Phosphatic hard tissues of marine invertebrates: Their nature and mechanical function, and some fossil implications

Chemical Geology Elsevier Publishing Company, Amsterdam - Printed in The Netherlands

PHOSPHATIC HARD TISSUES OF MARINE INVERTEBRATES: THEIR NATURE AND MECHANICAL FUNCTION, AND SOME FOSSIL IMPLICATIONS*

HEINZ A. LOWENSTAM Division o f Geological and Planetary Sciences, California Institute of Technology, Pasadena, Calif. (U.S.A.) (Received September 22,1971)

ABSTRACT Lowenstam, H.A., 1972. Phosphatic hard tissues of marine invertebrates: their nature and mechanical function, and some fossil implications. Chem. Geol., 9: 153-166. Phosphatic substances are described from hard tissues of species from seven classes of marine invertebrates. Francolite is identified in the species of one class and amorphous calcium- and ferric phosphates, apparently in the form of hydrogels, are recognized as precipitates of species from six other classes. Considering all known phosphatic substances in animal hard parts, it is found that amorphous constituents are far more common in invertebrates than crystalline compounds. The role o: amorphous precipitates in providing physical strength to hard parts is examined. Brief comparison is made between phosphate fixation in the phyla spectrum of recent and fossil invertebrates. INTRODUCTION Phosphates are vital as nutrients o f plants and perform various biochemical functions in all organisms. In the oceans the effect of biologic activity is reflected in the low phosphate contents of the waters in the upper 200 m and their seasonal fluctuations in the euphotic zone. Various groups of marine organisms have been reported to stabilize phosphates in their hard tissues. They include species of the cestods, inarticulate brachiopods, polychaete worms, polyplacophorans, gastropods, bivalves, malacostracans, holothurians and all classes of the vertebrates (M6rner, 1902; Clarke and Wheeler, 1922; Vinogradov, 1953; Watabe, 1956; McConnell, 1963; Von Brand et al., 1967; Low ~,~stam, 1968; Lowenstam and McConnell, 1968; Rhodes and Bloxam, 1971). As for the invertebrates, many references to phosphate-rich hard parts are based solely on data from chemical analyses (M6rner, 1902; Clarke and Wheeler, 1922; Vinogradov, 1953; Rhodes and Bloxam, 1971). This is true for the reported occurrences o f phosphates in most species o f the inarticulate brachiopods and for all species of the polychaetes, ~rContribution No,2068 from the Division of Geological and Planetary Sciences, California Institute of Technology.

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malacostracans and holothurians. Carbonate hydroxyapatite (dahllite) was reported from the corpuscles of a cestod species and a larval bivalve shell (Watabe, 1956; Von Brand et al., 1967). The identifications were based on diffraction analyses but lacked elemental determinations to tell whether or not the mineral was carbonate fluorapatite (francolite). What remains are samples of one articulate brachiopod species, three polyplacophoran species and three gastropod species, where there are elemental determinations and X-ray diffraction analyses on the phosphatic constituents from hard tissue sites (McConnell, 1963; Lowenstam, 1968; Lowenstam and McConnell, 1968). Francolite was reported from the the shell of the inarticulate brachiopod and in the tooth denticles of the polyplacophoran species. The phosphatic constituents in the gizzard plates of the gastropods were shown by means of X-rays to be amorphous. Only relative abundances of their elemental constituents were reported and the nature of these phosphatic substances was considered uncertain. There are thus few reliable data on the classes of marine invertebrates which precipitate phosphates in their hard tissues and on the nature of the phosphatic substances which are incorporated in them. Considering the importance of such information for the model of the phosphate cycle in the oceans and for tracing the history of the phosphateprecipitating biomass in the geologic past, this is rather surprising. In the course of systematic investigations of the minerals in the hard parts of marine organisms, phosphatic substances were found in species of seven classes of invertebrates. The results of this study and some of its implications are presented in the following.

