 years has yielded spectacular results. Efforts to capture and release animals, track their movements using radio transmitters attached to the shells, and remote cameras have produced a complex picture of their behavior within their habitat [2-4]. These animals are nektobenthic, meaning that they generally live just above the sea bottom, and make vertical migrations along the slope [2-4].

Another approach to throw light on the habitat and life history of these animals is the analysis of stable isotopes of carbon and oxygen preserved in the shell. The isotope ratios 18O/16O and 13C/12C, expressed as delta values (δ18O, δ13C) relative to a standard (PeeDee Belemnite, PDB), can provide information on the temperature of the water [5-7], and on the isotope composition of carbon in the dissolved inorganic carbon reservoir (δ13C) in which the shell formed [8, 9].

Analyses of oxygen and carbon isotopes are very useful in understanding living nautilus, but they are invaluable in reconstructing the life history and habitat of fossil nautilids and ammonites. Because direct observations of these animals are impossible, the use of oxygen and carbon isotopes offer important insights, provided that the shells are well enough preserved and have not suffered diagenetic alteration [10]. Combined with clues from facies distribution, faunal association, and fossil preservation, analysis of the oxygen and carbon isotope composition of the shells can shed light on their habitat and rate of the growth of these extinct organisms [11-15].

The use of these methods requires knowledge of the chemistry (δ18O of water, δ13C of dissolved inorganic carbon, DIC) and temperature of the water from the site the animals inhabit. Although such information is difficult to assemble for the geologic past, it is surprisingly absent in most studies of modern nautilus (only example: Nautilus pompilius from the Philippines and Fiji [16]). This is due to the fact that trapping for nautilus and collecting water samples at the same time is a difficult proposition and that the analysis of δ18O of water and δ13C of DIC requires subsequent gas-source mass spectrometry. As a result, one usually relies on published values from worldwide ocean databases, which may not be detailed enough for the specific sites where nautilus live.

While the δ18O of the shell allows us to reconstruct the water temperature and thus habitat depth of nautilus, the δ13C of the shell has been considered difficult to interpret as a proxy for paleoenvironment. This is because the δ 13C of the shell is both a function of carbon incorporated via the metabolism of the animal as well as a function of the dissolved inorganic carbon (DIC) [17, 18]. Elucidating the fraction of metabolic carbon in the δ 13C of the shell is of importance because it can permit the reconstruction of the DIC of the ancient oceans as a new proxy.

As documented previously, the isotopic composition of the shell changes during ontogeny [16, 19–24]. Do these changes coincide with changes in morphology? Such knowledge is of relevance when reconstructing the ecology of extinct cephalopods where the shells are sometimes not well enough preserved for isotope analysis. Although highly resolved morphological examination of nautilid conchs was difficult in the past, it is now possible owing to the advancement of tomographic methods. Using such methods, classical morphological parameters such as the whorl expansion rate and the whorl width index as well as the siphuncle position index at various ontogenetic points can be measured [25].

In this study, we aim to answer the following questions: (1) What is the habitat depth of Nautilus macromphalus through ontogeny? (2) What is the fraction of metabolic δ13C in the nautilid shell? (3) Are changes in δ18O and δ13C of nautilid shells reflected in changes in morphology?

Background

Based on studies of nautilus raised in aquaria, nautilus secretes its shell in oxygen isotope equilibrium with its environment [23]. Thus, the δ18O of the nautilus shell can be used as a reliable indicator of the temperature of the environment in which it lives. There are no indications of a “vital effect”. This applies to the septa secreted in the egg capsule as well as those secreted post-embryonically. In contrast, as noted above, the δ13C of the shell is a function of fractionation between it and the δ13C of the dissolved inorganic carbon (DIC), as well as the incorporation of metabolic carbon from food sources.

Septa are convenient targets of isotopic analysis. However, they do not form instantaneously but over a finite interval of time. This time interval increases over ontogeny. Landman and Cochran [26], in a review of nautilus growth rates, estimated that the length of septal secretion in early ontogeny, following hatching, may approach weeks, but at the onset of maturity, it may approach months. Thus, the isotope compositions of individual septa reflect time averages.

During embryonic development, the animal secretes its shell in an egg capsule attached to the sea floor. In aquaria, embryonic development takes as long as one year [27]. Following hatching, nautilus is a mobile animal. Based on previous studies, it is clear that it migrates vertically along the slope of the steep fore-reef [28]. These migrations may occur every day or every few days [29]. In addition, nautilus migrates laterally, sometimes for long distances. Saunders and Spinosa [4] recorded movements of as much as 150 km in 332 days around Palau, Western Caroline Islands. Thus, the isotopic compositions of septa reflect averages over space as well as time.

Furthermore, nautilus is long-lived, perhaps as long as 15 or even 20 years [26, 30], and the environment in which it lives may change over this time period. Therefore, the temperature and salinity recorded at a single marine station may not encompass the changes in the environment during the lifetime of the animal. On the other hand, prior studies [28] suggest that nautilus mostly lives below 50 m depth, except for rare reports [31]. Thus, seasonal variation in the water column, which is limited mainly to the mixed layer, may not be a determining factor in the interpretation of isotopic values.

Finally, analyses of the outer shell wall of nautilus have revealed a range of isotope values, probably reflecting depth migration [29]. Similarly, analyses through the thickness of an individual septum reveal patterns of isotopic variation, also probably reflecting vertical migration [16]. Based on these results, it appears that nautilus secretes its shell more-or-less continuously throughout its life at whatever depth it inhabits. Thus, not only is δ18O in nautilus shell material a reliable indicator of the environment in which it forms its shell (i.e., no vital effect), but shell secretion is continuous, unlike that, for example, in intertidal bivalves.

In summary, the oxygen isotope composition of septa secreted in the egg capsule during embryonic development represents a single depth and temperature over a period of as much as one year. In contrast, that of the post-hatching septa represent averages. These averages do not imply a single preferred depth, but instead integrate over time and space. In other words, they represent the depths (and temperatures) that nautilus inhabit, on average.

Materials and methods

Sample collection

Two specimens (one male and one female) of Nautilus macromphalus Sowerby (1849) were captured live in 2002 using traps baited with fish at a depth of 400 m off Nouméa, New Caledonia (Fig 1). The male specimen (AMNH 132423) is regarded as mature because it exhibits septal approximation (i.e., closer spacing of septa) and a black band at the aperture. These features are diagnostic of maturity [32]. The female specimen (AMNH 132380) is most likely submature considering that the septa are approximated, but the black band is absent. The specimens were sectioned along the median plane, and pieces were removed from each septum through ontogeny (with septum 1 as the first formed septum) for carbon and oxygen isotope analysis. The samples represented the entire thickness of a septum at any point, but the complete septum was not sampled or homogenized.

Fig 1

Study area and specimens.

Map of New Caledonia. Star indicates the locality at which the specimens were caught. Circles indicate two stations at which water samples were taken (A); Outer shell of Nautilus macromphalus (AMNH 132380; female) (B); Cross-section of N. macromphalus (AMNH 132380; female) (C); Outer shell of N. macromphalus (AMNH 132423; male) (D); Cross-section of N. macromphalus (AMNH 132423; male) (E). Scale bars = 10 mm. Samples were taken through ontogeny (the earliest formed septum as septum 1). Map was reproduced from OpenStreetMap (© OpenStreetMap contributors).

Water samples were collected in June, 2003, from two stations (Station 25 and 49; Fig 1A) near the site at which the nautilus specimens were captured. Samples (~1L) were taken every 50 m (to 400 m) and stored sealed in glass bottles in the dark for oxygen isotope measurement (see below). In addition, temperature and salinity of the water were measured every half-meter using a CTD.

Oxygen and carbon isotope analysis

Oxygen and carbon isotopes of the septal samples were measured at the University of California Santa Cruz Stable Isotope Laboratory. Pieces of each septum were broken off and sent to the laboratory. Prior to analysis, samples were ground to a size of 125–250 μm and 40–60 μg of sample were selected for analysis. Standards and samples were conditioned in a 60°C vacuum-roasted oven overnight to remove any residual water from the glass or samples. Samples were then analyzed for δ18O and δ13C via acid digestion using an individual vial acid drop ThermoScientific Kiel IV carbonate device interfaced to a ThermoScientific MAT-253 dual-inlet isotope ratio mass spectrometer (IRMS). Samples were reacted at 75°C in orthophosphoric acid (specific gravity = 1.92 g/cm3) to generate carbon dioxide and water. Non-condensable gases were pumped away, and the CO2 analyte was then cryogenically separated from water, finishing with the introduction of pure CO2 into the IRMS via the dual inlet.

Raw data were also corrected using a two-point calibration against samples of calibrated in-house granular Carrara Marble standard reference material (δ13C = 2.05 ± 0.1‰ and δ18O = -1.91 ± 0.1‰ VPDB) and granular NBS-18 limestone international standard reference material. The in-house Carrara Marble was extensively calibrated against NIST Standard Reference Material (NBS-19, NBS-18, and LSVEC) and further calibrated in intercomparison studies with international laboratories. Raw data were corrected for offset from the international standard PDB (PeeDee Belemnite) for δ18O and δ13C and corrected for instrument-specific source ionization effects. Aliquots of an external working standard (powdered Atlantis II calcium carbonate; δ18O = 3.42‰) were run "as-a-sample" to monitor quality control and long-term performance. Typical precision was 0.05‰ (1σ) for both δ18O and δ13C. All values were reported relative to VPDB.

A few samples of septa were sent to the University of Michigan Stable Isotope Laboratory for cross-comparison. These were of two types: intact pieces of septa and pieces that were ground before submission to the laboratory. Carbonate samples weighing a minimum of 10 micrograms of pure carbonate were reacted with anhydrous phosphoric acid in a Thermo Kiel IV preparation device coupled directly to the inlet of a Thermo MAT 253 triple collector isotope ratio mass spectrometer. 17O-corrected data were subsequently corrected for acid fractionation and source mixing by calibration to a best-fit regression line defined by two NBS standards, NBS 18 and NBS 19. Data were reported in ‰ notation relative to VPDB. Precision and accuracy of data were monitored through the analysis of a variety of powdered carbonate standards. Measured precision was maintained at better than 0.1 ‰ for both carbon and oxygen isotope compositions. These precisions comprise uncertainties of ~4% of the measured septal δ18O values and 9–18% for the δ13C values (S1 Table). The discrete water samples obtained at Station 25 and 49 (Fig 1A) were analyzed for δ18O of the water at the Keck Paleoenvironmental and Environmental Stable Isotope Laboratory. The water samples were not poisoned with HgCl2 after collection to prevent bacterial activity, which could alter the DIC and δ13CDIC. Thus, we do not report δ13CDIC values and, alternatively, we use literature values of δ13CDIC appropriate to the study site, as described in detail below. For oxygen isotope measurements, aliquots of samples were equilibrated with CO2; δ18Owater was determined using a ThermoFinnigan MAT 253 mass spectrometer. Values were reported relative to VSMOW with an internal precision of ±0.02‰.

