Spicular Morphology and Mineralogy in Some Pyuridae (Ascidiacea)

BULLETIN OF MARINE SCIENCE, 45(2): 243-252, 1989

SPICULAR MORPHOLOGY AND MINERALOGY IN SOME PYURIDAE (ASCIDIACEA)

H. A. Lowenstam

ABSTRACT A number of ascidians form mineralized spicules at the post-larval stage. Depending on the species, the spicules are located in one or several of the following body sites: test, body wall, branchial sac, gonads, or still other tissues. Spicules of several species of the stolidobranch genera Bathypera. Pyura, and Cu/eo/us in the family Pyuridae were examined in this study and compared with those of l-Ierdmania published earlier. Scanning electron microscopy shows much variability in spicular morphology at the genus level. Each genus except for Pyura has spicules of only one basic building plan, whereas in Pyura they not only usually differ between species, but within a species differently shaped spicules may be present at various tissue sites and in some instances even in the same tissue. Six different minerals are represented among the pyurid spicules examined. They are vaterite, calcite, amorphous calcium carbonate, amorphous calcium phosphate, crystalline carbonate hydroxylapatite (dahllite) and amorphous fluorite. The spicules of Balhypera. l-Ierdmania and Cu/eo/us are monomineralic, whereas in Pyura the mineralogy differs between as well as within species. In p, brad/eyi three minerals occur in one type of spicule. The Pyuridae, like the aplousobranch Didemnidae and Cystodytes species, form only calcium minerals. The Pyuridae, however, do not form aragonite, which is the only biomineralization product in didemnid and Cys- todytes spicules. The stellate spicules found in species of Pyura and Didemnidae are morphologically similar but distinct in their mineralogy. This has to be taken into account when one attempts to trace the evolution of the Pyuridae and Didemnidae by means of fossil spicules.

Mineralized spicules are commonly formed by ascidian representatives in the families Didemnidae and Pyuridae, by species in the genus Cystodytes of the family Polycitoridae and by a few species of the family Styelidae. SEM micrographs of ascidian spicules have become available only in recent decades and mainly encompass recent and fossil species of Didemnidae and Cystodytes (Lafargue and Laubier, 1980; Monniot, 1970; Monniot and Buge, 1971). Very few are available for the Pyuridae and none for the Styelidae. Ascidian spicules were generally stated to be calcareous and until about a decade ago, mineral determination by staining and modern techniques were limited to some didemnids and Cystodytes spicules (Schmidt, 1924; Prenant, 1925; Low- enstam, 1963; 1964). Aragonite, a metastable polymorph of crystalline calcium carbonate, was the only biomineralization product identified by these studies. This led to the generalization that ascidian spicules are aragonitic (Milliman, 1974). However, the identification of the crystalline carbonate polymorph vaterite as the spicular mineralization product in Herdmania momus (Lowenstam and Abbott, 1975) and published reports that the spicules of three Pyura species are siliceous (Kott, 1972; 1974) and those of one Culeolus species are composed of calcium phosphate (Fahlbusch, 1964) indicate that at least with respect to spicular mineralogy the Pyuridae may differ from the Aplousobranchiata. To further in- vestigate this possibility, the following survey of pyurid spicules was undertaken. This study of asci dian spicular morphology and mineralogy is based on samples from species of the four genera of Pyuridae known to form spicules: Bathypera, Herdmania. Pyura and Culeolus. The information on Herdmania, though pre-

243 244 BULLETIN OF MARINE SCIENCE, VOL. 45, NO.2, 1989 viously published, is included so that it can be compared with the other genera and be included in characterization of the spicule-bearing pyuridae as a whole.

MATERIALS AND METHODS

With one exception, the spicules reported in this study were from specimens preserved in formalin, and many were unbuffered when received. They came from the collections of the U.S. National Museum; the Queensland Museum, Australia; and the Scripps Institution of Oceanography, La Jolla, California. The spicule-bearing specimens of Bathypera sp. indet. which were dredged by the writer off Catalina Island, California, were preserved in 70% ethanol. The collection sites of the Bathypera, Cu/eo/us and some Pyura samples are noted in the text. Those of the Herdmania specimens are listed in the paper by Lowenstam and Abbott (1975). The Pyura species, whose locations are not listed in the text, were obtained from the western part of Victoria, Australia, Spicules were extracted from the different tissues either manually or in a 5.25% solution of sodium hypochlorite (Clorox). Electron microprobe data were obtained with a MAC V automated electron microprobe using spicules mounted in epoxy, polished and carbon-coated. Infrared spectra were obtained with a Perkin Elmer 180 spectrophotometer using KBr and paraffin wax pellets, Scanning electron micrographs (SEM) were obtained with an ETEC Autoscan.