EXTRACTION AND ANALYTICALPROCEDURES The hard tissues investigated in this study were extracted mechanically from specimens preserved in 70% alcohol. Aliquots of the samples were then treated with Clorox to digest most of the organic fractions. Powder camera X-ray diffraction patterns were obtained of the cloroxed and uncloroxed aliquots of the samples. When large samples were available, another cloroxed aliquot was heated to 500°C and a powder diffraction pattern was obtained of the heat-treated sample. Also, in the case of large samples, semi-quantitative spectroscopic determinations of their elemental compositions were made with a Jarrel-Ash 21-ft. grating spectrograph. In small and in most large samples one aliquot of the samples was used for semi-quantitative elemental determinations with an MAC 5-SM 3 electronprobe. Quantitative elemental determinations were obtained for some samples from wavelength scans of the electron probe. Infrared absorption spectra were obtained on aliquots of samples from specimens of Sternaspis scutulata and Molpadia intermedia. RESULTS Table I shows the species which yielded phosphatic substances, the phyla and class relations of the species and the hard tissues in which the substances were located. The table contains also the results of the X-ray diffraction analyses of aliquots of the skeletal

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substances which were mechanically extracted or cloroxed and of those which were heat treated. The samples listed in the table belong to species of inarticulate brachiopods, polychaete worms, polyplacophorans, gastropods, bivalves, malacostracans and holothurians. X-ray diffraction patterns were obtained only from shell samples of the inarticulate brachiopod and the gizzard plates of five species of tectibranchian gastropods. The pattern from the shells of Pelagiodiscus atlanticus is shown to be similar to that of dahllite. The minerals indicated by the diffraction patterns from the gizzard plates of the gastropod samples are: fluorite in S. lignarius, weddellite in S. cylindrellus and C. cylindracea, and monohydrocalcites in P. quadripartita and P. angasi. The presence of fluorite in gizzard plates of S. lignarius and of weddellite in S. cylindrellus was previously reported (Lowenstam, 1968; Lowenstam and McConnell, 1968). The purpose of investigating these samples further was to attempt identification of a second amorphous phosphate-rich constituent indicated in the earlier studies to be present on the basis of wave-length scans by the electron probe. The diffraction analyses of the untreated samples extend the distribution range of weddellite in hard tissues of invertebrates to a second species of tectibranch gastropods. Monohydrocalcite has been reported previously as a biologic precipitate from the statocoenia of the tiger shark (Carlstrom, 1963). The identification of CaCO3" H20 and CaCO3 • 0.65 H20 in gizzard plates of two tectibranch gastropods extends the biosynthesis range of monohydrocalcites to the invertebrates. No diffraction patterns were obtained from the untreated samples of the polychaetes, polyplacophorans, malacostracans, holothurians and six of the tectibranch species. Following heat treatments, aliquots of the sternal shields of the polychaete S. scutulata and of the mesodermal granules of the holothurian, M. interrnedia gave diffraction patterns which we have been unable to interpret so far. Those of the tube lining of the polychaete C. granulata showed a pattern of maghemite. Whitlockite patterns were obtained from the heat treated samples of the setae of S. scutulata, the gizzard plates of S. interruptus and ofA. culcitella, the gill supports of the bivalve, N. margaritacea. Dahllite plus calcite patterns were found in aliquots from gizzard plates of the two species of Philine and of the carapace of the malacostracan, P. bigelowi. The gizzard plates ofS. lignarius showed a pattern similar to dahllite plus fluorite after heating. Aliquots of the samples of the gizzard plates of S. cylindrellus, and of the three Cylichna species were only large enough to investigate their elemental compositions and hence were not subjected to heat treatment Table lI contains the data on the chemical composition of samples which were large enough for further study and the methods used for the elemental determinations. All but two of the samples are shown to have high phosphorus contents. Other major constituents are either calcium or iron, apparently in the ferric state as indicated by the orange-brown to reddish color of the samples which have high iron contents. The samples which are rich in calcium and phosphorus have intermediate concentrations of magnesium and usually low iron contents. Those with ferric iron and phosphorus as the major constituents contain on the average considerably lower magnesium contents and usually low calcium concentrations. Fluorine, manganese and barium, when semi-quantitatively or

