Skeletal chemistry of Nautilus and its taxonomic significance

BiochemicalSysterna~csand Ecology,Vol. 15, No. 4, pp. 461-474, 1987. Printed in GreatBritain.

0305-1978/87 $3.00+0.00 © 1987PergamonJournalsLtd.

Skeletal Chemistry of Nautilus and its Taxonomic Significance REX E. CRICK,* KEITH O. MANNt and JOHN A. CHAMBERLAIN, JR:~§ *Department of Geology, University of Texas, Arlington, TX 76019-0049, U.S.A.; tDepartment of Geology, University of Iowa, Iowa City, IA 52242, U.S.A.; :~Department of Geology, Brooklyn College of the City University of New York, Brooklyn, NY 11210, U.S.A.; §Osborn Laboratories of Marine Sciences, New York Aquarium, New York Zoological Society, Brooklyn, NY 11224, U.S.A.

Key Word Index--Nautilus; Cephalopoda; skeletal chemistry; strontium; magnesium; biochemical taxonomy; ontogenetic variation. Abstract--The strontium (Sr) and magnesium (Mg) chemistry of the shell wall and septum as well as the spherulitic-prismatic and nacreous layers of these structures was determined for Nautilus species: N. belauensis, N. macromphalus, N. pompilius and N. scrobiculatus. Each species of Nautilus exhibits greater variability and higher concentrations of Mg in juvenile portions of the shell than in more mature portions of the shell. This decrease in the variability and amount of Mg in the aragonite lattice suggests a physiochemical system which becomes more efficient with time relative to carbonate production. Statistically significant differences in the Sr and Mg content of spherulitic-prismatic and nacreous layers of the shell and septum indicate that these layers were formed from extracellular fluids of different compositions. Concentrations of Sr and Mg in aragonite of the shell wall are characteristic for each species and sufficiently invariant within species to allow species of Nautilus to be distinguished statistically on the basis of either the Sr or Mg content of the shell wall or the Mg content of septa.

Introduction The cephalopod genus Nautilus is the only survivor of 550 million years of cephalopod evolution which continues to use an external, camerated shell composed of calcium carbonate (CaCO3) for support and protection. Because Nautilus is the best modern analogy for the diverse array of extinct cephalopods, great interest has developed in the ecology, shell rnicrostructure, hydromechanics and certain aspects of the physiology of this unique invertebrate. One aspect of the physiology of Nautilus that needs clarification is the inorganic chemistry of the calcified or mineralized structures of the skeleton, including the shell, beak of the masticatory apparatus, and deposits within the renal appendages (uroliths). Knowledge of the concentration of elements important to the carbonate system--magnesium (Mg), strontium (Sr), calcium (Ca)--and details of their occurrence in calcified portions of Nautilus provides an indirect link to the physiology and genetics of the genus and its species [1, 2]. Moreover, knowledge of the chemistry of calcified portions (Received28 July 1986) 461

of Nautilus and of the physiochemical controls responsible for this chemistry will enable the use of this information as a first order approximation of the chemistries of extinct cephalopods whose shell material has survived in an unaltered state [3]. Early interest in the inorganic chemistry of Nautilus established the presence of Sr in the aragonite shell [4-6], but little else was known of the chemistry of the shell until Crick and Ottensman [3] reported the Mg, Ca, manganese (Mn) and Sr of limited number of shells of Nautilus. Further work by Crick et al. [7] established that during the last year of shell growth, four species of Nautilus (IV. belauensis, IV. macromphalus, N. pompilius and N. scrobiculatus) differ with respect to the Mg, Sr and Ca in the shell, and suggested that these differences reflect complex interrelationships among animal physiochemistry and shell structure. Analyses of the major inorganic and organic constituents of the shell, septum, beaks and uroliths of N. pompilius revealed that N. pompilius effectively discriminates against Mg and Sr in constructing its shell and septa, and that uroliths are composed of Mg oxalate dihydrate with nuclei of hydroxyapatite and do not function as Ca reset-

462

REX E. CRICK, KEITH O. MANN AND JOHN A. CHAMBERLAIN, JR.

voirs during calcification of septa [8]. Lowenstam et al. [9] have also recently reported concentrations of Sr and Mg in several portions of N.

belauensis. Our aim here is to determine whether chemical differences observed among species of Nautilus are the result of differences in the physiochemical systems of species with respect to CaCO3 production or the result of chemical or volumetric differences in the nacreous and prismatic layers of the shell. Towards these goals we have examined the physiochemical systems of four species of Nautilus taken from the Western Pacific (Fig. 1: N. belauensis, N macromphalus, N. pompilius and N. scrobiculatus) as these systems relate to the production of CaCO3. The location of these populations is given in Fig. 1. Our conclusions are based on the Mg, Sr and Ca chemistry of the shell wall and septa from early ontogeny through maturity, and from the nacreous and prismatic layers of the shell wall.

Analytical Results

macromphalus. However, during the first two weeks of capture, Sr concentrations increased by 45% and 29% for N. pompilius and N. macromphalus respectively. Mg concentrations increase 23% and 20% (interval 1, Fig. 2). Following this period, and over a period of 10 weeks, the increased Sr and Mg concentrations steadily decreased to precapture concentrations. Because the salinity and temperature of tank waters of the two aquarium facilities remained reasonably constant during periods of captivity (see Experimental section), these results indicate that the observed changes in shell chemistries illustrated in Fig. 2 are not random, but rather reflect the influence of one or more causal factors that cut across taxonomic lines. We feel that the most logical cause of the perturbation in shell chemistry is apparent physiological stress associated with capture, containment and transportation. The fact that two species maintained in different aquaria show the same trends in shell chemistry immediately following capture strongly supports this claim.

Precapture and Postcapture She//Chemistry Results of analyses to determine the effects of capture and postcapture environments on shell chemistry are shown in Fig. 2, The concentrations of Mg and Sr in precapture shell material varies only slightly for three specimens of Nautilus pomp~flus and one specimen of N.