RESULTS Bathypera spicules, which are located in papillae on the test surface, were studied from a co-type of the deep sea species B. ovoida (Ritter, 1907), specimens of an undetermined Bathypera sp. dredged from depths of 60 to 100 m off Shiprock adjacent to Catalina Island, California, and a specimen ofthe species B, splendens taken off South Shetland Island. As shown in Figure 1,11_9, the spicules of the three species have a common basic building plan, which is quite distinct from that of the known spicules of other pyurid genera. The common design is a castellate-like form with a somewhat expanded base. The principal species dif- ferences are the numbers, shapes and distribution of the spines on the upper spicule surface. Although the spicular surfaces in B. ovoida have been attacked to varying degrees by the formalin preservative, it is quite clear that the area inside the upper surface, bounded by five to six oblique outward-projecting spines, is smooth, whereas in Bathypera sp. indet. it is occupied by a very prominent, large, inclined spine which occurs in different spicules in various positions off center. By contrast the upper spicular surface in B. splendens is densely crowded by spines which decrease progressively in length from one side of the spicule to the other

(Fig. 1, 19), The spicular mineralization product in all three species is calcite. In Bathypera sp. indet. the spicules are magnesium calcite with 8.4 wt% MgC03• Herdmania momus is a highly varied pyurid species or species complex. It is common in shallow nearshore waters of subtropical to tropical seas and has occasionally been found in shallow warm temperate waters (Tasmania) and at depths below 500 m (Van Name, 1945; Tokioka, 1967). Mineralized spicules occur in the tunic, branchial sac, gonads and body wall. All have the same basic building plan: a fusiform axis fringed by rows of unidirectional fringing spines (Fig. 2, 111,2)' The tunic spicules differ, however, from the body spicules by being considerably shorter (about 0.1 the length), having fewer rows of fringing spines «0.5 the number) and having a mace-shaped spiny base that anchors them to the tunic surface (Lambert and Lambert, 1987). Both spicule types are mineralized by vaterite, the most metastable polymorph of crystalline calcium carbonate (Low- enstam and Abbott, 1975; Lambert and Lambert, 1987). Pyura saccljormis has antler-shaped spicules within the inner surface of the body wall, in the gonads, in the tentacles and most abundantly in the branchial sac (Fig. 2, 113) (Tokioka, 1967). The branchial sac also has large numbers of spherical spicules covered by many short thin spines (Fig. 2, 114,5)' The aperture LOWENSTAM: MORPHOLOGY OF ASCIDIANS 245

Figure 1. I,: Bathypera ovoida. co-type. Side view of a spicule, with partial resorption and broken surface spines. 12, I): Bathypera ovoida. co-type. Showing densely spaced spicules on tunic surface. Note spicular shapes variously modified by formalin resorption. I., Is: Bathypera sp. indet. Densely spaced spicules on tunic surface, showing blocking of contracted siphonal aperture by the long inclined spines on the spicular surfaces. 16: Bathypera sp. indet. Side view of tunic spicular. I,: Bathypera sp. indel. Top view of spicule showing central and fringing spines. Is: Bathypera splendens. Side view of tunic spicule. 19: Bathypera splendens. Top view of spicule, showing spine orientation and dimension.

wall contains ovoid to irregularly shaped granules. Infrared spectra of the antler- shaped and branchial granules show that the mineralization product is crystalline carbonate hydroxylapatite (dahllite) and a trace of amorphous calcium carbonate. Because of the intervening preservation in formalin, the question arises whether the original mineral was amorphous calcium phosphate (ACP).