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quantitatively determined, were found in most samples as trace constituents. After heating to 100°C most samples show a weight loss of 20-25%, indicating that these samples have high water contents. Infrared absorption spectra of the iron and phosphorus rich substances from S. scutulata and M. intermedia demonstrate the absence of structurally ordered hydroxyls. There are some exceptions to the chemical composition indicated for most samples. The tube linings of C. granulata have only iron as a major constituent, considerably smaller amounts of phosphorus and low magnesium contents and fluorine concentrations. Electron-probe analyses of consecutive layers of S. lignarius show that some layers are high in calcium and phosphorus and low in fluorine, whereas others have high calcium and fluorine contents and only a trace of phosphorus. In the gizzard plates ofS. cylindrellus the subunits underlying the weddellite-rich layers have on the average high phosphorus and iron contents and fairly high calcium concentrations. Their barium and manganese contents are on the average considerably higher than in any of the other samples. In the mesodermal granules ofM. intermedia calcium is present in intermediate amounts, whereas in other samples which are also rich in iron and phosphorus the calcium concentrations are fairly low. The shells of/°. atlanticus contain, apart from high concentrations of calcium and phosphorus, fluorine estimated to amount to about 3% by weight. The elemental determinations indicate that phosphatic substances are present in all but one of the samples investigated here. Integrating these data with those from X-ray diffraction analyses, we find that the shells ofP. atlanticus contain the carbonate fluorapatite mineral, francolite. Amorphous ferric phosphatic substances apparently in the form of hydrogels are indicated as the hardening agents of the sternal shields of the polychaete S. scutulata, a structural component of the mature teeth in the chiton C stelleri and the mesodermal granules of the holothurian M. intermedia. The hydrogel contained in the lower structural component of the gizzard plates from the gastropod S. cylindrellus is also rich in ferric iron and phosphorus. Yet it has intermediate concentrations of calcium and appreciable amounts of barium and manganese. Hence, there is the possibility that more than one amorphous substance may be present. Calcium phosphate hydrogels were found in the hard tissues of a variety of invertebrate samples. They include the gizzard plates of seven species of gastropods, the gill supports of the bivalve N. margaritacea, and the carapace of the malacostracan P. bigelowi. To judge from the whitlockite pattern of a heat treated sample, the setae of the polychaete S. scutulata, may also contain a calcium phosphate hydrogel. From the Ca- Mg/P ratios and the X-ray diffraction patterns of heat-treated samples, it appears that some of the calcium phosphate hydrogels are chemically similar to whitlockite, whereas others are chemically closer to dahllite. The data show that aside from phosphatic hydrogels, the gizzard plates ofS. lignarius contain also the mineral fluorite, those of S. cylindrellus and C. cylindracea the mineral weddellite and the two Philine species the monohydrocalcite minerals. The fluorite in S. lignarius is found in submicroscopic zones and also together with the phosphatic

PHOSPHATICHARD TISSUES OF MARINE INVERTEBRATES

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hydrogel. The monohydrocalcites appear to be evenly distributed in a substrate of phosphatic hydrogels in the two Philine species. The weddellite occurs locally in the two gastropod species in the outer micro-architectural unit and the phosphatic hydrogel in the underlying unit of their gizzard plates. The chemical composition of the carapace of the malacostracan P. bigelowi, indicates an excess of calcium relative to phosphorus. The diffraction pattern of the heat-treated aliquot of the sample shows calcite and dahllite. Hence, the carapace contains amorphous calcium carbonate in addition to the calcium phosphatic hydrogel. Considering the tube linings of the polychaete, C. granulata, the elemental determinations suggest that we are dealing with an iron-rich organic compound that contains some phosphorus and calcium. DISCUSSION The data obtained in this study contribute to the definition of the nature of phosphatic constituents which are found in hard tissues of some marine invertebrates. They document phosphate fixation by species in a wide range of invertebrate classes. A crystalline compound was found only in the shells of inarticulate brachiopod samples. The mineral is francolite. The phosphorus-rich constituents located in the hard tissues of all other invertebrates investigated are, by the evidence of X-rays, amorphous. Electron probe and elnmission spectroscopic analyses indicate that these constituents are either rich in iron or in calcium. The iron-rich constituents contain 3-12% CaO and about 3% MgO. The calcium-rich components contain from 3-11% MgO. The orange to deep wine-red color of the iron-rich constituents indicates that the iron is in the ferric state. High water contents for the samples are indicated by the loss of 20-25% in weight after heating to 100°C. The organic fractions determined from loss in weight after cloroxing or following further heating to 500°C, represent between 5-10% of all samples except for the malacostracan carapace. The chitin fraction of the samples from different carapace sites range from 40--85% by weight. Thin sections show under the microscope that the phosphatic constituents are localized in discrete layers and lenticular bodies in the chitinrich carapace. These data suggest that the amorphous phosphate-rich constituents are present in the form of hydrogels of ferric phosphates and calcium phosphates rather than as organically bound compounds. Their precise formulation will have to wait until it has been determined whether the constituents contain only H20 or also OH fraction. Structural components in animals are usually strengthened by crystalline compounds to provide protective armor, internal supports and attachment surfaces for soft parts. One would not expect a priori amorphous hydrogels of lesser hardness to perform similar functions. This is true in the polyplacophoran C. stelleri, where the phosphatic hydrogel strengthens that structural component in the tooth denticles which is subjected to lesser stress than the magnetite-bearing subunit which is primarily engaged in the scraping action for food on hard substrates. On the other hand, the phosphatic hydrogels in the gizzard