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Shell and Septum Chemistry Mean concentrations of Sr and Mg in parts per thousand in the shell and septa of the four species of Nautilus are reported in Table 1. Coefficients of variation (CV) are used as a measure of variation within and among species. Concentrations are categorized as representing septum/shell pairs 7 to last (oldest to youngest), and as septum/shell pairs 20 to last. This latter convention reflects the sampling procedure that was adopted to avoid problems associated with the extreme variability of early shell and septum chemistry, and to allow us to compare the chemistry of shells at approximately the same ontogenetic stages within and among species. A thorough analysis of septum/shell pairs 7 to last from one specimen of each species revealed that the Mg or Sr concentrations of the early septa and contemporary shell of three of the four species (N. belauensis, N. macromphalus and N. scrobiculatus) were higher and more variable than aragonite deposited in more mature portions of the shell (compare CVs of Table 1 and data of Figs 3A, B and D). The Sr and Mg chemistries of the shell wall and septa of N. pompilius behave differently than the other

SKELETAL CHEMISTRY OF NAUTILUS

463

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species (Fig. 3C). Mg concentrations can be as much as an order of magnitude more variable than Sr in either shell or septum aragonite in the same sample, and the Mg concentrations in the shell wall and septa of N. macrornphalus are the most variable. The variability of Sr in shell and septum aragonite is small (low CVs) with the exception of that in N. belauensis. In addition, the Mg and Sr concentrations of shell septa pairs of N. belauensis (Fig. 3A) and N. pompilius (Fig. 3C) show two additional patterns. First, the concentration of Sr of the septa of N. belauensis

increases by a factor of three between septa 7 and 9, and, although the Mg content of these early septa doubles, this increase is considerably less than increases in Mg content for N. rnacromphalus and N. scrobiculatus. The concentrations of Sr and Mg in the shell of N. belauensis shows a slight decrease over this interval. Second, the shell and septum chemistry of N. pompilius differs from the other three species by having a Mg concentration that varies, apparently randomly, throughout observed ontogeny while the Sr concentration

TABLE 1. MEANS AND STANDARD ERRORS OF Sr AND Mg FOR SEPTUM/SHELL PAIRS 7 TO LAST (FIRSTROW) AND 20 TO LAST (SECOND ROW) FOR FOUR SPECIES OF NAUTILUS Magnesium (ppm)

Strontium (ppm) Species*

Shell

Septum

Shell

Septum

NB [29] [16] NM [24] [11] NP [25~ [12] NS [25] [12]

2143±61.3 (15.4) 2326 ± 60.4 (10.4) 1697 ± 22.8 6.6) 1767 ± 31.5 5.9) 1618± 17.5 3.6) 1662+12.3 2.5) 1705+33.3 9.7) 1740±24.5 4.8)

2414± 149.7 (33.4) 2396± 64.8 (10.8) 1767± 24.5 ( 6.8) 1767± 35.9 ( 6.7) 1627± 64.8 (13,2) 1653± 12.3( 2.6) 1670± 18.4 ( 5.5) 1653± 23.6 ( 4.9)

182+ 7.5 (22.3) 180+ 5.6 (12.4) 505+ 74.8 (72,5) 202± 8.7 (14.4) 342+ 30.8 (45,0) 304+ 31.1 (35.5) 833+ 114.4 (67.7) 639±100.8 (54.7)

189+ 19.4 (55.2) 151 ± 6.8 (18.1) 981 ±155.2 (77.5) 206± 12.1 (19,5) 430± 37.9 (44.1) 449± 31.1 (30.0) 1256+ 100.3 (39.9) 10C~4± 70.7 (23.0)

*Species: NB, N. belauensls; NM, N. macromphalus; NP, N. pompilius; NS, N. scrobiculatus.Values in brackets are sample sizes and values in parentheses following standard errors are coefficients of variation (standard deviation as a percentage of the mean).

464

REX E. CRICK, KEITH O. MANN AND JOHN A. CHAMBERLAIN, JR.

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Septum/Shell Pairs FIG. 3. DISTRIBUTION OF Mg AND Sr IN SEPTUM AND SHELL WALL PAIRS THROUGHOUT ONTOGENY OF FOUR SPECIES OF NAUTILUS BEGINNING WITH THE 7TH SEPTUM/SHELL PAIR, Symbols are given in 3A. Mg and Sr concentrations for the septum of N scrobicu/atus (3D) begin at septum/shell pair 10 because earlier septa were destroyed during preparation.

SKELETAL CHEMISTRY OF NAUTILUS

465

remains essentially invariant. This observation is documented by the much higher CVs for Mg (Table 1). Analysis of random samples of septum/shell pairs between the apertural margin and growth increment 7 of these shells and those of additional specimens from each of the four species revealed that the patterns illustrated in Fig. 3 are characteristic within species. Further testing revealed that, if the sampling interval was restricted to septum/shell pairs 20 to last, as few as five random samples from this interval would produce the same chemical signature for Sr and Mg with only a slight increase in the standard error. This observation strongly suggests that the Sr and Mg chemistries of the shell and septa in the submature to mature portions of the conch are stable enough to be useful as biochemical indicators of species taxonomy for the genus. The results of these analyses are presented in Table 1 as the second row of values with smaller sample sizes (n) for each species. As expected, the mean concentrations of Sr and Mg changed little for those species with small CVs, and the CVs of all subsets are less than CVs based on analysis of the entire shell. Although the mean Sr and Mg concentrations of species with large initial CVs

changed the most (the most pronounced change was in N. macromphalus as a result of the extreme variability of these concentrations of the early shell and septa), we are confident that the Sr and Mg concentrations of shell/ septum pairs 20 to last are valid indicators of the shell chemistry of these species of Nautilus during submature/mature growth stages. The data of Table 1 were tested using the approximate t-test to first determine the effectiveness of sampling through the interval 20 to last vs 7 to last, and second to calculate the significance of differences in the chemistry of shell and septa among species. Test results for the t-test are presented in Table 2, and document the following observations. First, while mean ratios based on samples 7 to last (Table 1) appear to be very different among all species, the greater variance among samples for species always prevents at least one species pair from rejecting the null hypothesis of no difference among concentrations of Sr or Mg in shell or septal aragonite (Table 2, matrices A & B). Second, data from the sampling interval 20 to last are less 'noisy', yet shifts in means and changes in variance prevent the Sr content of shell and septal aragonite from being unique among all species. Third, the Mg content of