Pyura australis has stellate, antler-like and rod-shaped spicules (Fig. 2, II6_9). The stellate forms are mostly concentrated in the upper test surface layer, but they also occur in small numbers in the branchial sac. Antler-like spicules char- acterized by branching in only one plane occur abundantly in the branchial sac 246 BULLETIN OF MARINE SCIENCE, VOL. 45, NO.2, 1989

Figure 2. II,: Herdmania momus. Side view of mature tunic spicule, showing mace-shaped spiny base (somewhat eroded). II,: Herdmania momus. Partial side view of spicule, showing arrangement and shape offringing spines. 113-lIs: Pyura sacciformis. 113, antler-shaped spicules from branchial sac. II., spiny, spherical spicules from branchial sac. lIs, variously shaped spicular types together with antler-shaped and spiny spherical spicules from branchial sac. 116-119: Pyura australis. 116, antler- shaped and stellate spicules from branchial sac. 117, 118, stellate spicules. Note formalin-etched spine tips and surface. 119, spine-shaped spicule from branchial sac. but are also developed in the mantle and gonads in small numbers. The rod- shaped spicules, occasionally slightly curved, are found in small numbers in the apertures, tentacles and gonads. Infrared spectra show that the stellate spicules are composed of calcite and the antler-shaped ones of crystalline carbonate hydroxylapatite (dahllite). Our sample of the rod-shaped spicules was too small to determine their mineralogy. Since the Pyura australis specimens had been preserved in formalin, it remains to be determined using fresh material whether the stellate LOWENSTAM: MORPHOLOGY OF ASCIDIANS 247 and antler-like spicules were originally mineralized by amorphous calcium carbonate and ACP instead of the identified crystalline minerals. In Pyura bradleyi the spicules are either in the form of antlers, needle-like or flattened and variously shaped (Van Name, 1945). The antler-like forms which occur in most tissues and organs and in the branchial sac are found together with the needle-shaped spicules. Flattened, variously shaped spicules have been noted only in the distal part of the branchial siphon (Van Name, 1945). Spicules isolated by Clorox treatment from a specimen collected by the "Te Vega" Cruise at Punta Centinella, Santa Elena Bay, Ecuador, consisted only of antler- and needle-shaped forms (Fig. 3, IIII-4)' The antler-shaped spicules are shown by infrared spectra to consist of ACP and amorphous hydrous calcium carbonate. When heated to 500°C the ACP fraction converts to crystalline carbonate hydroxylapatite (dahllite). Cross- sections of broken branches from heat-treated antlers reveal distinct interlayering of large crystals in the central core and aggregates of spherical bodies peripheral to it. X-ray dispersive analyses show that the outer layers have only calcium as the major elemental constituent, indicating that they are the site of the amorphous calcium carbonate in the spicules, whereas the inner core area contains the ACP fraction as indicated by high calcium and phosphorus concentrations. Electron microprobe wavelength scans do not show any fluorine content, indicating that the crystalline apatite formed by heating of the antler-shaped spicules consists of the mineral dahllite. SEM micrographs of the needle-shaped spicules show that they are usually slightly curved and have very short broad-based pointed spines on their surface (Fig. 3, IIII. 3)' These spines point in opposite directions (Fig. 3, III4). Infrared spectra show that they, like the antlers, are composed of ACP and amorphous calcium carbonate. Electron microprobe wavelength scans show that the spicules contain very high fluorine levels amounting to slightly more than twice the con- centration of phosphorus. X-ray diffraction patterns of heated needles show the presence of fluorite in addition to calcite and crystalline hydroxylapatite. This clearly indicates that these spicules contain an amorphous fluorite compound. A specimen of Pyura stolomfera praeputialis, lacking the test, collected at Broken Hill, New South Wales, Australia, has antler- and irregularly shaped spicules (Fig. 3, IIIs_7)' They are abundant in the pharynx, but occur also sparingly in other tissues. The antler-shaped spicules tend to branch irregularly in several planes (Fig. 3, Ills, 6)' Infrared spectra together with elemental determination by electron microprobe wavelength scans indicate that the spicules consist of amorphous calcium carbonate, some calcite and smaller amounts of ACP. The spicules of Pyura stolonifera recovered off Cape Town, South Africa, are mineralized by ACP and to a lesser extent by amorphous hydrous calcium carbonate. Spicules extracted from the test of Pyura spinosa were found to vary from spherical or ovoid-shaped bodies with a granular surface to stellate-like forms with very short blunt spines (Fig. 3, Ills 9)' SEM micrographs show that the spicules have been subjected to various degrees of surface resorptions. The spicular mineralogy consists of calcite with smaller amounts of ACP. Spicules of a co-type of Pyura littoralis (Kott, 1985) from the intertidal zone on Cuvier Bay, Hunter Island, Tasmania, were also studied. The spicules occur in large numbers in the tunic. As shown in SEM micrographs, they are spherical in shape and have evenly distributed low warty surface ornamentations (Fig. 4, IVI). X-ray diffraction shows that they are composed of calcite. As seen in SEM micrographs, crushed spicules break with a conchoidal fracture, and only the spicule surface has a thin calcite crystal veneer (Fig. 4, IV2)' Infrared spectra of 248 BULLETIN OF MARINE SCIENCE, VOL. 45, NO.2, 1989