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plates of the tectibranch gastropods here investigated seem to provide the mechanical strength required for the crushing of shelled prey. The gizzard plates of some species contain in addition to the hydrogel, crystalline compounds in the form of fluorite, monohydrocalcites and weddellite. The mechanical wear of these gizzard plates is similar to those which, in other species, are hardened solely by phosphatic hydrogels. Amorphous phosphatic hydrogel with amorphous calcium carbonate also provide adequate strength to the exoskeletal carapace of the malacostracan P. bigelowi for body protection and suspension of soft parts. The mechanical function, if any, is more obscure in the hydrogelbearing sternal shield and setae in the deposit feeding polychaete S. scutulata. The same is true for the densely packed mesodermal granules of the holothurian deposit feeder M. intermedia. Thus, aside from these two invertebrates, hard tissues with amorphous hydrogels appear to perform commonly mechanical functions similarly to those containing crystalline compounds. Phosphate precipitates in the form of amorphous hydrogels have been found in hard tissues of polychaetes, polyplacophorans, gastropods, bivalves, malacostracans and holothurians. They may be present also in the cestods (Von Brand et al., 1%7). The crystalline phosphates, carbonate hydroxyapatite (dahllite) and carbonate fluorapatite (francolite) are known at present only from the invertebrate classes, polyplacophora, brachiopoda and bivalvia. This suggests that phosphatic precipitates of amorphous hydrogels are more widespread in biochemical systems of marine invertebrates than crystalline phosphates. Why this should be so is not clear at present. Attempts to relate this phenomenon to a common denominator points toward the more frequent occurrences of hydrogels in tissue sites which are enclosed by live cell layers, at least at the time of their precipitation or by thick organic layers. This is true for species of the polychaetes, polyplacophorans, gastropods, bivalves, malacostracans and holothurians. However, in the polyplacophorans there are other species where homologous tissue sites are mineralized by the crystalline compound francolite. The francolite precipitates are found in tropical species, whereas the amorphous hydrogel occurs in a subarctic to cold-temperate species. The exoskeletat precipitates of francolite in inarticulate brachiopod shells are less extensively sheathed by organic compounds than are the amorphous hydrogels in the exoskeletons of the malacostracan species. However, the differences in degree of insulation from the external environment contributes little to explain their mineralogic differences. The reported mineralization of the first larval shell (prodisoconch 1) by dahllite, of calcite in the second larval shell (prodisoconch 2) and of calcite and aragonite in the adult shell of the bivalve P. martensii must be related to the biochemistry of the cells involved in their precipitation. Prodisoconch 1 is secreted by the larvally operating shell gland. Prodisoconch 2 is laid down by the epithelial cells of the undifferentiated mantle, whereas in the adult stage the calcitic layer is precipitated by the epithelial cells of the middle lobe of the mantle and the aragonite layer by the extrapallial fluid of the mantle surface bounded by the pallial muscles (Watabe, 1956). There are no data on the biochemistry of the cells which precipitate phosphates either in the form of amorphous hydrogels or as crystalline compounds at different, analogous and even homologous tissue sites. Hence,