Morphological analysis

To examine the morphology of the two conchs of Nautilus macromphalus through ontogeny, we CT-scanned the specimens with a voxel size of ~0.66 mm at the Microscopy and Imaging Facility of the American Museum of Natural History. The CT-scans obtained were used to measure the following morphological parameters (Fig 2): conch diameter (dm), whorl width (ww), whorl height (wh), distance between the ventral edge of the siphuncle and the ventral edge of the conch (vd) and septal spacing (rotational angle). We took these measurements every 30° (rotational angle; starting at aperture) for dm, ww, and wh, while vd was measured at each septum. These parameters were used to calculate the whorl expansion rate [(dm1/dm2)2; WER], whorl width index (ww/dm; WWI), whorl height, and siphuncle position index (vd/wh; SPI). For details of these morphological parameters, see Tajika and Klug [25], and Tajika et al. [33]. Direct comparisons between WER, WWI, and the isotope values are difficult because they require estimation of the position of the aperture at the time of septal formation. According to Collins and Ward [32], the body chamber angle increases by approximately 15° after the formation of the last septum. Therefore, we estimated the position of the aperture by subtracting 15° from the observed body chamber length (compare with Ohno et al. [19]). In contrast, direct comparisons were possible between septal spacing, SPI, and the isotope values. We calculated Spearman’s rank correlation coefficients to determine if the morphological and isotopic values were correlated.

Fig 2

Cross-section of Nautilus macromphalus with measured morphological conch parameters.

ww = whorl width, dm = conch diameter, wh = whorl height, ww = whorl width, and vd = distance between the ventral edge of the siphuncle and the ventral edge of the conch.

Ethics statement

The specimens of Nautilus macromphalus (Mollusca: Cephalopoda) used in this study were captured live in a baited trap by Dr. Royal H. Mapes in 2002 before the species was given local protected status around New Caledonia in 2008, and before they were protected under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in 2016. The individuals were euthanized and retained for morphological study. The specimens are reposited at the American Museum of Natural History.

Results

Hydrography

Temperature (T) and salinity (S) profiles in the upper 400 m of the water column are shown in Fig 3A and 3B. The temperature profiles show surface water with ~23°C, extending over a ~30 m mixed layer. T values decrease to ~13°C at 400 m. Salinity is ~35.4 (as psu) in surface water, increases to 35.6 at ~150–200 m and then decreases to 35.1 at 400 m. A T-S plot of the data (Fig 3C) shows mixing among three water masses: 1) surface water (S~35.5; T~23°C), 2) subsurface salinity maximum water (S ~35.65; T~19°C, and 3) deep water (S ~35.1; T~13°C). These measurements represent a single snapshot of the water column whereas the lifetime of Nautilus macromphalus extends over at least 10 years [30]. However, annual seasonal variation in temperature and salinity is largely confined to the upper 50 m of the water column [34]. Previous studies of N. macromphalus indicate that it usually lives in deeper water [28], suggesting that seasonal variation may be of little importance in interpreting the isotopic signals in the shell.

Fig 3

Hydrography.

Temperature versus depth (A); Salinity versus depth (B); TS diagram (C).

Isotope hydrography

The water column isotope data are presented in S1 Table. The δ18Owater profiles are similar at the two stations (Fig 4), showing values increasing from ~0.85‰ at 50 m to values of 0.91–1.38‰ at the depth of the salinity maximum and then decreasing to 0.6–0.8‰ at ~400 m (S1 Table). The integrated water column (50–400 m) average δ18O is 0.93‰. As mentioned above, the water samples were not poisoned with HgCl2 and consequently, values of δ13CDIC were not reliable. Thus, we use literature values of δ13CDIC [35] to interpret the septal carbonate δ13C data, as described in detail below.

Fig 4

Isotope hydrography.

Oxygen isotope composition (δ18Owater; ‰ SMOW) versus depth.

C and O isotopes in the septa

The trends of C and O isotope data of the septa are similar in the two specimens (Fig 5; S1 Table). Both show lower δ18O values ranging from 0.32 to 0.73‰ in septa 1–7, increasing to 1.13 to 2.35‰ after septum 7. Both specimens show lower δ18O in the two most recently deposited septa. Values of δ13C progressively increase in the first 7–8 septa of each specimen, and then decrease for the next two septa and increase again through ontogeny, varying from -0.01 to 1.49‰. Results of the laboratory intercomparison are presented in Fig 5 and S1 Table. Given that different pieces of a particular septum were analyzed by the two laboratories, the general agreement is within 10–20%. One sample (AMNH 13423, septum 28) showed a value from Santa Cruz that was ~50% greater than that from Michigan, but when the ground sample was re-analyzed at Michigan, it agreed within 0.10‰ for both δ18O and δ13C (S1 Table). We consider this comparison further below in the context of reconstructing the habitat depth of Nautilus macromphalus from the oxygen isotope data.

Fig 5

Septal C and O isotopes.

δ13C (‰ VPDB) (A); δ18O (‰ VPDB) (B). Filled markers represent the data produced at the University of California, Santa Cruz. Unfilled markers represent the data produced at the University of Michigan.

Morphology

The morphological parameters in both specimens of Nautilus macromphalus are shown in Fig 6. Remarkable changes occur between pre- and post-hatching in all morphological parameters. Septal spacing increases and then decreases rapidly at hatching and it decreases moderately thereafter until septum 14 in the female (AMNH 132480) and septum 17 in the male (AMNH 132423). In the female specimen, the septal spacing fluctuates with a more or less constant mean value of 24° (Fig 6A). In contrast, the male specimen shows an increase after septum 17 and the angle remains the same (24°) until it starts decreasing at septum 25 (Fig 6B). Septal spacing decreases to a lower value at the last septum (male: 22.6°; female: 20.6°).

Fig 6

Morphological parameters in Nautilus macromphalus.

Septal spacing for AMNH 132380 (female) (A); septal spacing for AMNH 132423 (male) (B); siphuncle position index (= vd/wh) for AMNH 132380 (female) (C); siphuncle position index (= vd/wh) for AMNH 132423 (male) (D); whorl expansion rate [= (dm1/dm2)2] for AMNH 132380 (female) (E); whorl expansion rate [= (dm1/dm2)2] for AMNH 132423 (male) (F); whorl width index (= ww/dm) for AMNH 132423 (male) (G); whorl width index (= ww/dm) for AMNH 132423 (male) (H).

The siphuncle position index increases rapidly until septum 14 with a plateau between about septa 5 and 8 (Fig 6C and 6D). Then, it shows a gradually decreasing trend until the end of ontogeny. A somewhat rapid decrease in the siphuncle position index is visible before the attainment of maturity in both the female and male.

The whorl expansion rate (WER) shows a sharp decrease until the point of hatching (~30 mm), which is followed by an increase (Fig 6E and 6F). During the juvenile stage, the female specimen exhibits a higher WER than the male (about 3.2), which gradually decreases until a diameter of ~ 90 mm. Thereafter, WER increases until the end of ontogeny. The male shows a WER of ~ 3.0 after hatching until a diameter of ~ 80 mm after which it starts increasing. At about 110 mm, WER becomes stable until the end of ontogeny. The whorl width index (WWI) rapidly decreases until the end of the embryonic stage, and then the decrease becomes moderate. The moderate decrease in WWI stops at about 75 mm in both specimens. While the female exhibits a stable WWI after a conch diameter of 80 mm, the male shows an increasing trend. WWI abruptly decreases at the end of ontogeny in the female and male.

Discussion

Oxygen isotopes in the septa: Habitat depth

Previous studies have used the classic equation relating the δ18O of molluscan aragonite to the temperature and δ18O of the water in which it was secreted. For example, the equation of Grossman and Ku [6], as modified by Hudson and Anderson [36], such that the δ water values are corrected for the difference between the PDB and SMOW scales, is:

As noted in the Background section, Landman et al. [23] analyzed the shells of two specimens of Nautilus belauensis that grew in aquaria with measured water temperatures. The eggs were laid in the aquarium and developed for nearly one year. They hatched and lived for several months after hatching. The calculated water temperatures from the oxygen isotope analysis of the shells showed good agreement with the measured temperatures in the aquarium tanks, demonstrating that they were secreted in isotopic equilibrium with seawater, both before and after hatching. Indeed, the results reported by Landman et al. [23] provided a foundation for the paleotemperature interpretation of the oxygen isotope record of fossil nautilids and ammonites.

In the present study, we have measured the depth profiles of both temperature and δ18O of the water in which the nautilus were caught. We can thus rewrite Eq 1 to calculate the δ18O of precipitated biogenic aragonite (δ18Oar) as a function of the two measured variables, and, in effect, as a function of depth in the water column (S1 Table):

Fig 7 shows how the calculated δ18Oar varies with depth in the water column. We use the linear relationship between depth and δ18O of precipitated aragonite to determine the depths of precipitation of each septum. The results reveal shallower depths for septa deposited pre-hatching. Indeed, average δ18O values for the pre- and post-hatching septa (S1 Table; Fig 5) yield average depths of 140 ± 8 m and 370 ± 25 m, respectively (Fig 8), with corresponding water temperatures of ~21°C and ~15°C, respectively. The shaded areas in Fig 8 indicate the estimated errors in depth calculated from the 95% confidence interval of the regression line in Fig 7. Although the estimated error produces some variation in depth (~±30 m), the two specimens show the same pattern during ontogeny. The shallower depth for septa deposited pre-hatching within the egg capsule is consistent with prior studies [16, 21–23, 37] that show warmer temperatures and shallower depths for egg incubation in modern nautilids.

Fig 7

Calculated δ18Oaragonite vs. depth.

Dashed lines indicate the 95% confidence interval.

Fig 8

Reconstructed habitat depths of Nautilus macromphalus from New Caledonia.

Filled markers represent the data produced at the University of California, Santa Cruz. Unfilled markers represent the data produced at the University of Michigan. Shaded areas represent the estimated error range calculated with the 95% confidence interval in Fig 7.

We indicate in Fig 8 the difference in the calculated depth that results from our intercomparison of δ18O values measured at different laboratories (S1 Table). In general, the difference is small, although in one case (AMNH 132423, septum 28), the averages of the δ18O measurements of the septal piece and ground sample run at the Michigan Stable Isotope Facility are 1.39‰ and 2.02, yielding depths of 258 m and 357 m, respectively. The measurement from the Santa Cruz Isotope Facility of this septum (2.07‰) yields a depth of 365 m. The multiple measurements from the different labs likely reflect variations related to our sampling method (small pieces of each septum). The variation is consistent with the observation of Oba et al. [16], Ohno et al. [19], and Zakharov et al. [37] who documented variations of 0.3–0.6‰, 0.9‰, and 0.4‰, respectively in individual septa from Nautilus pompilius collected in the Philippines. As noted in the Background section, septa do not form instantaneously but over a time interval that increases through ontogeny [21, 26, 38]. The differences in δ18O in single septa are consistent with movements of nautilus through the water column over time.