TABLE 2. RESULTS OF STUDENT'S t-TEST TO DEMONSTRATE THE EFFECTIVENESS OF SAMPLING THE INTERVAL 20 TO LAST SEPTUM/SHELL PAIRS VS 7 TO LAST AND TO DETERMINE IF CONCENTRATIONS OF Sr AND Mg ARE SIGNIFICANTLY DIFFERENT AMONG SPECIES OF NAUTILUS WITH RESPECT TO THE CHEMISTRY OF THE POST 20TH SHELL/SEPTUM PAIRS A - - Strontium (7 to last)

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NB

NM

NP

NB NM NP

-10.67 4.86

5.21 --2.60

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3.86

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5.04 -3.96 -2.91 --

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Values underlined indicate that the Sr or Mg chemistry of corresponding pairs are significantly different with less than a 5% chance of error at greater than 33 d.f. (7 to last) and 21 d.f. (20 to last). The key to species is given in the caption to Table 1. Species abbreviations (NB, NM, NP, NS) as in Table 1.

466

septum/shell samples from the interval 20 to last are significantly different among all species pairs and provide a chemical means of distinguishing species. The significance of this last statement will be addressed in a later section. Chemical Effects of Crystal Habit The aragonite shell of Nautilus is constructed of two forms of CaCO3 common to mollusk shells (nacreous and prismatic aragonite) [10]. Price and Hallam [6] reported the Sr content of the nacreous layer of the shell wall of two specimens of N. pompilius to be greater than that of the prismatic layer. Nothing is known however, of the variation of the Sr content of these layers with ontogeny, or of their Mg content, or how these concentrations differ among species of Nautilus. Towards this end, the Sr and Mg content of these layers were analyzed separately to test the possibility that these structural layers have chemistries different enough to bias test results based on whole shell chemistries. Significant differences in the chemistry of these layers would indicate that the carbonate for each layer was produced from fluids of different chemistries. The composition of the fluid medium for carbonate production can be controlled either by isolation (division of extrapallial space as in some bivalves), by localized secretions of mantle cells used for the formation of each layer [1], or perhaps, as suggested by Petit [11] for some bivalves, by separation of prismatic and nacreous aragonite by the periostracum during crystal growth. Structurally, prismatic aragonite forms two layers of the shell: the outer spheruliticprismatic layer and the thinner, inner prismatic layer. Nacreous aragonite lies between these layers and, although more variable in thickness, the volume of nacre exceeds that of total prismatic aragonite in a ratio of approximately 3:1. A septum of Nautilus is composed predominantly of nacreous aragonite situated between an adapical spherulitic-prismatic layer of aragonite and an adoral prismatic layer, both of which are very thin and occasionally discontinuous over the surface of the septum [10]. The volume ratio of nacre to total prismatic aragonite in the septum varies between 15:1 and 10:1. To determine the degree to which the chemistry of the shell is influenced by the

REX E. CRICK, KEITH O. MANN AND JOHN A. CHAMBERLAIN, JR.

chemistry of these crystal habits, contiguous outer prismatic and nacreous layers of the shell wall at five arbitrary septum/shell sample sites were mechanically separated and analyzed for Sr and Mg. Because of the extreme thinness of the inner prismatic layer of the shell wall, this layer was not analyzed. For the same reason, it was only possible to sample and analyze the nacreous layer of the septum. These results are presented in Table 3 with the same conventions as in Table 1. In theory and if both prismatic layers were analyzed together with the nacreous layers, the combination of Sr or Mg concentrations in the proper proportion of nacreous to prismatic layers of the shell should duplicate (within one standard error) the mean ratios of species in Table 1. That they do not (cf. Tables 1 and 2) can be attributed to the mechanics of separating the layers. The data of Table 3 illustrate several trends in the carbonate chemistry of the nacreous and prismatic layers of the shell and the nacreous layer of the septum. First, the most general trend documented by these data is that the chemistry of the prismatic and nacreous layers of the shell wall and nacreous layer of the septum of Nautilus scrobiculatus behaves inversely to that of the remaining three species. That is, trends observed in the chemistry of these layers in N. belauensis, IV. macromphalus and N. pompilius are generally opposite to those in N. scrobiculatus. Second, the concentrations of Sr in layers of the shell wall are much less variable than concentrations of Mg in the same samples. This trend is reversed in the septal nacre of N. belauensis and IV. scrobiculatus. Third, with exception of N. scrobiculatus, the Sr content of prismatic aragonite of the shell wall is greater than that of the contiguous nacreous layer while the concentration of Mg in these layers is greater for N. macromphalus, N. pompilius, and N. scrobiculatus, and less for N. belauensis. Fourth, with regard to the chemistries of nacreous layers of the shell wall and the septum, the Sr content of septal nacre is greater than the Sr concentration in the nacre of the shell walls of N. belauensis, N. macromphalus and N. pompilius. The Mg content of these layers is less in septal nacre than in shell nacre only for N. belauensis and N. macromphalus. The septal nacre of N. scrobiculatus contains a very high

SKELETAL CHEMISTRY OF NAUTILUS

467

TABLE 3. MEANS AND STANDARD ERRORS OF Sr AND Mg FOR PRISMATIC AND NACREOUS LAYERS OF THE SHELL AND THE NACREOUS LAYER OF SEPTA OF FOUR SPECIES OF NAUTILUS, AND RESULTS OF STUDENT'S t-TEST TO DETERMINE DIFFERENCES AMONG CHEMISTRIES OF LAYERS AND VARIOUS STRUCTURES

Shell Prismatic Species

Septum Nacre

Nacre

Sr

Mg

Sr

Mg

Sr

Mg

180± 5.1 (6.9) 209+10.2 (12.0) 321 + 27.0 (20.6) 870±64.4 (18.1)