Figure 3. III,-III.: Pyura bradleyi. III" antler- and needle-shaped spicules from branchial sac. III2' antler-shaped spicule from branchial sac. III), needle-shaped spicule from branchial sac. III., partial view of a needle-shaped spicule showing distribution pattern, shape and orientation offringing spines. 111,-111,:Pyura slalanifera praepulialis. III, •• antler-shaped spicules from pharynx. I1I'b' irregularly shaped spicules from pharynx. III6' antler-shaped spicule. III" irregularly shaped spicule. IIIs, III9: Pyura spinosa. Ills, spherical and ovoid spicules from test. Note formalin-etched spine tips. 1119, stellate-like spicule from test. Note varying degrees of formalin erosion on spines. the powders from whole spicules show an amorphous hydrous calcium carbonate pattern. Hence it is clear that except for the calcitic surface veneer, the spicules are composed mainly of amorphous calcium carbonate. Some of the deep-sea pyurids of the genus Cu/eo/us are known to form spicules in their branchial sac lining (Herdman, 1884). Spicules were extracted from a specimen of Cu/eo/us murrayi recovered on the U .S.S. Eltanin cruise 23, at station 1673 between depths of 4,866 and 4,881 m and also from an unidentified species LOWENSTAM: MORPHOLOGY OF ASCIDIANS 249

Figure 4. IV" IV,: Pyura littoralis, co-type. IV" surface view of spherical spicule. IV" interior view of broken specimen. Note conchoidal fracture of spicule core and crystalline calcitic surface veneer. IV), IV.: Culeolus sp. IV), antler-shaped spicules from branchial sac. IV., surface detail of an antler- shaped spicule. IV" IV.: Culeolus murrayi. Antler-shaped spicules from branchial sac. collected by the R. V. Melville on the abyssal plain off Baja Califomia Norte at a depth of 3,880 m. As seen in Figure 4, IV3-6, the spicules of both species are antler-like, with the flattened branches aligned in the same plane. Hence both have the same basic building plan but differ from each other in overall dimensions and in their branch shapes. Infrared spectra show that the spicules of both species are mineralized by hydrous ACP.