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the question why phosphate precipitates in marine invertebrates are more commonly in the form of amorphous hydrogels remains to be explained. Consideration is given next to the contribution of the present study to a more precise characterization of the groups of marine invertebrates which are involved in hard tissue fixation of phosphates, their possible significance in phosphate extraction from sea water and possible evolutionary changes in the distribution of phosphate fixation among marine animals during the last 6 • 10 s y. Reports on the occurrence of phosphatic shells among inarticulate brachiopods include a number of chemical analyses from species of two genera (Clarke and Wheeler, 1922; Vinogradov, 1953; Rhodes and Bloxam, 1971). Precise crystalochemical determinations accompanied by chemical analyses have been reported only for shells of one Lingula species. They indicated that the shell is mineralized by francolite (McConnell, 1963). Unpublished data indicate that the same mineral is found also in the shells of a Discinisca species (Caskren). The present study adds mineralogic data on samples of the eurybathic species, P. atlanticus. The X-ray diffraction patterns obtained from these samples show a dahllite pattern. On the basis of finding appreciable amounts of fluorine by wave length scans with the electron-probe it is concluded that the mineral laid down in the shells is similarly francolite. P. atlanticus is known to be common from bathyal and abyssal depths to 5530 m in the oceans (Zezina, 1964). Shallow water species of Lingula, Glottidia and Discinisca are also widely distributed in the sea and have locally sizeable populations. This indicates that the volume of phosphate fixation by inarticulate brachiopods should be significant. Considering the Annelida, the flexible tubes of two polychaete species have been reported to be rich in phosphorus (Clarke and Wheeler, 1922). The calcium and magnesium contents of the tubes were shown to be low as compared to the phosphorus concentrations. Hence Clarke and Wheeler (1922) were uncertain whether the phosphorus was part of an organic compound or in the form of a phosphate mineral. A similar case was noted here for the inner lining of the arenaceous tubes of Cystenoides granulata (Table II) except that in this species the lining is Fe rich and its P, Ca and Mg contents are low by comparison An amorphous hydrous ferric oxide gel may be present, but if this should be the case the mineral would then constitute a minor constituent of the organic rich lining of the tubes. Sternaspis scutulata, investigated in the present study, provides the first indication that phosphate mineralization of hard tissues takes place in marine annelids. The sternal shield of this polychaete species were found to be extensively mineralized by amorphous, hydrous ferric-phosphate and its setae by an amorphous, hydrous calcium phosphatic substance. Microscopic examination of two other species of the family Sternaspidae, S. forsor from Southern California and St. scutulata from off Lobito, Angola, West Africa show that their sternal shields have the same orange red color as those in Sternaspis scutulata. The Sternaspidae are widely distributed in the oceans. Hence, it would appear that appreciable volumes of ferric phosphate hydrogels are precipitated by members of this annelid family in the oceans. Turning to the mollusca, the polyplacophorans are considered first. Earlier studies

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(e.g., Lowenstam, 1967) have shown that the denticles of the mature teeth of three species contain a microarchitectural unit which is mineralized by carbonate fluorapatite (francolite). The present study indicates that an "amorphous" iron phosphate hydrogel forms a major constituent of one of the microarchitectural units of the denticles in the mature teeth of Cryptochiton stelleri. This species is common in the North Pacific in subtidal waters. As in all chitons the anterior-most teeth are continuously shed, following wear by scraping on rocky substrates for food. There is thus not only a noticeable tie-up of phosphorus in the teeth of the standing crop of this species but also a continuous transfer of the phosphorus-bearing compound to the marine sediments. Adding to this the francolite fraction of the three earlier noted tropical species, which are widely distributed in the West Atlantic and lndo Pacific oceans, the volume of phosphorus precipitated and transferred by chitons to the sediment seems sizeable. The largest number of samples investigated in this study belong to tectibranch species of the molluscan class gastropoda. Calcium rich, and in one species, Fe-rich, amorphous phosphatic hydrogels were found to be the sole or major constituent of their gizzard plates. The present study qualifies more precisely the previously reported occurrence of an amorphous hydrogel rich in P in two species of the genus Seaphander. (Lowenstam, 1968; Lowenstam and McConnell, 1968). It adds the occurrence of an amorphous hydrogel to one other species of this genus and extends its presence to species of three other tectibranch gastropods. The electron probe data and Xoray diffraction patterns of heattreated samples indicate that the Ca-rich hydrogels have Ca + Mg/P ratios which in some species are similar to those of the carbonate apatite mineral, dahllite, whereas in others they resemble those of whitlockite. The iron-rich sample noted in one species contains, moreover, high Ca, Ba and intermediate concentrations of Mn in some layers, and hence, there may be in this case more than one amorphous hydrogel phase present. It has been reported earlier that the gizzard plates of two species of Scaphander contain fluorite (CaF2) and one species from the same genus, the calcium oxalate dihydrate mineral, weddellite (CaC204 • 2 H 2 0 ) (Lowenstam, 1968; Lowenstam and McConnell, 1968). In the present study, weddellite was found also in the gizzard plates of one species of Cylichna and monohydrocalcites (CaCO3 • H20; CaCO3 • 0.65 H20) were located in the gizzard plates of two species of Philine. In two species of Scaphander, one species of Actaeocina and two species of C),lichna, amorphous hydrogels rich in P and Ca or Fe constitute the sole strengthening agents of their entire gizzard plates, or of some of their micro-architectural units. Species of the tectibranch genera Scaphander, Philine and Cylichna are found in all oceans and locally have large populations. Moreover, species of Scaphander and Philine are known to range to depths in excess of 3000 m and Cylichna to between 6820 and 6850 m (Wolff, 1970). Hence, the total volume of phosphorus tied up by the gizzard plates of these tectibranch gastropods may be sizeable. It was noted earlier that there is one published record of phosphate mineralization of a hard tissue site in the mollusca class bivalvia. Watabe (1956) determined with the aid of electron diffraction that the first larval shell (prodisoconch 1) is mineralized by the carbonate apatite mineral, dahllite. The present study establishes for the first time that