2634±96.3 (8.9) 1784±26.3 (3.6) 1662 ± 12.3 (1.8) 1663±24.5 (3.6)

155± 3.6 (5.7) 180± 3.6 (5.0) 240 ± 16.3 (16.8) 1100±15.8 (3.5)

(p.p.m.) NB NM NP NS

2118±58.6 (6.8) 1750+26.5 (3.5) 1880 ± 39.4 (5.1) 1584±15.8 (2.5)

197±12.4 (I 5.3) 199±10.0 (12.1 ) 216 ± 5.8 (6.5) 228±13.6 (14.6)

2013+55.1 (6.7) 1689±26.3 (3.8) 1539 ± 19.3 (3.1) 1785± 8.8 (1.1)

Strontium Nacre

Nacre NB NM NP NS

Prismatic

NB

NM

NP

--

4.88 --

7.46 4.26 --

NS

NB

3.72 -3.30 -10.86 --

-0.86

(A)

NM

NP

NS

1.44 -7.25 10.12

(8)

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--

5.28 --

2.98 -2.73 --

(C)

7.90 4.92 6.41 --

Magnesium Nacre

Nacre NB NM NP NS

Prismatic

NB

NM

NP

NS

--

2.42 --

--4.68 -3.48 --

-9.77 -9.25 -7.20 --

(D) Pris. NB NM NP NS

NB

NM

NP

NS

1.28 0,65 3.42 8.93 (E) --

(F)

-0.09 --

-1.25 -1.36 --

--1.45 1.50 -0.65 --

Values in parentheses beneath each mean is the coefficient of variation (standard deviation expressed in percentage of the mean) for that mean. Matrices A, C, D, and F compare Sr or Mg of nacreous and prismatic layers among species. Matrices B and E compare Sr or Mg of nacreous and prismatic layers within species. Underlined test values document relationships that are significantly different with less than a 5% change of error at 10 d.f. The key to species is given in Table 1.

concentration of Mg relative to the other species while the Sr content of the septal nacre is less than the shell nacre. These relationships are summarized in Table 4 as ratios of Sr/Mg. Only Nautilus macrornphalus has a reasonably consistent balance between Sr and Mg concentrations in layer of prismatic and

nacreous layers in the shell wall and the nacreous layer of the septum. The remaining species show considerable variation in the Sr/ Mg ratios of one or more of these layers. These trends are graphically illustrated in Fig. 4, and show that N. belauensis and N. scrobiculatus have considerably different associations of Sr

468

REX E. CRICK, KEITH O. MANN AND JOHN A. CHAMBERLAIN, JR.

TABLE 4. RATIOS OF Sr/Mg ILLUSTRATING DIFFERENCES IN THE RELATIVE CONCENTRATIONS OF Sr TO Mg IN SPHERULITICPRISMATIC AND NACREOUS LAYERS OF THE SHELL WALL AND NACREOUS LAYER OF THE SEPTUM Shell

NB NM NP NS

Prismatic Sr/Mg

Nacre Sr/Mg

Septum Nacre Sr/Mg

10.75 8.79 8.71 6 94

11.18 8.08 4.49 2.05

16.99 9.91 6.92 1.51

Key to species is given in Table 1.

and Mg in prismatic and nacreous layers of the shell wall and septum. The pattern of these associations in the shell wall and septum of N. macromphalus and N. pompilius are similar but significantly different. On the basis of these data, it could be assumed that the general shell or septum chemistry of each of these species would be significantly different.

Although volume relationships are suspect due to difficulties of sampling, the values used to calculate the mean ratios are accurate within analytical precision. These data were tested for differences among the mean concentrations of Sr and Mg of species using the approximate ttest. The results of the t-tests are presented in the lower half of Table 3 and document that the Sr content of shell nacre and prismatic layers (Table 3, matrices A and C) and the Mg c()ntent of shell nacre (Table 3, matrix D) are significantly different among all species pairs. A comparison of the Mg or Sr content of the nacreous and prismatic layers within species shows that, with respect to both Mg and Sr, that only Nautilus pompilius and N. scrobiculatus have concentrations that are significantly different among these layers (Table 3, matrices B and E). The implications of these test results are that: (a) the Sr content of nacre and prismatic shell are significantly different among all species, but that the additive combination of the two layers

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FIG. 4. SUMMARY OF THE BIVARIATE RELATiONSHiP AMONG SPECIES OF NAUTILUSWITHRESPECT TO THE Mg AND Sr CONCENTRATIONS OF SHELL SPHERULITIC-PRISMATIC AND NACREOUS LAYERS, THE NACREOUS LAYER OF THE SEPTUM, AND THE MEANS OF THESE ELEMENTS IN THE SHELL AND SEPTUM Continuous lines enclose these bivariate relationships for each species,

SKELETAL CHEMISTRY OF NAUTILUS

469

may not be significant (Table 2) owing to differences in the volumes of these layers; ,(b) the Mg content of prismatic aragonite in the shell is not significantly different among species because of the high variability of concentrations of this element in this layer; and (c) the nacreous layer exerts a greater influence over the chemical signature of the shell of Nautilus than does the prismatic layer because the larger volume of the nacreous layer masks concentrations in prismatic layers. The ratio of Sr/Mg in these layers for each species serve to summarize differences observed between layers and among species (Table 4). Sr/Mg ratios for prismatic aragonite have a much narrower range than Sr/Mg ratios for the nacre of the shell or septum. The similarity of Sr/Mg ratios for N. macromphalus and N. pompilius illustrates the reason that these species could not be distinguished chemically on the basis of Mg content of the prismatic layer and points out the influence of Mg in determining the general shell chemistry with respect to minor and trace elements. Table 5 lists the results of tests to determine differences among Sr or Mg concentrations in nacreous layers of the shell and septa within four species of Nautilus. These results show that the Sr and Mg concentrations of the shell and septum nacre of each species are significantly different as was suggested by the mean concentrations of Sr and Mg (Table 3). These differences can only result from aragonite produced from fluids containing these ratios because the production of CaCO3 is an extracellular process and requires that the elemental ratios of the CaCO3 faithfully reflect those of the producing fluid.