DISCUSSION The Pyuridae investigated here differ in spicular morphology at the genus level. In Bathypera and Culeolus the spicules within each species have the same gross morphology, while in Pyura as many as three distinct spicular forms may be found (e.g., P. bradleyi). Morphologic differentiation of spicular form occurs in species in which the spicules occur at more than one body site. In some cases, the same spicule morphology, for example the antler-shaped, may occur at several body sites. On the other hand, there are species which have two distinct spicular forms at the same body site. The tunic spicules of Herdmania momus, following nucleation and early growth, migrate from the blood vessels within the tunic to the tunic surface from which they ultimately protrude (Lambert and Lambert, 1987). Spicule migration also occurs in the polycitorid Cystodytes lobatus (Lambert, 1979), suggesting that this 250 BULLETIN OF MARINE SCIENCE, VOL. 45, NO.2, 1989 may be a widespread phenomenon among ascidians. Similar studies should be undertaken for the pyurids which have several spicule types at the same body site to determine where they originated. The stellate spicules found in Pyura australis (Fig. 2, 1178) and the stellate-like spicules of Pyura spinosa (Fig. 3, 1119) resemble many of the didemnid spicules. Hence they could be easily confused with the latter, if sedimentary occurrences are identified solely on morphological grounds. Their distinct mineralogic composition makes it possible to distinguish between the two ascidian species provided the original mineralogy is preserved. Pyura gibbosa also has stellate spicules (Kott, 1985). Identification oftheir mineralogy should determine whether stellate pyurid and didemnid spicules are generally distinguishable on mineralogic grounds. The functional significance of spicular designs is obscure. Only in Herdmania momus and in Bathypera sp. indet. does it appear that the spicules function as protective devices. In H. momus, the protruding tunic spicules provide the body surface with a physical deterrent, and their organic sheath may add a chemical irritant as well (Lambert and Lambert, 1987). The prominent, inclined spine on the spicular surface of Bathypera sp. indet. seems to serve as a protective device. In Figure 1, 14,5, the densely crowded spicules on the tunic surface form a grill- like obstruction over the retracted siphon aperture, which may prevent the en- trance of possible intruders such as amphipods. Six different Ca++minerals are represented among the pyurid spicules examined here. They are amorphous calcium carbonate, vaterite, calcite, amorphous calcium phosphate (ACP), crystalline carbonate hydroxylapatite (dahllite) and amorphous fluorite. No evidence for siliceous spicules was found. The spicules in Bathypera, Herdmania and Culeolus are all monomineralic. Calcite is formed by all Bathypera species, vaterite by Herdmania momus and ACP by Culeolus species. In sharp contrast, Pyura species form from one to as many as three different minerals. Mineral distribution in spicules differs according to species. As elaborated above, the two differently shaped spicules of P. australis are further differentiated by each having a different mineral, whereas the two morphologically distinct spicules of P. sacci/ormis both contain two identical minerals. P. bradleyi illustrates the extreme case where the antler-shaped spicules are composed of two minerals and the needle-shaped spicules have three minerals. One of them is represented by amorphous fluorite. This unusual mineral is also known to occur in the spicules of certain nudibranches (Mollusca) where they occur together with amorphous monohydrocalcite (Lowenstam and McConnell, 1968). The proliferation in spicular mineralogy among Pyura species coincides with the development of distinct spicular morphological types and the proliferation of spicule development at many more body sites than is found in the other genera. Comparison with other spicule-bearing ascidians shows that in general the spicule morphologies in the Pyuridae are considerably more varied, and with one exception, the stellate forms are clearly distinct from those found in the Didem- nidae and Cystodytes species. The Pyuridae, like the Didemnidae and Cystodytes, form only calcium minerals, but do not form aragonite. In the Didemnidae and Cystodytes species this crystalline metastable carbonate mineral is the only spicular biomineralization product. Thus in terms of their mineralization products, the Pyuridae are certainly a most unusual group. The evolutionary significance of these observations, including the fact that some pyurids form phosphate minerals, cannot at this stage be ascertained. The occurrence of stellate spicules in the Didemnidae has long been known, whereas their development in pyurid species has somehow escaped the attention LOWENSTAM: MORPHOLOGY OF ASCIDIANS 251

of sedimentary petrographers. Hence stellate spicules when found in recent sediments and in geologically older sedimentary rocks were automatically assigned to the Didemnidae. Based on this assumption the Didemnidae have been traced back to the late Jurassic-early Cretaceous (Bonet and Benveniste-Vehl.squez,1971). It is shown here that stellate spicules from didemnids and two pyurid species are mineralogically distinct, so that it becomes necessary to pay greater attention to the original mineralogy and the trace element chemistry when tracing the evolutionary history of these two ascidian orders back in geologic time. It has been pointed out that unbuffered formalin, the usual preservative of spicule-bearing ascidians, causes modification of the spicular morphology to varying degrees and introduces uncertainty as to whether spicular Ca++minerals were originally deposited in the amorphous or crystalline state. It should also be noted that long-term storage of spicule-bearing ascidians in unbuffered formalin leads to partial breakdown of mineralized spicules or their total dissolution. Hence, it is suggested that ascidians with mineralized spicules or other mineralization products should be stored in 70% ethanol.