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phosphate mineralization of hard tissues occurs also in the post-larval stage of bivalves. The gill supports of adults ofNeotrigonia margaritacea contain a hydroge[ rich in Ca and P, with a Ca/P ratio similar to that of the crystalline phosphate mineral, whitlockite, The exoskeletons of a variety of marine malacostracan arthropoda have been analyzed for their elemental composition (Clarke and Wheeler, 1922: Vinogradov, 1953). Most of the samples indicated high Ca and medium to low P concentrations. Only the stomatopod samples were found to contain equal amounts of CaCO3 and Ca3P208 (Schmidt, 1845: Clarke and Wheeler, 1922). X-ray diffraction analyses of decapod carapaces by Chave (1954) and in our own laboratory showed only patterns of magnesian calcites but did not reveal the nature of the phosphatic component. The present study of a carapace of the stomatopod Pseudosquilla bigelowi, though rich in phosphorus shows that the phosphate component is present in the amorphous state. The data are too limited to ascertain whether malacostracan species in the sea do not also precipitate crystalline phosphate minerals in their carapace. The average P2Os content of all carapaces investigated so far amounts to about 3% by weight. However, the malacostraca are common surface-water dwellers and extend in the oceans to 5000 m in depth, ttence, they constitute an important biologic agent of phosphate fixation in the sea. Our data call further attention to a phosphatic constituent of hard tissues in holothurians which, like all Echinodermata, are widely considered to synthesize solely magnesian calcites. The species belongs to the same order, Molpadonia, from which M6rne (1902) had earlier reported in another species mesodermal granules that are rich in Ire and P and contain appreciable amounts of Ca. Our investigations suggest that the granules are composed of a Ca-bearing hydrogel of ferric phosphate. Species of this order are widely distributed in the oceans and range from littoral to abyssal depths (6000 m; Clark, 1920). In the Puget Sound area, where our samples ofM. interrnedia were obtained, this species has locally sizeable populations. More species will have to be mineralogically investigated and better estimates on their population size are needed before it will be possible to assess their importance as phosphate-precipitating agents in the sea. The data presented here indicate that phosphate fixation in hard tissues of marine invertebrates encompasses, apart from the generally recognized crystalline compounds, dahllite and francolite, a number of hydrogels which are rich in either Ca and P or Fe and P. Phosphatic hydrogels were previously known only from the "calcareous" corpuscles of a cestod worm (Von Brand et al., 1967). They are shown here to constitute the sole or principal strengthening agents of certain hard tissues in species of polychaetes, polyplacophorans, gastropods, bivalves', malacostracans and holothurians. These occurrences fill in much of the gap in phyla representation of phosphate-precipitating animals between the brachiopoda and vertebrata (Fig.l). Several of the newly added groups that are involved in phosphate fixation are common constituents of the marine biomass. Hence, it would appear that the biologic constituents of the marine biomass which concentrate phosphorus in hard tissue sites is considerably more diversified than has been recognized in the past. Estimates of total volume of phosphorus fixation in skeletal hard parts will have to wait until we have better data on all of the organisms