Discussion General Considerations We have demonstrated that there are real and consistent differences in certain aspects of the trace element chemistry of the shell wall and septum proper and of the prismatic and nacreous layers of these structures for four species of Nautilus. Factors that may be responsible for these differences can be grouped into three major categories. The first concerns crystallographic aspects of the incorporation of inorganic ions into CaCO3. This factor can be ignored as a potential explanation for differences in minor and trace element concentrations observed among species because we are dealing with only the aragonite polymorph of CaCO3. The second category includes environmental factors, especially temperature, which can affect the composition of shell CaCO3. While it has been demonstrated that temperature affects trace and minor element concentrations in shell CaCO3 of many mollusks from shelf environments [1, 12, 13], two factors act to diminish the influence of environment on the trace and minor element content of the shell of Nautilus: (a) the 200 or so meters of water that commonly separates Nautilus from surface waters serves to buffer the ambient environment of Nautilus from seasonal fluctuations in water temperature or salinity; and (b) the activity coefficients of Mg, Sr and Ca of seawater vary in proportion to changes in salinity. Since the Sr/Ca and Mg/Ca ratios of seawater are invariant within the range of 25%0 to 40%0 salinity [14, 15], each population of Nautilus experiences the same relative concentrations of Mg, Sr and Ca regardless of geographic location

TABLE 5. TEST VALUES OF STUDENTS t-TEST FOR DIFFERENCES AMONG Sr OR Mg CONCENTRATIONS IN THE NACREOUS LAYERS OF THE SHELL AND SEPTA WITHIN FOUR SPECIES OF NAUTILUS A - - Strontium

B - - Magnesium Shell NB

Septa NB NM NP NS

NM

NP

NS

5.09 -2.42 4.93 --4.50

NB Septa SB NM NP NS

Shell NM

NP

NS

-3.66 -2.46 -2.33 3.18

Matrix A: comparison of Sr content of nacre of shell and septa. Matrix B: comparison of Mg content of nacre of shell and septa. All test values are significant with less than 5% chance of error at 10 d.f, Species key is given in Table 1.

470

(Fig. 1). The third factor concerns the physiological and biochemical processes of the animal, and involves both the micro-environment in which shell CaCO3 is produced and the transfer of materials from this environment to the site of crystal formation [1, 2]. There is, of course, some influence of the external environment on physiological functions as they effect shell formation, and potentially some contamination of extracellular fluids by seawater, but there is no evidence that these circumstances have a significant effect on the chemistry of shell CaCO3 [16]. Furthermore, it is reasonable to expect these types of influences to affect all species or populations equally causing similarities in shell chemistries among species and populations. Such similarities are not consistent with our findings. Ions which form the basis of CaCO3 crystals of the shell and septum of Nautilus (and mollusks in general) pass from the mantle cavity, which contains the external medium and food, to the tissue and blood sinus, and finally into one or more compartments (extrapallial spaces) between the growing surface of the shell wall and the mantle epithelium. Here such ions constitute the inorganic portion of an extracellular fluid commonly referred to as extrapallial fluid. The concentration of inorganic ions in shell and CaCO3 is a direct function of the concentration of these ions in the tissue and blood and the rate at which these ions are removed from the external environment [1, 2, 16, 17]. The regulation of the composition of extracellular fluids by the mantle cells using selective discrimination for certain ions is genetically controlled and expected to vary with species [1, 18]. Such differences have been observed among extant bivalve genera and related species [1] and among extinct congeneric species of nautiloid cephalopods [3]. Different concentrations of trace elements in shell CaCO3 of the four extant congeneric species of Nautilus would therefore not be unexpected.

Shell Structure Differences in the minor and trace element content of prismatic and nacreous layers indicate that these layers were precipitated from extracellular fluids of different compositions. It is most probable that, like some bivalves with

REX E CRICK, KEITH O, MANN AND JOHN A, CHAMBERLAIN, JR

more than one extrapallial space, the mantle epithelium of Nautilus supports two separate extrapallial spaces; one for the production of the outer prismatic layer and one for the nacreous layer. The uneven distribution and varying thickness of the inner prismatic layer suggests that it may have developed under less controlled circumstances; perhaps over a large area similar to septal formation. Price and Hallam [6] observed similar differences in two specimens of N. pompilius and three specimens of the cuttlefish Sepia. Unlike the data of Price and Hallam, our data show no cycles which might be attributable to seasonal changes in the external environment. Our data do reveal that the chemistry of nacreous aragonite exerts a major influence on the chemical characteristics of both the shell and septum of Nautilus by virtue of the greater volume of this layer relative to prismatic aragonite. The nacre has the additional characteristic of having significant different concentrations of both Mg and Sr among species, while the prismatic layer is significantly different among species only with respect to Sr. This is particularly well illustrated by comparison of the Sr/Mg ratios of Table 4 where the range of ratios among species is much greater for the nacre of the shell than for the prismatic aragonite. The range of the Sr/Mg ratios for the nacre of the septum is even greater.