For the septa deposited post-hatching, the depth calculated from δ18O is consistent with the trapping depth of the specimens in New Caledonia (400 m). However, given that the time of secretion of each septum increases through ontogeny [21, 26, 38], the calculated depths and temperatures based on analyses of the entire septal thicknesses represent temporal averages. The exact time for septal formation is not clear, but observations in aquaria and inferences based on radiometric data in N. macromphalus suggest 2–3 weeks for juvenile and > 10 weeks for sub-adult/adult individuals [30, 39].

Temporal variation is also evident in the outer shell of N. macromphalus from New Caledonia, as shown in the detailed measurements by Secondary Ion Mass Spectrometry (SIMS) by Linzmeier et al. [29] in a specimen from the same collection as the specimens in the present study. The results show that the animal migrated over depths representing a range of δ18Oar 2.5‰. Using ultrasonic telemetry techniques, Dunstan et al. [2] have shown that N. pompilius in Australia can range over depths bounded by the upper limit of temperance tolerance (~25°C) and the implosion depth of the shell (~800 m). The 2.5‰ range in δ18O measured by Linzmeier et al. [29] brackets the range of our calculated δ18Oar (translating to 50 to 400 m depth), but could include deeper depths as the temperature decreases and the calculated δ18Oar increases with depth to ~3.5‰ at 800 m.

It is notable that the δ18O of the last two septa in both specimens from New Caledonia reflect shallower depths. The male is mature and the female is submature. This is expressed in the approximation of the last two septa in both specimens. It is likely that the migration to shallower and warmer water of the female is linked to selection of hatching sites for incubation. It is also possible that, with respect to both sexes, mating preferentially occurs in shallower water.

We compare our results with the data of Davies et al. [40] on a specimen of Nautilus macromphalus from the same collection as our specimens. Davies et al. [40] measured both conventional δ18O and carbonate clumped isotopes (Δ47). They measured δ18O in selected post-hatching septa, with values ranging from 0.73 to 1.47‰ VPDB. The lower values were observed in the most recently deposited septa, similar to our observations. Davies et al. [40] used an estimated δ18Ow of 0.5‰ to calculate growth temperatures of ~12–14°C. Recalculating their results using our measured higher δ18Ow yields estimated depths of precipitation ranging from ~160–265 m with temperatures of ~17.48–20.60°C. Interestingly, these values are more comparable, although still lower than the range of clumped isotope temperatures of ~17–31°C calculated by Davies et al. [40] on the same specimen.

As noted above, the age of maturity of Nautilus macromphalus is at least 10–12 years, and the period of septal formation increases over ontogeny from weeks in early ontogeny to months at the onset of maturation [30]. Given that our water samples were collected at one time, the septa we analyzed did not form in the exact seawater we sampled. Previous studies documented temporal and seasonal variation of seawater temperature and chemistry at/near New Caledonia [34, 41]. Van Den Broeck et al. [34] documented a seasonal change in temperature and salinity of ~3°C and ~0.3 (psu), respectively, in the upper 50 m. At deeper depths, the seasonal change in temperature and salinity was ~1°C and ~0.1 (psu). Linzmeier et al. [29] presented a composite profile of water column temperature near New Caledonia based on World Ocean Atlas data [41] and showed similar seasonal variation of ~4°C in the upper 50 m and <1°C in deeper water. Seasonal variation of surface water δ18O at New Caledonia has been estimated from seasonal SST and surface salinity variation to be 0.16‰ VPDB [42] and is presumably less in deeper waters. Thus, seasonal fluctuations in temperature and salinity (and δ18Owater) at the study site are likely restricted to surface and near-surface water (<50 m). Inasmuch as N. macromphalus generally lives below depths of 50 m [28], variations in the upper 50 m are less significant.

Habitat depth of Nautilus macromphalus versus other species of Nautilus

As noted above, few studies of modern nautilus combine C and O isotope measurements of the shell with information about the temperature and isotope composition of the water column. Oba et al. [16] presented results of δ18O in the septa and outer shell wall of Nautilus pompilius collected in Fiji and the Philippines, along with corresponding δ18O measurements of the water. Temperature profiles of the same stations were included for the Philippines in Hayasaka et al. [43] and for Fiji in Hayasaka et al. [44]. We draw on these data and use our approach to calculate the δ18O of precipitated aragonite as a function of depth in the water column for the two sites. We note that Oba et al. [16] used ad hoc temperature equations to calculate the temperatures of precipitation. This was pointed out by Landman et al. [23], and here we apply Eq 1 to a reevaluate the data of Oba et al. [16]. The results (Fig 9A and 9B) show that pre-hatching septa were deposited in water depths of ~170–230 m (~20–22°C) in Fiji and 185–230 m (~20–22°C) in the Philippines. Post-hatching septa were deposited at depths of 300–430 m (~13–16°C) in Fiji and 310–400 m (~14–16°C) in the Philippines. Our average depth for pre-hatching septa in N. macromphalus from New Caledonia (~140 m at ~21°C) is slightly shallower than the depths in the Philippines and Fiji. On the other hand, the average depth of post-hatching septa in New Caledonia (370 m) is similar to that of N. pompilius from both the Philippines and Fiji. Taken at face value, the data suggest that N. pompilius in Fiji and the Philippines hatch at slightly deeper water than in New Caledonia, but that adults live at or range over similar depths (temperatures) at all three sites. However, the temperature recorded in the pre-hatching stage at all three sites is the same (~20–22°C), suggesting that water temperature rather than depth is the controlling factor in egg-laying and incubation.

Fig 9

Reconstructed habitat depth of Nautilus pompilius from the Philippines and Fiji.

Depth vs. septal number in the Philippines (A) and Fiji (B), based on the δ18Oar vs. depth relationships. Data from Oba et al. [16] and Hayasaka et al. [43, 44].

Carbon isotopes in modern nautilus and fossil nautilids: Identifying carbon sources

The carbon isotope signature of molluscan aragonite is more difficult to interpret than that of oxygen in that it is only very weakly dependent on temperature [20, 23, 37]. It is a function of the isotopic composition of carbon incorporated via the metabolism of the animal as well as that of carbon in the dissolved inorganic carbon (DIC) reservoir [17]. This dependence may be expressed as: where ε is the δ13C fractionation between aragonite and DIC (+2.7 ± 0.6‰, dominated by HCO3- [45]), Cmeta is the fraction of metabolic carbon incorporated into the shell, and δ13Car, meta, DIC are the δ13C values corresponding to the shell, metabolic carbon, and DIC, respectively. In the present study, we use literature values of the δ13C of the DIC to interpret the fraction of metabolic carbon as recorded in the δ13C of the septa. The δ13C of oceanic DIC has been mapped broadly but has changed with time owing to the influence of increasing inputs of fossil fuel CO2 (with low δ13C) into the atmosphere, surface ocean, and upper water column. Measurements of δ13CDIC in the Pacific show these changes, documented by Ko et al. [35] along a meridional transect using snapshots based on sampling in 1994 and 2008. At the latitude of New Caledonia, the δ13CDIC in 2008 was ~1.2‰ in the upper ~100 m, decreasing to ~0.8‰ at 400 m [35]. In applying Eq 3 to our nautilus δ13C data, we use δ13CDIC = 1.2‰ for septa formed pre-hatching (depth ~140 m estimated from δ18O) and 0.8‰ for septa formed post-hatching. The value of δ13Cmeta is not known precisely, but Crocker et al. [46] reported measurements of δ13C for siphuncular organic material in six wild-collected specimens of Nautilus macromphalus from New Caledonia. Data were not tabulated in Crocker et al. [46], but interpolating values from their plot of δ13C vs. septal number shows that in individual specimens and in the six specimens taken as a whole, there was little difference between the siphuncular material formed pre-hatching (overall average ± 1sd = -16.6 ± 2.2‰ VPDB) and post-hatching (overall average ± 1sd = -16.5 ± 1.4‰ VPDB). The overall average ± 1sd for all material is -16.6 ± 1.7‰ VPDB. This result is comparable to that of Pape [47], who measured -17.4‰ on siphuncular material from a specimen of Nautilus pompilius from the Philippines. Given that the values are not significantly different pre- and post-hatching, we use an overall average value (±1sd) of -17‰ ± 2‰ VPDB for δ13Cmeta to calculate values of Cmeta using Eq 3 (Fig 10).

Fig 10

Reconstructed fraction of metabolic carbon in Nautilus macromphalus.

Shaded areas indicate the estimated uncertainty in Cmeta given the average ±1sd of δ13Cmeta (-17 ± 2‰).

The pattern of change in septal δ13C observed in both specimens of Nautilus macromphalus shows higher values of Cmeta in septa formed pre-hatching (septa 1–7) compared with those formed post- hatching: averages ± 1sd of 21.8 ± 2.5% vs. 14.2 ± 2.4% in AMNH 132423, respectively (Fig 10; S1 Table). Both specimens show an increase in Cmeta to 24–27% in septa 9 and 10. We interpret this to result from an accelerated rate of growth shortly after hatching. Our results are comparable to those of Chung et al. [18] who calculated Cmeta values for specimens of nautilus based on previously published data on septal and DIC δ13C values. They calculated that Cmeta decreased from ~30% to ~10% through ontogeny. The average values of Cmeta for post-hatching nautilus septa are comparable to the low values, generally less than 10%, observed in other marine mollusks such as bivalves and gastropods [8]. In contrast, values of Cmeta for another group of shelled cephalopods, extinct ammonites, are estimated to be considerably greater than those of modern nautilids. For example, Tobin and Ward [48] compared δ13C in bivalve and ammonite shells from the Lopez de Bertadano Formation of Seymour Island (Antarctica) spanning the K/Pg boundary. They used the δ13C of bivalves to calculate the δ13C of the DIC (assuming 10% metabolic C in the bivalves), and then used Eq 3 to calculate the average Cmeta values ranging from 31.8–36.8% in ammonites. Landman et al. [49] used the detailed δ13C data of Sessa et al. [50] measured on foraminiferas, gastropods, bivalves, and ammonites from the Upper Cretaceous Owl Creek Formation (Mississippi, USA) to calculate the Cmeta of 29% for Eubaculites sp.

Given that modern nautilids appear to incorporate less metabolic carbon in their shells than did the extinct ammonites, it is interesting to consider whether the δ13C of extinct shelled nautilids can be used to estimate the δ13C of the DIC in which the nautilids lived. We consider two examples, both involving Eutrephoceras dekayi, an extinct nautilid often compared with modern nautilus [14, 33, 51]. In both cases, preservation of the shell microstructure of the samples was assessed using the Preservation Index of Cochran et al. [10] and determined to be excellent (PI = 5). One sample of E. dekayi from the Owl Creek Formation included in Sessa et al. [50] showed a value of δ13C of 0.76‰. Assuming Cmeta = ~-15% and δ13Cmeta = ~-17‰, application of Eq 3 yields a value of δ13CDIC of 0.7‰. This compares favorably with the δ13C values of benthic foraminifera at the site (0.8–0.9‰), which predominantly reflect the δ13C of the DIC.