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Fig. 1. Phyletic distribution of phosphatic hard tissues. involved in this process and on their population sizes for the oceans as a whole. One may ask what is the source of the phosphorus which becomes stabilized in hardtissue precipitates of marine organisms and what is the fate of those phosphate-bearing compounds after death. It has been generally assumed that in marine animals phosphorus required for their vital processes and for the formation of phosphatic minerals in their hard tissues comes directly from the food. A few experimental data indicate, however, that dissolved, labeled phosphorus similar to labeled calcium is taken up by sea water by some marine invertebrates (Bevelander, 1952; Rao and Goldberg, 1954). The experiments performed on carbonate-precipitating molluscs showed that the labeled phosphorus was initially localized in their mantle tissue and later incorporated in the organic matrices of the carbonate minerals of the shells, whereas the labeled calcium was ultimately concentrated largely in the carbonate crystals. There is thus the possibility that dissolved phosphorus in sea water may be also incorporated in phosphatic precipitates of marine invertebrates. Experiments are needed to determine whether this is so, and where this can be demonstrated, what proportion of the phosphorus in the minerals is derived from sea water compared to that derived from the food. As to the po'st-mortem fate of the

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phosphatic mineral precipitates from hard tissues, it is well known that francolite-bearing shells of inarticulate brachiopods and carapace remains of crustaceans, originally strengthened by phosphatic hydrogels, are found in recent marine sediments and sedimentary rocks alike. It remains to be determined whether the phosphatic hard tissues of the other invertebrate classes reported here also become incorporated after death in marine sediments and add to the noted depletion of phosphorus in the surface waters of the sea. The widespread distribution of phosphate hard parts in the phyla spectrum of recent invertebrates invites brief comparison with the known phyla distribution of phosphatic skeletons in the geologic past. The distribution relationships are graphically shown in Fig. 1, including the approximate time-stratigraphic ranges for each group of organisms. In the Paleozoic there are representatives among the coelenterata and bryozoa with partially or entirely phosphatic hard parts extending the phyla range to lower structural grades and implied lower biochemical complexity as compared to the present day biota. In the Cambrian there are a number of phosphate-precipitating organisms which, with the exception of the ostracoda, cannot as yet be safely placed taxonomically. The hyolithelminths may be either related to the mollusca or more likely to the annelids. Stenothecopsis, which has been alternately assigned to the paraconodonts, phoronids, entoproctids or molluscs, may well constitute the inner zoacial lining of bryozoan polyps which had an organically sheathed outer skeleton (Lowenstam, 1972). The ubiquitous conodonts, which range through all of the Paleozoic and possibly through part of the Mesozoic, are taxonomically still an enigma. The ostracoda precipitated phosphatic shells in the Cambrian, but not later. This indicates a shift in skeletal phosphate fixation in the classes of the Arthropoda following the Cambrian. There are no Cambrian records of phosphate precipitating Echinodermata or Vertebrata. Hence, arthropods were the most advanced structural grade of skeletal phosphate fixation, at least until the Ordovician. There is thus an indication that skeletal phosphate fixation shifted in the phyla spectrum within the last 6 • 108 years from lower structural grades and inferred lower biochemical complexity to more advanced biochemical systems. ACKNOWLEDGEMENT Dr. H. Lemche called the writer's attention to the mineralized gizzard plates in tectibranch gastropods and supplied samples of several species for this study. Dr. J.B. Kirkegaard suggested the mineralogic investigation of the sternal shields of Sternaspis species and provided a sample from one species for study. Dr. J.E. Smith provided me with space and collections at the Plymouth Marine Laboratory and Drs. J.H. Mcpherson, R. Menzies and P. Hopner with various samples. Dr. C. Hubbs made possible my participation in the Magdalena Bay Expedition where some of the samples investigated here were obtained. R. Squires and the late J. Hall called my attention to the mesodermal granules in the holothurian Molpodia intermedia and collected some of the samples. The infrared absorption spectra, referred to in this study, are part of an extensive survey of

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biologically precipitated a m o r p h o u s substances which is being u n d e r t a k e n j o i n t l y with Dr. G.G. Rossman. The technical assistance was provided by M. Dekkers, K. Potter, B. Bingham and A. Chodos. This research was s u p p o r t e d by National Science F o u n d a t i o n Grant GB - 6707X1.

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