Maturation of the Biomineralization System Three species (Nautilus belauensis, N. macromphalus, and N. scrobiculatus) show considerable differences in the Sr and Mg content of the early and late portions of the shell. This is most pronounced in the shell and septal Mg content of N. macromphalus. Ontogenetic variation is most erratic in the Mg of the shell and septa of N. scrobiculatus, and only moderately evident in the Sr and Mg of septa of N. belauensis (Fig. 3). The Sr content is fairly stable within all species (except for the already noted early variation of Sr in the septa of N. belauensis). There are two possible explanations for ontogenetic variation in Sr and Mg concentration: (a) age-related change in biomineralization rate; and (b) maturation of the biomineralization system. In the first case, Sr and Mg concentrations vary with the rate at which new carbonate is accreted to the shell, and hence ultimately are

SKELETAL CHEMISTRY OF NAUTILUS

functions of growth rate. In the second case, the maturing physiochemical system achieves increasing control over its output. In our view, the former idea is at best a weak one. There is no firm evidence suggesting that variable growth rates influence shell chemistry in the necessary manner in either mollusks or other animals. But even if growth rate operates in the required manner for some animals, this is manifestly not the case for Nautilus. Aquariumbased measurements of Nautilus growth rate ([19, 20]; Ward, P. D., personal communication), and growth determination in wild specimens [21-23] show that apertural growth rates in Nautilus: (a) remain constant over most of ontogeny or decline slightly with age; (b) decline appreciably with the onset of sexual maturity; or (c) temporarily decline after major shell breakage while the break is under repair. Of these three alternatives, only the last seems remotely constant with the wide fluctuations in trace element content observed among the specimens included in Fig. 2. But the plausibility of this interpretation fades when it is noted that our specimens lack the prominent scarring always observed in cases of extensive shell damage. We infer from this that variable growth rate is not the primary agent responsible for the inter-specific differences in trace element chemistry found in our data. The pronounced variability of Mg in the shell and septa of Nautilus macromphalus and N. scrobiculatus and to a much lesser degree in the early septa of N. belauensis (Fig. 3) must, in our view, reflect a physicochemical system which 'matures' with ontogeny relative to its ability to reduce the level of Mg in skeletal CaCO3. Nautilus scrobiculatus never achieves the control over levels of Mg achieved by N. macromphalus by the 20th septum/shell wall pair, whereas the only slightly variable nature of Mg and Sr in early septa of N. belauensis ceases after the 10th septum/shell wall pair. The Mg content of shell and septa of N. pompilius is also variable, but much less so, and essentially fluctuates without a trend until late maturity (Fig. 3C). In each case these trends or fluctuations reflect differing amounts of Mg incorporated in the nacre of either shell wall or septum. The actual cause for the differing amounts must lie with the inability of mantle cells to exclude the Mg ion from the

471

extracellular fluids, and, with exception of N. pompilius, the Mg content decreases with ontogeny although the Mg contents of shell wall and septa of N. scrobiculatus never reach the stability of the other species. It should be noted, however, that the Mg content of both shell wall and septa of N. scrobiculatus is considerably higher than the other species. Ontogenetic Variation in Mg This leads to the consideration of why a secular decrease in the amount of Mg in skeletal CaCO3. We feel the answer lies with the kinetics of crystal chemistry and structure. The major inorganic constituents of the skeleton of Nautilus are Ca, Sr and Mg in order of relative abundance and the ionic radii of these elements are 1.0, 1.13 and 0.72 respectively. While aragonite is polymorphous with calcite, the structure of aragonite is stable for ions larger than Ca ( > 1.0 radius), whereas the calcite structure is stable for ions smaller than Ca (<1.0 radius). This stability factor for aragonite is caused by its structure which consists of planar trigonal CO3 groups with Ca ions in positions of hexagonal close packing; a situation which gives aragonite its pseudo-hexagonal character. The importance of this in this context is that each CO3 group lies between six Ca atoms and is arranged such that each 0 is linked to three Ca ions. This arrangement produces a radius ratio (radius cation/ radius anion) with 0 of approximately 0.71 which is intermediate to the coordination numbers 6 and 8. Similar radius ratios with O for Sr and Mg are 0.81 and 0.51 with corresponding coordination numbers of 8 and 6 [24]. Thus the Sr ion more easily substitutes for the Ca ion in the aragonite structure than does the Mg ion. Further, the inclusion of Mg in the lattice of aragonite at Ca sites distorts the orthorhombic structure as the lattice 'adjusts' to the smaller Mg ion and consequently high Mg aragonite will be more unstable than low Mg aragonite [24]. The most efficient means of producing a shell wall and septum of predominantly nacre (pseudo-hexagonal crystals) is to exclude as much Mg as possible and to use available Sr when necessary. An important consideration in this reasoning is the relationship between the relative ratio of Ca, Mg, and Sr in world oceans, 1.0:3.2:0.02 [24]

472

(i.e. for every atom of Ca in the world ocean there are 3.2 atoms of Mg and only 0.02 atoms of Sr) and the ratios of averages of Ca, Mg and Sr concentrations for Nautilus, 1.0:0.0003:0.002. The body fluids of mollusks are essentially isosmotic with sea water, and in order for Nautilus to produce CaCO3 with these ion ratios, quantities of Mg on the order of 104 times the amount of Ca required for CaCO3 production must be excluded from the makeup of extrapallial fluids. Why Nautilus discriminates against Sr is not clear, although the larger ionic diameter of Sr would tend to slightly distort the aragonite lattice and make Sr less desirable as a skeletal component. Groups of lower evolutionary grade with skeletons of aragonite commonly do not discriminate against Sr relative to Ca; e.g. taxa as diverse as planktonic foraminifera [25] and coelenterates [26] commonly have an order of magnitude greater concentrations of Sr in shells and tests than does Nautilus. Most important, however, is the magnitude of the exclusion of Mg from the skeleton of Nautilus. It seems reasonable to assume that genetic differences in -'~ physiochemical systems might be more pronounced with respect to Mg than St. This inference is indirectly supported by our data.