Another example is from the work of Cochran et al. [52], which documents the δ13C of a shell of Eutrephoceras dekayi collected at a fossil methane seep deposit in the Upper Cretaceous Baculites compressus ammonite Zone in the Pierre Shale of South Dakota. The outer shell of the specimen was sampled sequentially to determineδ18O and δ13C through ontogeny. Values of δ13C varied from 0.15‰ to -0.32‰, generally decreasing through ontogeny. The corresponding calculated values of δ13CDIC range from -0.6‰ to -0.0‰ (average -0.24‰). The lower calculated values of δ13CDIC at this site are consistent with the influence of the anaerobic oxidation of methane with low δ13C impacting the DIC reservoir in near-surface sediments. The presence of seep fluids in the immediate overlying water column at the site is supported by 87Sr/86Sr ratios in the shells of ammonites and nautilids that differ from coeval seawater values [52], as well as by patterns of δ13C in the shells of B. compressus and other ammonites [13, 53]. Future work is needed to determine whether the δ13C of fossil nautilid shells can reasonably be used as a proxy for δ13C of paleo-DIC. This may permit the reconstruction of paleoenvironments over long geological periods ranging from the Carboniferous [54] to the Miocene [55].

Morphology and isotope signals

Elucidating the ecology of fossil cephalopods, including their habitat and migratory behavior is of great importance to better understand the mechanisms of their evolution and extinction. As mentioned, however, these aspects can only be studied based on indirect evidence. In both fossil and modern nautilids, the hard part (i.e., conch) is the most accessible material and so the morphology of the conch is the focus of most studies. Stable isotopes are also a useful tool to reconstruct ecology, particularly habitat, when the original aragonitic shell is preserved. However, the conch of cephalopods is susceptible to diagenetic changes and, as a result, the original shell material is often altered. If there is a link between isotope signals and morphological changes, then cephalopods that do not preserve their original shells can provide valuable information.

Our results reveal that changes in morphological parameters are apparent at some ontogenetic stages. The most conspicuous morphological changes occur at the point of hatching (i.e., a conch diameter of about 30 mm corresponding to septum 8) as expressed in septal spacing, whorl expansion rate (WER), whorl width index (WWI), and siphuncle position index (SPI) (Fig 6). These morphological changes most likely occur in all species of modern nautilids according to the results of recent studies [25, 56]. The point of hatching also coincides with changes in δ18O and δ13C, as observed in the two specimens of Nautilus macromphalus we studied.

Another conspicuous morphological change occurs in late ontogeny—known as the morphogenetic count down [25, 57]. A direct comparison of the patterns of septal spacing and δ18O reveals that the onset of the morphogenetic countdown, expressed as an approximation of the last two septa, approximately coincides with a decrease in the value of δ18O in both specimens, indicating a preference for more shallow water (Figs 5B, 6A, and 6B) [37]. Other morphological parameters (WER and WWI) are more difficult to compare with the changes in isotopes because of the uncertainty of the conch diameter at which a particular septum formed. As mentioned in the Materials and Methods section, although we estimated the body chamber length at the time each septum formed using the length of the body chamber as an adult, the estimated position of the aperture may not be exact. Bearing that in mind, the inferred conch diameters at which each septum formed are presented in S1 Table. The estimated conch diameters at which each septum formed are presented in S1 Table. This allows for a comparison through ontogeny between WER, WWI, on the one hand, and the isotope values, on the other hand. It is notable that the change in WER and WWI, corresponding to septum 29, approximately coincides with a change in δ18O and δ13C (Table 1).

Table 1

Comparison of the onset of maturity and changes in isotope composition of the septa.

	Septal number at which a significant change occurs in late ontogeny
	AMNH 132380 (female)	AMNH 132423 (male)
δ18O	28	30
δ13C	28	30
Septal spacing	29	31
Siphuncle position index	29	31
Whorl expansion rate	29	27
Whorl width index	29	29

Septal numbers at which a significant change occurs in late ontogeny. The septal numbers are indicated in Fig 6.

A conspicuous change in δ13C occurs at septum 13. Although there are no obvious changes in most conch parameters at septum 13, the change in δ13C appears to coincide with the onset of a decreasing trend of WER (Fig 6E and 6F). It is not clear whether this is a mere coincidence or a biological/ecological signal. In addition, we note several other minor changes in morphological parameters, namely in WER and WWI (Fig 6). The changes in δ18O and δ13C that correspond to the septal numbers at which these morphological changes occur are difficult to evaluate. We calculated Spearman’s rank correlation coefficients between SPI, septal spacing, and isotope signals (Table 2). The results indicate that the values of δ18O and δ13C are not correlated to SPI and septal spacing with one exception—septal spacing in AMNH 132423. Given these results, we conclude that the morphological changes are not clearly reflected in the values of δ18O and δ13C during middle ontogeny [i.e., after hatching until the onset of morphogenetic countdown (sensu Seilacher and Gunji [57])]. As demonstrated by previous researchers [29], conventional methods of analyzing δ18O and δ13C usually require significant amounts of aragonite for each sample and involve time-averaging (i.e., multiple days/months of growth are averaged), and therefore, may have masked detailed changes in morphology. Highly resolved sampling may shed new light on the relationship between morphology and stable isotopes [29, 58]. Another potential approach to explore the relationship between morphology and isotope values is specimens with sub-lethal injuries. Shell fracture and repair are relatively common in modern nautilus and are manifested by changes in septal spacing [59, 60]. It is possible that injured specimens also change their migratory behavior pattern to avoid predators, which could be reflected in the isotope signatures. In addition, it is worth mentioning that ammonoids may exhibit a different relationship between morphological and isotope changes than nautilids. As suggested by a previous study [51], ammonites likely responded to changes in environment more rapidly than nautilids due to differences in their respective metabolic rates. Therefore, further investigation with additional specimens of nautilids and ammonoids is valuable.

Table 2

Spearman’s rank correlation coefficients between morphological parameters septal spacing and siphuncle position index (SPI) and isotopic values (δ18O and δ13C).

Correlation coefficient		δ13C	δ18O
AMNH 132380	SPI	-0.1971	0.0465
	Septal Spacing	-0.2632	-0.4068
AMNH 132423	SPI	0.0714	-0.0273
	Septal Spacing	-0.4403	-0.3494
p-value		δ13C	δ18O
AMNH 132380	SPI	0.4315	0.8547
	Septal Spacing	0.2902	0.0938
AMNH 132423	SPI	0.7583	0.9079
	Septal Spacing	0.0472*	0.1210

*Statistically significant (p < 0.05)

Conclusions

We analyzed δ18O and δ13C in the septa of Nautilus macromphalus from New Caledonia. We also analyzed water samples from two sites near where the nautilus specimens were collected to determine the temperature-salinity-δ18O profiles as a function of depth. We summarize our conclusions:

We reconstructed the habitat depth of N. macromphalus from New Caledonia as ~140 m (~21°C) pre-hatching and ~370 m (~15°C) post-hatching. The pre-hatching habitat depth of N. macromphalus differs slightly from that for N. pompilius from the Philippines (185–230 m) and Fiji (170–230 m), as recalculated from the data of Oba et al. [16]. Nevertheless, the pre-hatching temperature seems to be the same in all three groups (20–22°C). The post-hatching depth is also similar among the three sites. In addition, there is no difference between the female and male of N. macromphalus from New Caledonia. At maturity, both specimens of this species show a change to lower values of δ18O, reflecting migration to shallower (warmer) water, possibly related to mating or the search for egg-laying sites. However, as emphasized in the Background section, these estimates represent averages and do not necessarily imply a single preferred depth, but instead are integrated over time and space.

Using published data and our results, we estimate that the average fraction of metabolic carbon in N. macromphalus is ~21% before hatching and ~15% after hatching. These values are significantly lower than those inferred for fossil ammonites. In addition, we suggest that our results about metabolic carbon may be useful in estimating the δ13C of the DIC in ancient oceans.

We carried out morphometrics for N. macromphalus using computed tomography to calculate four conch parameters (septal spacing, septal position index, whorl expansion rate, and whorl width index). Changes in isotope values (δ13C and δ18O) and changes in morphology coincide at the point of hatching and at the onset of maturity. Although both the conch parameters and isotope values fluctuate during middle ontogeny, there does not seem to be a clear correlation between them. This suggests that minor changes in the environment barring injuries, are not reflected in the morphology of the conch after hatching until the onset of the morphogenetic countdown. Further investigation is needed to discern more precisely the exact relationship between isotope signals and morphological changes in ectocochleate cephalopods.

Raw data of δ18O, δ13C, and morphological parameters and calculated fraction of metabolic carbon.

(XLSX)

Click here for additional data file.

3 Feb 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Geerat J. Vermeij

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf"

2. Thank you for stating the following in the Funding Section of your manuscript:

“AT was supported by a Grant-in-Aid for JSPS Research Fellow, Grant-in-Aid for Young Scientists (grant nrs. 20J00376 and 21K14028). NHL and JKC were supported by the Norman D. Newell Fund (AMNH).”

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“AT was supported by a Grant-in-Aid for JSPS Research Fellow, Grant-in-Aid for Young Scientists (grant nrs. 20J00376 and 21K14028). NHL and JKC were supported by the Norman D. Newell Fund (AMNH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

4. We note that [Figure 1] in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure(s) [#] to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an ""Other"" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

Additional Editor Comments:

The two reviewers raise truly important issues regarding your study. It may prove very difficult to address yourselves to all the problems, but at the very least you should acknowledge the problems even if you can't fix them or solve them. On a personal level, I have been a skeptic of isotopic studies like this for some of the reasons cited by Reviewer 2, especially the question when shell secretion actually takes place. If you do resubmit this paper, I shall have it critically reviewed again.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dr. Tajika and colleagues present an excellent study that combines the investigation of morphological variation and stable isotope variability through ontogeny within Nautilus macromphalus from New Caledonia. These data extensively explore the depth-metabolism-morphology covariation in an extant externally shelled cephalopod. The manuscript and supporting information are well presented and clear. I have a few general comments to help improve the manuscript and some more specific suggestions for sentences and figure changes.

I believe that this study requires a short ethics statement given the locally protected status of Nautilus macromphalus and the PLoS expectations for ethics statements related to cephalopods (https://journals.plos.org/plosone/s/animal-research). Mostly a statement on the timing of collection and the purpose of collection would suffice. I should reiterate that I believe the work these authors have done has been conducted ethically, but given the journal requirements, this statement should be added.