ACKNOWLEDGMENTS

This paper is dedicated to the late D. P. Abbott who introduced me to the fascinating world of ascidian spicules. Our joint unexpected discovery of vaterite as the spicular mineral in Herdmania momus led to the present study: to explore whether other pyurids might produce the same calcium carbonate polymorph. Don helped this information would lead to a clearer understanding of pyurid evolution in which he was so keenly interested. Unfortunately, Don died before this study came to hoped completion. D. P. Abbott was instrumental in obtaining the majority of the specimens from museum collections. He and P. Kott further contributed additional samples for this study. A. A. Chodos performed the electron microprobe analyses. M. Dekkers and J. Muramoto-Nicholson skillfully extracted pure samples and obtained the infrared spectra. SEM micrographs were taken by S. Weiner and J. Muramoto-Nicholson. X-ray diffraction data were obtained in P. Duyez's laboratory. Caltech Division of Geological and Planetary Sciences 4696.

LITERATURE CITED

Bonet, F. and N. Benveniste-Velasquez. 1971. Fossil and livingascidian species. Rev. Inst. Mexicanol Petroleo 3: 8-35. Fahlbusch, K. 1964. Die Stellung der Conodontida im biologisehen System. Palaeontographica 123, Abt. A, 127-201. Herdman, W. A. 1884. The presence of calcareous spicules in the Tunicata. Proc. Geol. Soc. Liverpool 5: 42-45. Kott, P. 1972. The ascidians of South Australia II. Eastern sector of the Great Australian Bight and Investigator Strait. Trans. R. Soc. S. Aust. 96, pt. 4, 165-196. --. 1974. The ascidian fauna of Western Part, Victoria and a comparison with that of Port Phillips Bay. Mem. Nat. Mus. Viet. 37: 53-96. --. 1985. The Australian Ascidiacea. Part I. Phlebobranchia and Stolidobranchia. Mem. Qd. Mus. 23: 1-440. Lafargue, F. and L. Laubier. 1980. Lignee evolutive chez les Didemnidae des cotes de France. Valeur systematique des spicules. Ann. Inst. Oceanogr. Nouy. Ser. 56(1): 21-44. Lambert, G. 1979. Early post-metamorphic growth, budding and spicule formation in the compound ascidian Cystodytes lobatus. BioI. Bull. 157: 464-477. -- and C. C. Lambert. 1987. Spicule formation in the solitary ascidian Herdmania momus. J. Morph. 192: 145-159. Lowenstam, H. A. 1963. Biologic problems relating to the composition and diagenesis of sediments. Pages 137-195 in The earth sciences: problems and progress in current research. Uniy. Chicago Press, Chicago, Illinois. --. 1964. Sr/Ca ratio of skeletal aragonites from the recent biota at Palau and from fossil gastropods. Pages 144-132 in H. Craig, S. L. Miller and G. 1. Wasserburg, eds., Isotopic and cosmic chemistry. North Holland, Amsterdam. -- and D. Abbott. 1975. Vaterite: a mineralization product of the hard tissues of a marine organism (Ascidiacea). Science 188: 363-365. -- and D. McConnell. 1968. Biologic precipitation of fluorite. Science 162: 1496-1498. 252 BULLETINOFMARINESCIENCE,VOL.45, NO.2, 1989

Milliman, J. D. 1974. Marine carbonates. Springer Verlag, New York. 375 pp. Monniot, F. 1970. Les spicules chez les Tunicier Aplousobranches. Arch. Zool. expo gen. (Ser. 3) 3: 205-336. -- and E. Buge. 1971. Les spicules d'Ascidies fossiles et actuelles. Ann. Paleont. 57(2): 93-105. Prenant, M. 1925. Contribution a l'etude cytologique du calcaire. II. Sur les conditions de formation des spicules de Didemnidae. Bull. BioI. Fr. Belg. 59: 403-435. Ritter, W. E. 1907. The ascidians collected by the United States Fisheries Bureau steamer Albatross on the coast of California during the summer of 1904. Univ. California Pub!. Zoo!. 4: I-52. Schmidt, K. J. 1924. Die Bausteine des Tierkorpers in polarisiertem Licht. Friedrich Cohen Verlag, Bonn, 478 pp. Tokioka, T. 1967. Pacific Tunicata of the United States National Museum. Smithsonian Inst. U.S. Nat!. Mus. Bull. 251: 1-242. Van Name, W. G. 1945. The North and South American ascidians. Bull. Amer. Mus. Nat. Hist. 84: 1-476.

DATEACCEPTED: September 7, 1988.

ADDRESS: Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, California 91/25.