Taxonomic Implications Tables 1 and 3 and Fig. 4 clearly demonstrate that the chemistry of the skeletons of Nautilus species differ chemically in many ways. Most distinctive is the higher content of Sr in the shell wall and septum of N. belauensis and the much higher Mg content of all but the spheruliticprismatic layer of the shell wall of N. scrobiculatus, N. macromphalus and N. pompilius are not as similar as proximity suggests in Fig. 4. The Mg and Sr concentrations of the various layers and structures of N. macromphalus are similar, whereas the same concentrations in N. pompilius cover a much wider range; especially with respect to Sr. Work of a similar nature using skeletons of Upper Carboniferous (Westphalian D/Desmoinesian) nautiloid cephalopods preserved in bitumen [3], demonstrated that three species of the orthoconic nautiloid Mitorthoceras Gordon could be distinguished on the basis of the Mg content of the shell wall. The Sr content of the shell wall among the species of Mitorthoceras was not significantly different, and the

REX E. CRICK, KEITH O. MANN AND JOHN A. CHAMBERLAIN, JR.

average Sr content for the genus not much less (1488+52) than that of Nautilus. Brand [27] reported differences in Sr and Mg concentrations among genera of various taxa preserved in the Kendrick Shale (Upper Carboniferous, Westphalian B/Atokian), but did not attempt to distinguish among species. Unpublished data of Crick on Jurassic and Cretaceous ammonites and belemnites show similar patterns among species and genera with respect to both Sr and Mg in shell walls, septa, and rostra (in the case of belemnites). Detailed work involving the paleobiochemistry of molluscan shell proteins [28] concluded that observed differences in the genetically controlled makeup of shell protein should result in similar differences in the ionic chemistry of shell CaCO3.

Experimental Specimens of Nautilus be/auensis Saunders 1981 (Palau) were supplied by Chamberlain and by W. Bruce Saunders (Bryn Mawr College). Specimens of N, macrompha/us (New Caledonia), N. pomp/flus (Philippines) and N, scrob/cu/atus (New Guinea) came from the collections of Chamberlain, Crick, and Peter D. Ward (Universib/ of Washington). A total of 16 shells were analyzed: five shells each of IV. be/auensis and N. macrompha/us; four shells of IV. pompi//us; and two shells of IV. scrob/cu/atus. The small sample size for N. scrobicu/atus reflects the extreme rarity of this species [Saunders, W. B., personal communication]. All specimens were mature adults (final septum approximated) with between 30 and 35 chambers. The known geographic distributions of the species and water salinity for their preferred depth range are indicated in Fig. 1. Specimens were prepared for sampling by sectioning the shells in the plane of symmetry to expose the septa. Potential contaminants (surface films, organic membranes, epibionts, etc.) were removed by air abrasive from external and internal surfaces of shells and septa. For purposes of comparing the chemistry of contemporaneously deposited shell and septal aragonite, a sampling scheme was used which allowed the correlation of samples separated in space but not in time. For each septum sampled, a strip of shell was removed between curvilinear growth lines corresponding to angles of 95° and 105° adoral from each septum (Fig. 5). This was done because the body chamber of a species of Nautilus subtends approximately 100° of the shell spiral during ontogeny, and X-radiography of living Nautilus reveals that: (a) the shell aperture lies at a point approximately 810 to 85 ° adoral of the last formed septum at the initiation of septum formation; and (b) that septal formation ceases at a point approximately 105° to 110° adoral of the last-formed septum [19, 20]. We chose the interval between 85 ° and 105° as a conservative estimate of the amount of shell aragonite deposited during the formation of a coeval septum. The first 7 septa and corresponding shell material do not form part of this work because there is some evidence to indicate that all of this material was formed prior to hatching [21, 29, 30]. We will

SKELETALCHEMISTRYOF NAUTILUS

FIG. 5. DIAGRAM ILLUSTRATING HOW SHELL SAMPLES WERE SELECTEDRELATIVETO AN APPROXIMATELYCOEVALSEPTUM.The 20° segment along the venter representsthe approximate volume of shell producedduring growth of the septum. report data from this embryonic portion of the phragmocone elsewhere in the context of the chemistry of egg fluids. The shells we sampled came from animals with mixed histories. Some derive from animals that died prior to collection, others came from animals that died or were sacrificed shortly after capture, and yet others belonged to animals that spent considerable time in captivity. We felt it necessary to investigate the possibility that observed differences in shell or septum chemistry were the result of these disparate histories. To test this possibility, shell material was analyzed from Nautilus pompilius and N. macromphalus specimens with precapture and postcapture histories. Three specimens of N. pornpilius were maintained at the New York Aquarium in a 14 000 liter recirculating seawater tank at 30%0 salinity and 20°. The single specimen of N. macromphalus was held in an aquarium (University of California at Davis) until death and experienced similar environmental conditions as specimens of N. pompilius [Peter Ward, personal communication]. The leading edge of the shell of each/V, pompilius specimen was notched upon arrival and thereafter at 14 day intervals until death. At death, samples of postcapture shell material were taken along the venter at notched intervals, and samples of precapture shell material were also removed along the venter of the shell using the same approximate sampling intervals as used on the postcapture portion of the shell. We recognize the potential discrepancy between the length of the precapture and the postcapture intervals, but the data suggest that error induced by this sampling scheme does not significantly alter the resulting comparisons. The time interval represented in these uniformly spaced precapture samples is not necessarily equal to 14 days since there is some evidence from observations of Nautilus in its natural environment [22, 23] and in captivity [20] that growth rate decreases with age and that there may be some disparities in growth rates between wild and captive animals.