I would like to see slightly more nuanced phrasing related to the depth estimates throughout the manuscript. Later in the text it is clearly stated that these estimates are averages given septal formation duration, but it would be helpful to state early-on that “depth estimates average X days growth”. There is also an implicit assumption that septal formation rate is constant at all depths, which is probably fine given inferences from apertural growth and variance observed in subsampling septa by other authors. In addition, the time averaged within each septa likely varies with growth, but the possible depth range (or swimming velocity) also likely scales with size. Changing the time-at-depth distribution coupled to the time averaging could change the observed mean depth habitat in a free-swimming organism like Nautilus especially during the morphogenic count down (Linzmeier, 2019).

One potential area of improvement would be to do a “time averaging” to the morphological parameters and compare these averaged values to the resultant δ18O and δ13C. It is unclear to me from the methods description if the quantification is done at a point along the shell or an average for a region. Currently the comparison between the isotope and morphological data is done broadly and descriptively but quantitative comparison (e.g. regression results) could be explored. For this to be fruitful, major changes in morphology and isotope ratios at the hatching and morphogenetic count down would likely need to be trimmed from the regression. Remaining morphological parameters and isotope results could show a statistically significant, but low R2 regression. Doing this isn’t entirely necessary for publication but could show more subtle changes than currently described in the manuscript.

I would be interested in seeing some speculation about potential covariation in morphology and δ18O within shells that exhibit sub-lethal injuries. I would expect there to be a depth change to either avoid predation during healing or to compensate for hydrostatic pressure during repair. Some notes on what ammonoids would be most likely to show morphology-geochemistry covariation would also be useful and likely based on morphological variance.

Overall, this is a well-presented manuscript and I look forward to seeing it published. The data and methods presented are useful and provide a good framework for exploring the paleobiology of cephalopods. If the authors have any questions or would like any clarification on my comments, do not hesitate to contact me.

- Ben Linzmeier (blinzmeier@southalabama.edu)

Line by line comments (No line numbers so copied text is first followed by indented comment):

Using δ13C of the shells and published data

I would suggest rephrasing to “Using δ13C of shell carbonate and published data on metabolic carbon” or something similar.

morphological parameters and the changes in δ13C and δ18O during ontogeny do not coincide except at hatching and at the onset of maturity

It may be useful to relate this to an interpretative conclusion.

a complex picture of their habitat

May be useful to rephrase to “complex picture of their behavior within their habitat.”

This is due to the fact that trapping for nautilus and collecting water samples at the same time is a difficult proposition.

I think this is only half the story because the analysis of δ18O of water and δ13C of DIC are both more difficult in some ways than the measurement of δ18O and δ13C of carbonates using gas-source mass spectrometry.

This is because the δ13C of the shell is both a function of carbon incorporated via the metabolism of the animal as well as from a function of the dissolved inorganic carbon (DIC).

Incorporate some references in this paragraph. You have them cited later in the paper, but putting them here would be useful, too.

Highly resolved morphological examination of nautilid conchs was difficult in the past but is now possible owing to the advancement of tomographic methods.

Add some more specificity here about the scale and type of morphological change that can now be resolved.

ground to a powder before isotope measurement

Can you add more specifics here? I assume mortar and pestle for grinding rather than using a rotary tool.

Water samples were also collected in June, 2003

Remove “also” here.

were also analyzed for δ18O of the water and δ13C

Remove “also” here.

The CT-scans obtained were used to measure the following morphological parameters (Fig. 2): conch diameter (dm), whorl width (ww), whorl height (wh), distance between the ventral edge of the siphuncle and the ventral edge

Being a bit more specific here would help bolster your discussion of the influence of time averaging on the comparison of morphology to isotope composition. From what is here, I do not know if each measurement is discrete or if you have a continuous collection of data (I assume discrete given the figures).

However, we note that nautilid septa represent time-averaged growth increments, which increase over ontogeny [19, 30, 31].

Clarify this statement a bit more. Do you mean the time averaged within each septum increases though ontogeny? I assume that is what you mean.

Thus, the calculated depths and temperatures based on analyses of the entire septal thicknesses represent temporal averages.

Add a mention of the approximate time averaged within a single analysis.

Detailed measurements by SIMS of δ18O in the outer shell of

It would be useful to write out “Secondary Ion Mass Spectrometry” for this acronym somewhere within the paper.

Given that our water samples were collected at one time, the septa we analyzed did not form in the exact seawater we sampled.

There are also concerns about along-reef movement. Some telemetry datasets show this information. In addition, the δ18O differences between the collection sites you show should be discussed somewhere. The spatial heterogeneity of the δ18O should also be addressed slightly. I assume submarine groundwater discharge may be important?

Previous studies documented temporal and seasonal variation of seawater temperature and chemistry at/near New Caledonia.

Data from the World Ocean Atlas (Locarnini et al., 2013) also shows some of this seasonality, although with less spatial resolution.

The value of δ13Cmeta is not known precisely, but Crocker et al. [39] and Pape [40] reported measurements of -17‰ for siphuncular organic material in nautilus, and we use that value in estimating Cmeta using eqn. 2.

Given the paucity of data, it may be useful to report what the range in δ13Cmeta would need to be in order to produce no metabolic change in the results of equation 2.

Both specimens also show an increase to 24–25% in Cmeta in septa 9 and 10.

It is likely important to consider variation in δ13Cdiet coinciding with the loss of the egg-yolk source of carbon and transition to other foods. From what I understand, you are assuming a constant δ13C of DIC and diet to calculate the metabolic rate.

They calculated that Cmeta decreased from ~30% to ~10% through ontogeny. The average values of Cmeta for post-hatching nautilus septa are comparable to the low values, generally less than 10%, observed in other marine mollusks such as bivalves and gastropods [8].

Chung et al., 2021 also mention that “the Cresp values before septum 10 are biased due to uncertain δ13Cdiet and δ13CDIC values, rendering the evaluation of ontogenetic variation in metabolism in the egg stage difficult.” Some discussion of pre-hatching δ13Cdiet and δ13CDIC may relate to the permeability of the egg case or changes in the δ13C of the diet given large contrasts between the yolk and post-hatching diet as explored in ammonites (Linzmeier et al., 2018).

Future work is needed to determine whether the δ13C of fossil nautilid shells can reasonably be used as a proxy for δ13C of paleo-DIC.

It may be good to point out the potential time intervals for some of the preserved nautilid carbonates here. I expect you may want to refer to the Buckhorn Asphalt?

Indeed, many studies of extinct cephalopods examine conch morphology.

Include some more references here.

Our results reveal that the changes in δ18O and δ13C also coincide with

Potentially reduce the use of “also” in this paragraph and potentially throughout the manuscript. It is not terribly distracting, but potentially unnecessary in some places.

We estimated the body chamber length at the time each septum formed using the length of the body chamber at adult.

Ohno et al., 2014 use a slightly more complicated approach to compare the δ18O of septa to shell wall and increase the size of the body chamber during growth given the results of a regression across many shells. It is unlikely to change the results with this adjustment, but may be worth mentioning here. In addition, the body chamber length varies periodically with the septal formation cycle, reaching a maximum size immediately before the precipitation of the septa during mural ridge formation which adds additional complexity to the septum-shell wall correlation (Ward et al., 1981). It’s a sticky problem to solve, but you could potentially do some sort of moving window/expanding window averaging to test sensitivity in the future.

Another conspicuous morphological change occurs in late ontogeny—known as the morphogenetic count down

It is probably worth citing (Zakharov et al., 2006) somewhere in this paragraph because they report similar changes in the δ18O of Nautilus during the morphogenetic count down.

resolved sampling may shed new light on the relationship between morphology and stable isotopes, although such methods also suffer from time-averaging to some degree

The math of the time averaging combined with swimming is incorporated into Linzmeier, 2019. For instance, Figure 5 in that paper shows the expected reduction in variance associated with averaging 23 days/analysis compared to random replicate sampling with 2 days/analysis. So the septal δ18O minimize expected depth variance even more than what is modeled. Differing vertical swimming velocities through ontogeny could also further interact with the time averaging.

Figure Comments.

Figure 1.

It may be useful to outline the possible Nautilus habitat around New Caledonia using implosion depth as the limiting factor.

Figure 3.

It would be useful to add curves from the World Ocean Atlas data product to show how these data relate to what are available there (including the seasonal component of variability). In addition, it would be useful to plot these with the full possible water depth distribution of Nautilus macromphalus linked to the Y axis. You could have panel C as its own figure and combine A and B with Figure 4 to have slightly different aspect ratios for the figures.

Figure 4.

I think the mean line is not necessarily appropriate for this figure given the number of levels that the mean is only of one analysis. It could either be dropped or using LOESS, or polynomial fit may be more appropriate to summarize the data given some depths with single observations.

Figure 5.

Noting the analytical precision on these figures would be useful.

Figure 6.

I find the inset plots slightly distracting. Given the body of the paper it may be useful to separate these into an additional figure that could be focused on only post-hatching variability. It would leave a fair amount of blank-space on this figure, but that could be adjusted by using a log y axis?

Figure 7.

I would like to see the R2, p-value and a 95% confidence interval around the regression.

Figure 8.

It would be useful to incorporate some sort of error estimation around these lines to illustrate the combined effects of error from the δ18O analyses and regression models.

Figure 9.

Because you compare the depths of the N. macromphalus to these, I would like to see subtle grey lines showing these data as a point of reference.

Figure 10.

Adding an annotation based on comments from Chung et al., 2021, about uncertainty in the pre-hatching metabolic rate given uncertainty in diet and DIC would be useful. The assumptions of the calculation may be violated for these data.

Works cited

Linzmeier, B.J., 2019, Refining the interpretation of oxygen isotope variability in free-swimming organisms: Swiss Journal of Palaeontology, v. 138, p. 109–121, doi:10.1007/s13358-019-00187-3.

Linzmeier, B.J., Landman, N.H., Peters, S.E., Kozdon, R., Kitajima, K., and Valley, J.W., 2018, Ion microprobe–measured stable isotope evidence for ammonite habitat and life mode during early ontogeny: Paleobiology, v. 44, p. 684–708, doi:10.1017/pab.2018.21.

Locarnini, R.A. et al., 2013, World Ocean Atlas 2013, Volume 1: Temperature. (S. Levitus, Ed.): NOAA Atlas NESDIS 73, 40 p.

Ward, P., Greenwald, L., and Magnier, Y., 1981, The Chamber Formation Cycle in Nautilus macromphalus: Paleobiology, v. 7, p. 481–493.

Zakharov, Y., Shigeta, Y., Smyshlyaeva, O., Popov, A., and Ignatiev, A., 2006, Relationship between δ13C and δ18O values of the Recent Nautilus and brachiopod shells in the wild and the problem of reconstruction of fossil cephalopod habitat: Geosciences Journal, v. 10, p. 331–345, doi:10.1007/BF02910374.

Reviewer #2: Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail?

Partly.