473 Preparing samples of the prismatic and nacreous layers of the shell for analysis required the unfortunate but unavoidable loss of a small portion of each layer. In an effort to insure that nacreous or prismatic layers were not contaminated with one another, it was necessary to remove a portion of the layer being sampled, thereby reducing its volume. This resulted in a slight but apparently significant underestimation of volume ratios and prevented detailed comparisons of the chemistry of these layers. This reduced volume did not, however, affect the precision or accuracy of elemental concentrations of these layers. Because the general ratios of nacre to prismatic aragonite were determined by direct measurement on prepared surfaces prior to separation, it was possible to make observations regarding the relative differences in Sr and Mg concentrations of the various layers and to draw general conclusions regarding how these chemistries affect the overall chemistry of the shell or septum. Samples of all crystalline material were ground to a particle size less than 150 m and heated for 30 rain at 300" to volatilize the organics. Following the removal of organics, samples were weighed, dissolved in an aqueous solution containing 2% HCI and 0,1% La, and analyzed for Sr and Mg by flame atomic absorption spectrophotometry (Perkin-Elmer 403 and Varian 975PT). The high concentration of Ca in the shell and septum required that Ca concentrations of these structures be calculated by differences with concentrations of remaining major and minor elements. The majority of the results presented here are based on data contained in Mann [31] and the remainder are available on request from Crick. In the following tables and discussion, we depend on the discerning power of statistics to assist in evaluating the significance of comparisons of Mg and Sr chemistries within and among species, populations, and the shell and septum. We use the standard error as a measure of dispersion about the mean because the ratios of Table 1 are themselves based on means produced by the analytical method of the spectrephotometers. The coefficient of variation (CV) is used to compare percent variability in chemistries among structures, species, and populations. Each category of data within species was tested for significant departures from normality (all are normally distributed) and Bartlett's test for homogeneity of variance was used to determine the equality of variances among categories of data. Bartlett's test was used instead of analysis of variance because perusal of the data revealed a wide range in variances within categories among species. In all cases, Bartlett's test rejected the null hypothesis of equal variance at a significance level of 99%. Because of the heterogeneity of variances among species, an approximate t-test related to the Behrens-Fisher test was used instead of Student's t-test. This particular test calculates an approximate t-test value for which the critical value is calculated as a weighted average of the critical values of t based on the corresponding degrees of freedom of the two samples. Procedures for the calculation and application of this and other tests are explained fully in Sokal and Rohlf [32, 33]. The significance of test results was evaluated at the 95% significance level and reported as the probability P < 0.05 of rejecting a true null hypothesis. Ackno~dedgements--We wish to thank W. Bruce Saunders (Bryn Mawr College) and Peter D. Ward (University of Washington) for their unselfish sharing of unpublished data,

474

REX E. CRICK,KEITHO. MANN AND JOHN A. CHAMBERLAIN,JR

observations and collections. Pat Cowen typed the manuscript. Certain parts of the text formed the Masters thesis of Keith Mann in the Department of Geology, The University of Texas at Arlington. The research was supported under two research awards to REC from The Organized Research Fund of the University of Texas at Arlington.

P. and Skirrow, G., eds) Vol. 1, p. 365. Academic Press, London. Wada, K. and Fujinuki, T. (1976) in The Mechanisms of Mineralization in the Invertebrates and Plants (VVatabe, N. and Wilbur, K. M, eds) p. 175. University of South Carolina Press, Columbia. Crenshaw, M. A. (1982) in Biological Mineralization and Demineralization (Nancollas, G. H., ed.) p. 243. Springer, Berlin. Wilbur, K. M. and Owen, G. (1964) in Physiology of Mollusca (Wilbur, K. M. and ¥onge, C. M, eds) Vol. 1, p. 211. Academic Press, New York. Ward, P. D., Greenwald, L. and Magnier, Y. (1981) Paleobiology 7, 481. Ward, P. D. and Chamberlain, J. A., Jr (1983) Nature (London) 304, 57. Cochran, J. S., Rye, D. M. and Landmann, N H. (1981) Paleobiology 7, 489. Saunders, W. B. (1983) Paleobiology 9, 280, Cochran, J. S. and Landmann, N. H. (1984) Nature (London) 308, 725. Mason, B. and Moore, C. B. (1982) Principles of Geochemistry Wiley, New York. Graham, D. W., Bender, M. L., Williams, D. F. and Keigwin, L. D., Jr (1982) Geochim. Cosmocbim, Acta 46, 1281. Amiel, A. J., Friedman, G. M. and Miller, D. S. (1973) Sedimento/ogy 20, 47. Brand, U. (1981) Chem. Geol. 32, 1. Degens, E. T., Spencer, D. W. and Parker, R. H. (1967) Comp. Biochem. PhysioL 20, 553. Oba, T. and Tanabe, K. (1983) Kagoshima Univ. Research Center South Pacific Occasional Papers 1, 26. Taylor, B. E. and Ward, P. D. (1983) Palaeogeogr Palaeoclimatol. PalaeoecoL 41, 1. Mann, K. O. (1983) Unpublished M.Sc. thesis. University of Texas at Arlington, Arlington. Sokal, R. R. and Rohlf, F. J. (1969) BiometrF W. H. Freeman, San Francisco. Sokal, R. R. and Rohlf, F. J. (1973) Introduction to Biostatistics. W. H. Freeman, San Francisco.

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References 1. Wilbur, K. M. (1972) in Chemical Zoology (Florkin, M. and Scheer, B., eds) Vol. VII, Mollusks, p. 103. Academic Press, New York. 2. Wilbur, K. M. (1976) in The Mechanisms of Mineralization in the Invertebrates andP/ants (Watabe, N. and Wilbur, K. M., eds) p. 79. University of South Carolina Press, Columbia. 3. Crick, R. IF. and Ottensman, V. M. (1983) Chem. Geol. 39, 147. 4. Lowenstam, H. A. (1963) in Isotopic and Cosmic Chemistry (Craig, H., Miller, S. L. and Wasserburg, G. J., eds) p. 114. North-Holland, Amsterdam. 5. Hallam, A. and Price, N. B. (1966) Nature (London) 212, 25. 6. Price, N. B. and Hallam, A. (1987) Nature (London) 215, 1272. 7. Crick, R. E., Mann, K. O. and Ward, P. D. (1984) Geology 12, 99. 8. Crick, R. E., Burkart, B., Chamberlain, J. A. Jr. and Mann, K. O. (1985) J. Mar. Biok Assoc. U.K. 65, 415. 9. Lowenstam, H. A., Traub, W. and Weiner, S. (1984) Paleobiology 10, 268. 10. Mutvei, H. (1972) Bull Geol. Inst. Univ. Uppsala 3, 237. 11. Petit, H. (1980) Tissue Cell 12, 13. 12. Pilkey, O. H. and Goodell, H. G. (1964) Geol. Soc. Am. Bull. 75, 217. 13. Dodd, J. R. (1987) J. Paleontol. 41, 1313. 14. Brewer, P. G. (1975) in Chemical Oceanography (Riley, J. P. and Skirrow, G., eds) Vol. 1, p. 139. Academic Press, London. 15. Wilson, T. R. S. (1975) in Chemical Oceanography (Riley, J.

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