There are a number of important details explained in the discussion section that I would like to see explained earlier, particularly details relevant to making sense of the methods. For example, the methods state that the authors did not do high resolution isotopic analyses of growth increments but rather low spatial resolution sampling of septa. This raises all sorts of questions that should not be left until the end, such as whether this scale is appropriate to the question. That should be clear to the reader in the methods and not something to save for the discussion. How much time is represented by a single septum? Nautiloids are also known to migrate vertically and horizontally daily and should experience a range of environmental conditions. Do nautiloids grow new shell only when at certain preferred depths? What if they live most of the time in deeper waters but form new shell when they are more metabolically active in shallower waters? How do the authors know this isn’t the case? Or is the isotopic record of the nautilus shell averaged across environments (i.e., if they grow more or less continuously)? If the isotopic proxy for environment is averaged and not representative of a single environment, is it valid to use the regression equation for depth vs. predicted d18O values? The authors may be right in everything they did, but they do not make a strong enough justification that their approach works based on the methods section alone. The discussion section answers some of this, but not all of it, and the reader should have some confidence the methods are appropriate before they see the results.

There are many questions about the authors’ attempt to analyze d13C of sampled waters. There is no information on how much time elapsed between water sample collection and analysis. Samples can often be stored for some time if they have been treated properly. However, the methods say only that the samples were stored in the dark. This would not have prevented bacterial alteration of DIC and d13CDIC in the water samples. In the discussion, oddly enough, the authors admit they did not dose the samples with HgCl2 as required to stop bacterial activity. I’m puzzled why this didn’t happen in the first place, why it isn’t mentioned in the methods just the discussion, why the analyses were done at all after the early misstep, and why the authors reported the data knowing the data can not be interpreted as d13CDIC.

The authors used literature values of d13CDIC, but, as before, very little is explained in the methods about how were the data selected from Ko et al. (2014). Was a single value used? If so, why one? These answers are provided but only in the discussion. If it’s more efficient to leave the writing as is, fine, but at least refer the reader to “x, y, and z are explained in the discussion.”

The sampling of septa is a low-resolution approach but it seems appropriate to the scale of the question, which is correlation with habitat and morphological change over years.

The estimation of the fraction of metabolic carbon incorporated into the shell from equation 2 (reported from another previously published study – reference should be Crocker 39 and not Pape 40, by the way) relies on several poorly known parameters. One that stands out, besides d13CDIC, is the single value of -17 per mil for organic matter in nautilus. This is one measurement from one species at one point in ontogeny. I looked at the Crocker paper in depth, and I can not figure out why -17 per mil was selected. That paper presented data from 6 nautilus specimens, with different values between and within specimens. Variation within specimens can be as high as 6 per mil. There is no mention of -17 per mil in the text of the paper, so this number will have to be justified.

Conclusions are presented in an appropriate fashion and are supported by the data?

The main finding is that nautilus hatch in shallower, warmer waters and move to deeper depths post-hatching is supported by the use of d18Oshell as a proxy for depth.

Hope is also expressed that even though fossil nautilids can’t often be studied geochemically to reconstruct their reproductive habits/habitats, morphology could serve as a proxy for geochemical changes related to life habits. Other statements about this, however, are inconsistent. In one place, the authors state that changes in geochemistry concide with morphological parameters. Elsewhere, they state that geochemical and morphological changes are “within the range of fluctuation and thus difficult to correlate.” Later, they state that “detectable morphological changes are not clearly reflected” in the geochemistry. Which is it? This message is very muddled, and the significance for future work is not clear.

The article is presented in an intelligible fashion and is written in standard English?

Mostly. The aims of the paper are stated very clearly.

There are a few parts that could be written more clearly. For example, what is “presence of septal approximation”? Define specialized terms for non-specialists.

Equation 2 is missing some parentheses.

There’s a typo in Fig. 6E. One of the septum numbers is missing.

There may be issues with the references. The authors refer to Pape 40, and there is a Pape 40, but they clearly meant Crocker 39. A standard reference check would not catch that. References should be rechecked carefully.

Please show the abbreviated equations in the Fig. 6 caption in the parentheses instead of the acronyms. There’s no point in showing the acronym for the term since the term is already spelled out in the same sentence. However, what a reader may have trouble recalling are the equations for each index. It’s very frustrating as a reviewer or reader to have do a scavenger hunt to be confused about what the terms mean and then have to search the text. In this case, I had to open three windows side by side to figure it out, one for this figure, one for the text definitions of indices, and a third for the figure showing the measurements used in the indices. Ideally, any reader should be able to look at a graph and caption and get it.

Also in Fig. 6, should septal position index be siphuncle position index? The former is not defined in the methods, but the latter is.

Also in Fig 6A and B, the septal spacing graph is hard to compare to the other graphs because x-axis is different. That makes it difficult to look at the graphs an intuitively know whether all the morphological changes are happening at the same point in ontogeny or not.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Benjamin J. Linzmeier, PhD

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Jun 2022

POINT-BY-POINT RESPONSES TO REVIEWS

Comments regarding journal requirements:

“1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf"”

RESPONSE: We corrected the figure citations, the size of headings, and the legend in Table 1.

“2. Thank you for stating the following in the Funding Section of your manuscript:

“AT was supported by a Grant-in-Aid for JSPS Research Fellow, Grant-in-Aid for Young Scientists (grant nrs. 20J00376 and 21K14028). NHL and JKC were supported by the Norman D. Newell Fund (AMNH).”

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“AT was supported by a Grant-in-Aid for JSPS Research Fellow, Grant-in-Aid for Young Scientists (grant nrs. 20J00376 and 21K14028). NHL and JKC were supported by the Norman D. Newell Fund (AMNH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.”

RESPONSE: We removed the funding statement from the manuscript. The current funding statement is good as it is.

” 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.”

RESPONSE: All data are available in S1 Table and thus we would like to update the Data Availability statement.

“4. We note that [Figure 1] in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:”

RESPONSE: We reproduced the map in Fig 1 using OpenStreetMap, which is open data licensed under the Open Data Commons Open Database License (ODbL) by the OpenStreetMap Foundation (OSMF). We added the copy right information to the figure caption.

Comments from Editor:

“The two reviewers raise truly important issues regarding your study. It may prove very difficult to address yourselves to all the problems, but at the very least you should acknowledge the problems even if you can't fix them or solve them. On a personal level, I have been a skeptic of isotopic studies like this for some of the reasons cited by Reviewer 2, especially the question when shell secretion actually takes place. If you do resubmit this paper, I shall have it critically reviewed again.”

RESPONSE: We revised the manuscript as outlined below. Previous studies have suggested that the secretion of septa is more or less continuous (Ward 1987; Oba et al. 1992; Linzmeier et al. 2016). Although some issues remain, as discussed below, we are explicit in acknowledging and dealing with them. We think that the conclusions we reach are robust.

Comments from Reviewer #1:

“I believe that this study requires a short ethics statement given the locally protected status of Nautilus macromphalus and the PLoS expectations for ethics statements related to cephalopods (https://journals.plos.org/plosone/s/animal-research). Mostly a statement on the timing of collection and the purpose of collection would suffice. I should reiterate that I believe the work these authors have done has been conducted ethically, but given the journal requirements, this statement should be added.”

RESPONSE: We added Ethics Statement in the Materials and Methods section.

“I would like to see slightly more nuanced phrasing related to the depth estimates throughout the manuscript. Later in the text it is clearly stated that these estimates are averages given septal formation duration, but it would be helpful to state early-on that “depth estimates average X days growth”. There is also an implicit assumption that septal formation rate is constant at all depths, which is probably fine given inferences from apertural growth and variance observed in subsampling septa by other authors. In addition, the time averaged within each septa likely varies with growth, but the possible depth range (or swimming velocity) also likely scales with size. Changing the time-at-depth distribution coupled to the time averaging could change the observed mean depth habitat in a free-swimming organism like Nautilus especially during the morphogenic count down (Linzmeier, 2019).”

RESPONSE: We added more information on the time required to produce each septum. As Landman and Cochran (1987) explained, the time to secrete a septum increases throughout ontogeny. In early ontogeny, a septum may take weeks to be secreted. In later ontogeny, a septum takes months. This pattern is well established, but the actual times are difficult to pin down. The pattern can be inferred from aquarium observations, but the precise timing in nature is more difficult to ascertain.

“One potential area of improvement would be to do a “time averaging” to the morphological parameters and compare these averaged values to the resultant δ18O and δ13C. It is unclear to me from the methods description if the quantification is done at a point along the shell or an average for a region. Currently the comparison between the isotope and morphological data is done broadly and descriptively but quantitative comparison (e.g. regression results) could be explored. For this to be fruitful, major changes in morphology and isotope ratios at the hatching and morphogenetic count down would likely need to be trimmed from the regression. Remaining morphological parameters and isotope results could show a statistically significant, but low R2 regression. Doing this isn’t entirely necessary for publication but could show more subtle changes than currently described in the manuscript.”

RESPONSE: We produced new data on the siphuncle position index (SPI) for each septum and made new graphs. This allowed us to make a direct comparison between septal spacing, SPI, and isotopic values. Using the data, we carried out statistical tests to determine if there is a correlation between them. Accordingly, we produced Table 2 to show the results. It is important to emphasize that the two observed morphological changes indicative of hatching and the onset of maturity are, indeed, manifested in changes in the isotope signature.

“I would be interested in seeing some speculation about potential covariation in morphology and δ18O within shells that exhibit sub-lethal injuries. I would expect there to be a depth change to either avoid predation during healing or to compensate for hydrostatic pressure during repair. Some notes on what ammonoids would be most likely to show morphology-geochemistry covariation would also be useful and likely based on morphological variance.”

RESPONSE: It is a very interesting suggestion, and we added some additional notes on these points at the end of the discussion section. However, the shells that form the basis of this study do not show any significant shell breaks and, therefore, cannot be used to investigate this issue. This topic is the subject of a future study.

“I would suggest rephrasing to “Using δ13C of shell carbonate and published data on metabolic carbon” or something similar.”

RESPONSE: We rephrased the expression, following the suggestion.

“morphological parameters and the changes in δ13C and δ18O during ontogeny do not coincide except at hatching and at the onset of maturity

It may be useful to relate this to an interpretative conclusion.”

RESPONSE: We added some notes on this point to the Conclusions section.

“a complex picture of their habitat

May be useful to rephrase to “complex picture of their behavior within their habitat.”

RESPONSE: We rephrased the expression, following the suggestion.

“This is due to the fact that trapping for nautilus and collecting water samples at the same time is a difficult proposition.

I think this is only half the story because the analysis of δ18O of water and δ13C of DIC are both more difficult in some ways than the measurement of δ18O and δ13C of carbonates using gas-source mass spectrometry.”

RESPONSE: We added this point to the text.

“This is because the δ13C of the shell is both a function of carbon incorporated via the metabolism of the animal as well as from a function of the dissolved inorganic carbon (DIC).

Incorporate some references in this paragraph. You have them cited later in the paper, but putting them here would be useful, too.”

RESPONSE: We added some references to this part.

“Highly resolved morphological examination of nautilid conchs was difficult in the past but is now possible owing to the advancement of tomographic methods.

Add some more specificity here about the scale and type of morphological change that can now be resolved.”

RESPONSE: We added some text to specify the morphological changes.

“ground to a powder before isotope measurement

Can you add more specifics here? I assume mortar and pestle for grinding rather than using a rotary tool.”

RESPONSE: Septal samples were removed using clippers. The samples were then ground down using a mortar and pestle.

“Water samples were also collected in June, 2003

Remove “also” here.

were also analyzed for δ18O of the water and δ13C

Remove “also” here.”

RESPONSE: We removed “also” from the sentences.

“The CT-scans obtained were used to measure the following morphological parameters (Fig. 2): conch diameter (dm), whorl width (ww), whorl height (wh), distance between the ventral edge of the siphuncle and the ventral edge

Being a bit more specific here would help bolster your discussion of the influence of time averaging on the comparison of morphology to isotope composition. From what is here, I do not know if each measurement is discrete or if you have a continuous collection of data (I assume discrete given the figures).

RESPONSE: We added more details of the data collection.

However, we note that nautilid septa represent time-averaged growth increments, which increase over ontogeny [19, 30, 31].

Clarify this statement a bit more. Do you mean the time averaged within each septum increases though ontogeny? I assume that is what you mean.”

RESPONE: We rephrased the sentence as suggested.

“Thus, the calculated depths and temperatures based on analyses of the entire septal thicknesses represent temporal averages.

Add a mention of the approximate time averaged within a single analysis.”

RESPONSE: The exact time of septal formation of N. macromphalus in the wild is not known. We added some estimates from previous studies using aquarium observation and radiometric methods.

“Detailed measurements by SIMS of δ18O in the outer shell of

It would be useful to write out “Secondary Ion Mass Spectrometry” for this acronym somewhere within the paper.”

RESPONSE: We spelled out the acronym.

“Given that our water samples were collected at one time, the septa we analyzed did not form in the exact seawater we sampled.

There are also concerns about along-reef movement. Some telemetry datasets show this information. In addition, the δ18O differences between the collection sites you show should be discussed somewhere. The spatial heterogeneity of the δ18O should also be addressed slightly. I assume submarine groundwater discharge may be important?”

RESPONSE: As discussed in the manuscript, we go over at length the fact that nautilus is a mobile animal. It migrates vertically and horizontally, so isotopic values reflect averages. In addition, the septa themselves are time averaged. We explore the implications of these facts more fully in the manuscript. We added details in the Isotope hydrography section comparing the two water stations. In fact, they are quite similar when �  18O is plotted against salinity (plot added to supplemental information). To our knowledge there are no data on submarine groundwater discharge in New Caledonia and it is difficult to discern its possible impact on the �  18O profiles.

“Previous studies documented temporal and seasonal variation of seawater temperature and chemistry at/near New Caledonia.

Data from the World Ocean Atlas (Locarnini et al., 2013) also shows some of this seasonality, although with less spatial resolution.”

RESPONSE: We added the reference. However, it is worth noting that seasonal variation is most likely to affect the upper 50 m, which is not the preferred habitat of nautilus.

“The value of δ13Cmeta is not known precisely, but Crocker et al. [39] and Pape [40] reported measurements of -17‰ for siphuncular organic material in nautilus, and we use that value in estimating Cmeta using eqn. 2.

Given the paucity of data, it may be useful to report what the range in δ13Cmeta would need to be in order to produce no metabolic change in the results of equation 2.”

RESPONSE: We have refined the calculation of �  13Cmeta using the data of Crocker et al. They measured siphuncular �  13C through ontogeny in two wild caught N. macromphalus specimens from New Caledonia. We averaged �  13C values from pre- and post-hatching siphuncular material, and the values ae essentially identical in both specimens. This is now discussed in the text and used to refine the calculations of % metabolic carbon in the specimens using eqn. 2.

“Both specimens also show an increase to 24–25% in Cmeta in septa 9 and 10.

It is likely important to consider variation in δ13Cdiet coinciding with the loss of the egg-yolk source of carbon and transition to other foods. From what I understand, you are assuming a constant δ13C of DIC and diet to calculate the metabolic rate.”

RESPONSE: See the response above. The similarity in �  13C in pre- and post-hatching siphuncular material suggests that the �  13Cdiet doesn’t vary pre- and post-hatching. The yolk and Nautilus prey after hatching essentially represent marine organic matter. What changes is the amount of metabolic carbon incorporated into the shell in the egg and immediately post hatching. The simplest explanation for the data is that the metabolic fraction (and likely growth rate) is high in the early stages of growth in the egg (septa 1-5), deceases toward hatching (septa 7,8) then increases immediately after hatching, before finally reducing to about 15% through the remainder of ontogeny.

“They calculated that Cmeta decreased from ~30% to ~10% through ontogeny. The average values of Cmeta for post-hatching nautilus septa are comparable to the low values, generally less than 10%, observed in other marine mollusks such as bivalves and gastropods [8].

Chung et al., 2021 also mention that “the Cresp values before septum 10 are biased due to uncertain δ13Cdiet and δ13CDIC values, rendering the evaluation of ontogenetic variation in metabolism in the egg stage difficult.” Some discussion of pre-hatching δ13Cdiet and δ13CDIC may relate to the permeability of the egg case or changes in the δ13C of the diet given large contrasts between the yolk and post-hatching diet as explored in ammonites (Linzmeier et al., 2018).”

RESPONSE: See response above re: pre- and post-hatching �  13Cdiet. In fact, Chung et al. used the Crocker et al. data to obtain the �  13Cdiet (they get -16.5‰). Their �  13CDIC is from Aulclair et al. who give “~0.5‰” with no reference. The �  13CDIC may be different in the egg case, but we don’t have the information to address that, and the simplest explanation for the �  13C of septa formed in the egg, as noted above, is variation in the fraction of C incorporated into the shell via metabolism. Moreover, our link to fossil nautilids is restricted to post-hatching shell material as a potential indication of ambient �  13CDIC.

“Future work is needed to determine whether the δ13C of fossil nautilid shells can reasonably be used as a proxy for δ13C of paleo-DIC.

It may be good to point out the potential time intervals for some of the preserved nautilid carbonates here. I expect you may want to refer to the Buckhorn Asphalt?”

RESPONSE: We added a mention on this point.

“Our results reveal that the changes in δ18O and δ13C also coincide with

Potentially reduce the use of “also” in this paragraph and potentially throughout the manuscript. It is not terribly distracting, but potentially unnecessary in some places.”

REFERENCES: We also removed most of the “also” occurrences in the manuscript.

“We estimated the body chamber length at the time each septum formed using the length of the body chamber at adult.

Ohno et al., 2014 use a slightly more complicated approach to compare the δ18O of septa to shell wall and increase the size of the body chamber during growth given the results of a regression across many shells. It is unlikely to change the results with this adjustment, but may be worth mentioning here. In addition, the body chamber length varies periodically with the septal formation cycle, reaching a maximum size immediately before the precipitation of the septa during mural ridge formation which adds additional complexity to the septum-shell wall correlation (Ward et al., 1981). It’s a sticky problem to solve, but you could potentially do some sort of moving window/expanding window averaging to test sensitivity in the future.”

RESPONSE: Ohno et al. (2014) suggested a positive correlation between shell diameter and body chamber angle. However, they did not take into account the fact that modern nautilids grow the aperture by 15° on average after the formation of last septum (Collins and Ward 1987). Indeed, their data show that (most likely) adult specimens possess a longer body chamber angle, while the body chamber length of immature specimens seems constant with some variation. Therefore, their method is most likely not appropriate. As for the latter point, it is true that the body chamber length changes during ontogeny, which produces another uncertainty about the estimate of the aperture position. We added this point to the manuscript.

“Another conspicuous morphological change occurs in late ontogeny—known as the morphogenetic count down

It is probably worth citing (Zakharov et al., 2006) somewhere in this paragraph because they report similar changes in the δ18O of Nautilus during the morphogenetic count down.”

RESPONSE: We added the reference.

“resolved sampling may shed new light on the relationship between morphology and stable isotopes, although such methods also suffer from time-averaging to some degree

The math of the time averaging combined with swimming is incorporated into Linzmeier, 2019. For instance, Figure 5 in that paper shows the expected reduction in variance associated with averaging 23 days/analysis compared to random replicate sampling with 2 days/analysis. So the septal δ18O minimize expected depth variance even more than what is modeled. Differing vertical swimming velocities through ontogeny could also further interact with the time averaging.”

RESPONSE: We added the missing reference. We decided not to add the details to the text because it is slightly off the context.

“Figure 1.

It may be useful to outline the possible Nautilus habitat around New Caledonia using implosion depth as the limiting factor.”

RESPONSE: It would be interesting to indicate the habitat of Nautilus macromphalus near New Caledonia using implosion depth. However, it is slightly off the topic of this manuscript and is highly speculative due to lack of knowledge about horizontal migration. We also think that excessive information that is not directly related to the main point of this manuscript could be distracting for readers, and thus we decided not to add this information.

“Figure 3.

It would be useful to add curves from the World Ocean Atlas data product to show how these data relate to what are available there (including the seasonal component of variability). In addition, it would be useful to plot these with the full possible water depth distribution of Nautilus macromphalus linked to the Y axis. You could have panel C as its own figure and combine A and B with Figure 4 to have slightly different aspect ratios for the figures.”

RESPONSE: To our knowledge, no studies have been attempted to detect the possible habitat depth of Nautilus in New Caledonia using ultrasonic telemetry techniques. There are only a few studies that examined δ18O of Nautilus from New Caledonia. But, as mentioned in the manuscript, the information on water chemistry and temperature is missing in those studies. For this reason, we attempt to refine this aspect in our present paper. Therefore, we do not think that it is appropriate to indicate speculative depth in the figures. Also, there is ample evidence (including your own composite plot) suggesting that seasonal variation in temperature is small below 50 m. Finally, the dataset documenting the depth distribution of �  18Owater is extremely limited (i.e., virtually non-existent) for this area, and we believe it is preferable to focus on the new data rather than the conventional 0.5‰, assumed to be uniform with depth.

“Figure 4.

I think the mean line is not necessarily appropriate for this figure given the number of levels that the mean is only of one analysis. It could either be dropped or using LOESS, or polynomial fit may be more appropriate to summarize the data given some depths with single observations.”

RESPONSE: As the reviewer points out, there is uncertainty about the temperature in the area/ habitat of Nautilus. We are aware that it may not be completely accurate to use the mean value but using LOESS or polynominal fit does not solve this problem. Thus, we decided to keep the mean value.

“Figure 5.

Noting the analytical precision on these figures would be useful.”

RESPONSE: The analytical precision is cited in the relevant Methods section and is small relative to the measured values. A more significant component of uncertainty is the agreement between laboratories. We have now added a comparison of �  13C and �  18O of select septa measured by two isotope laboratories and discuss the “uncertainty” produced by these replicate measurements.

“Figure 6.

I find the inset plots slightly distracting. Given the body of the paper it may be useful to separate these into an additional figure that could be focused on only post-hatching variability. It would leave a fair amount of blank-space on this figure, but that could be adjusted by using a log y axis?”

RESPONSE: As the reviewer pointed out, the visibility may have not been high enough. However, we would like to keep the graphs of morphological parameters in a single figure. We found that using a log-transformed axis does not improve the visibility. Thus, following the suggestions of Reviewer #2, we changed the inset plots in a way that both graphs share the same x-axis to increase the visibility. In addition to these changes, we carried out statistical analyses for septal spacing and SPI and produced Table 2 so that readers will not need to visually compare the results.

“Figure 7.

I would like to see the R2, p-value and a 95% confidence interval around the regression.”

RESPONSE: We updated Fig. 7, following the suggestions.

“Figure 8.

It would be useful to incorporate some sort of error estimation around these lines to illustrate the combined effects of error from the δ18O analyses and regression models.”

RESPONSE: We added shaded areas to Fig 8, which represents the error estimate calculated with the 95% confidence interval of the regression. We also added some new data points of depths calculated with the δ18O values that we obtained from the University of Michigan for inter-lab comparisons.

“Figure 9.

Because you compare the depths of the N. macromphalus to these, I would like to see subtle grey lines showing these data as a point of reference.”

RESPONSE: We think that doing this will make the data difficult to read for N. pompilius, and we prefer not to add the data that are already provided in Fig. 9.

“Figure 10.

Adding an annotation based on comments from Chung et al., 2021, about uncertainty in the pre-hatching metabolic rate given uncertainty in diet and DIC would be useful. The assumptions of the calculation may be violated for these data.”

RESPONSE: See the comments above on �  13C in diet based on actual siphuncular �  13C from wild-caught Nautilus from New Caledonia (Crocker et al.). They show no difference pre- or post-hatching, suggesting that at least the “diet” has the same �  13C in both stages. The metabolic rate is certainly different as expressed in the higher fraction of metabolic carbon in the pre-hatching septal �  13C. We have added upper and lower bounds on the calculated metabolic fraction to the tables and Fig. 10 based on the average +/- 1sd of the Crocker data.

Comments from Reviewer #2:

“There are a number of important details explained in the discussion section that I would like to see explained earlier, particularly details relevant to making sense of the methods. For example, the methods state that the authors did not do high resolution isotopic analyses of growth increments but rather low spatial resolution sampling of septa. This raises all sorts of questions that should not be left until the end, such as whether this scale is appropriate to the question. That should be clear to the reader in the methods and not something to save for the discussion. How much time is represented by a single septum?”

RESPONSE: We added this information with references to the Material and Methods section. As noted above in response to reviewer 1, we added more information on the time required to produce each septum. As Landman and Cochran (1987) explained, the time to secrete a septum increases throughout ontogeny. In early ontogeny, a septum may take weeks to be secreted. In later ontogeny, a septum takes months. This pattern is well established, but the actual times are difficult to pin down. The pattern can be inferred from aquarium observations, but the precise timing in nature is more difficult to ascertain.

“Nautiloids are also known to migrate vertically and horizontally daily and should experience a range of environmental conditions. Do nautiloids grow new shell only when at certain preferred depths? What if they live most of the time in deeper waters but form new shell when they are more metabolically active in shallower waters? How do the authors know this isn’t the case? Or is the isotopic record of the nautilus shell averaged across environments (i.e., if they grow more or less continuously)? If the isotopic proxy for environment is averaged and not representative of a single environment, is it valid to use the regression equation for depth vs. predicted d18O values? The authors may be right in everything they did, but they do not make a strong enough justification that their approach works based on the methods section alone. The discussion section answers some of this, but not all of it, and the reader should have some confidence the methods are appropriate before they see the results.”

RESPONSE: It is true that modern nautilids migrate vertically and traverse different environments. Thus, the data presented in our paper are time-averaged. Nevertheless, as mentioned, aquarium experiments and radiometric data suggest that the formation of septa is more or less continuous. We added some additional explanation regarding the formation of septa and time-averaging to the manuscript. Linzmeier et al. (2016) and Oba et al. (1992) documented that shell is secreted continuously or how else could they entrain variation due to migration?

“There are many questions about the authors’ attempt to analyze d13C of sampled waters. There is no information on how much time elapsed between water sample collection and analysis. Samples can often be stored for some time if they have been treated properly. However, the methods say only that the samples were stored in the dark. This would not have prevented bacterial alteration of DIC and d13CDIC in the water samples. In the discussion, oddly enough, the authors admit they did not dose the samples with HgCl2 as required to stop bacterial activity. I’m puzzled why this didn’t happen in the first place, why it isn’t mentioned in the methods just the discussion, why the analyses were done at all after the early misstep, and why the authors reported the data knowing the data can not be interpreted as d13CDIC.”

RESPONSE: The samples were collected by our New Caledonian colleagues, and it was not possible to poison them using HgCl2. Nevertheless, we tried to measure DIC and �  13C of DIC but as noted originally in the text, the samples did not reliably preserve the original �  13C signature. We have now eliminated those data and subsequent discussion of them from the text and table.

“The authors used literature values of d13CDIC, but, as before, very little is explained in the methods about how were the data selected from Ko et al. (2014). Was a single value used? If so, why one? These answers are provided but only in the discussion. If it’s more efficient to leave the writing as is, fine, but at least refer the reader to “x, y, and z are explained in the discussion.”

RESPONSE: Re: �  13CDIC, we have added a link in the Methods section to the Isotope Hydrography Results and there to the Discussion explaining the choice of �  13CDIC values used in the calculation of the fraction of metabolic C incorporated into the septal aragonite.

“The sampling of septa is a low-resolution approach but it seems appropriate to the scale of the question, which is correlation with habitat and morphological change over years.”

RESPONSE: Yes. Although sampling septa is not a high-resolution approach, we think that it is reasonable to estimate the habitat depth and morphological change. To make things clearer, we added more information about the time-averaging and potential difficulty about estimating the position of the aperture.

“The estimation of the fraction of metabolic carbon incorporated into the shell from equation 2 (reported from another previously published study – reference should be Crocker 39 and not Pape 40, by the way) relies on several poorly known parameters. One that stands out, besides d13CDIC, is the single value of -17 per mil for organic matter in nautilus. This is one measurement from one species at one point in ontogeny. I looked at the Crocker paper in depth, and I can not figure out why -17 per mil was selected. That paper presented data from 6 nautilus specimens, with different values between and within specimens. Variation within specimens can be as high as 6 per mil. There is no mention of -17 per mil in the text of the paper, so this number will have to be justified.”

RESPONSE: We have expanded the rationale for our selection of the value of �  13Cmeta. We used the data from Crocker et al. to calculate average values of pre- and post-hatching siphuncular material in the six N. macromphalus reported by them. Although there is variation among the specimens, the average siphuncular �  13C, either in individual specimens or overall, is essentially the same and given in the text. We calculate an overall average of -16.6 ± 1.7‰ for the metabolic �  13C. Given all the uncertainties in the data, we round this to -17 ± 2‰ to calculate Cmeta from the shell �  13C data in our specimens. A range in �  13Cmeta of -15 to -19‰, based on the calculated standard deviation, is also plotted in Fig. 10. The value of �  13Cmeta calculated using the Crocker et al. N. macromphalus data is comparable to that measured in N. pompilius 32 years later by Pape (-17.4‰), but admittedly the latter is only a single measurement.

“The main finding is that nautilus hatch in shallower, warmer waters and move to deeper depths post-hatching is supported by the use of d18Oshell as a proxy for depth. Hope is also expressed that even though fossil nautilids can’t often be studied geochemically to reconstruct their reproductive habits/habitats, morphology could serve as a proxy for geochemical changes related to life habits. Other statements about this, however, are inconsistent. In one place, the authors state that changes in geochemistry concide with morphological parameters. Elsewhere, they state that geochemical and morphological changes are “within the range of fluctuation and thus difficult to correlate.” Later, they state that “detectable morphological changes are not clearly reflected” in the geochemistry. Which is it? This message is very muddled, and the significance for future work is not clear.”

RESPONSE: We meant that the morphological and geochemical changes coincide ONLY at the point of hatching and maturity and that the correlation is not clear in the rest of ontogeny. We clarified the text to emphasize this point.

“The aims of the paper are stated very clearly.There are a few parts that could be written more clearly. For example, what is “presence of septal approximation”? Define specialized terms for non-specialists.”

RESPONSE: We added an explanation for the somewhat technical terms.

“Equation 2 is missing some parentheses.”

RESPONSE: The equation has been modified but was correct as written.

“There’s a typo in Fig. 6E. One of the septum numbers is missing.”

RESPONSE: We corrected the typo.

“There may be issues with the references. The authors refer to Pape 40, and there is a Pape 40, but they clearly meant Crocker 39. A standard reference check would not catch that. References should be rechecked carefully.

Please show the abbreviated equations in the Fig. 6 caption in the parentheses instead of the acronyms. There’s no point in showing the acronym for the term since the term is already spelled out in the same sentence. However, what a reader may have trouble recalling are the equations for each index. It’s very frustrating as a reviewer or reader to have do a scavenger hunt to be confused about what the terms mean and then have to search the text. In this case, I had to open three windows side by side to figure it out, one for this figure, one for the text definitions of indices, and a third for the figure showing the measurements used in the indices. Ideally, any reader should be able to look at a graph and caption and get it.”

RESPONSE: As noted above, both Crocker et al. and Pape are relevant and are referenced in the discussion of the estimation of �  13Cmeta. We retain both references. We removed the acronym in the caption for Fig. 6 and added abbreviated equations.

“Also in Fig. 6, should septal position index be siphuncle position index? The former is not defined in the methods, but the latter is.”

RESPONSE: We corrected the labels in Fig. 6.

“Also in Fig 6A and B, the septal spacing graph is hard to compare to the other graphs because x-axis is different. That makes it difficult to look at the graphs an intuitively know whether all the morphological changes are happening at the same point in ontogeny or not.”

RESPONSE: We corrected the inset plots so that both graphs can share the same x-axis.

27 Jun 2022

Refining the habitat of Nautilus macromphalus based on knowledge of the δ18O and δ13C of the shell and the temperature and δ18O of the water column in New Caledonia

PONE-D-21-35042R1

Dear Dr. Tajika,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Geerat J. Vermeij

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The paper is now accepted except that I insist on a shortening of the title. I recommend: Isotopic evidence concerning the habitat of Nautilus macromphalus in New Calednonia.

Reviewers' comments:

1 Jul 2022

PONE-D-21-35042R1

Isotopic evidence concerning the habitat of Nautilus macromphalus in New Caledonia

Dear Dr. Tajika:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Geerat J. Vermeij

Academic Editor

PLOS ONE