Polymorphic crystalline structure of fish otoliths

JOURNAL OF MORPHOLOGY 218:l-28 (1993)

Polymorphic Crystalline Structure of Fish Otoliths R.W. GAULDIE Hawaii Institute of Geophysics, School of Ocean and Earth Science and Technology, Uniuersity of Hawaii, Honolulu, Hawaii 96822

ABSTRACT Although most otoliths of teleost fishes contain aragonite, a detailed survey of the otoliths of several species confirms that other crystalline forms o f calcium carbonate occur. Otoliths of Hoplostethus atlanticus, Pagrus major, Macruronus novaezelandiae, Merluccius australis, Congiopodus coriaceus, Kathetostoma giganteum, Argentina elongata, Rhombosolea tapirina, Neophrynichthys latus, Coelorinchus aspercephalus, Paranotothenia microlepidota, and Gonorhynchus gonorhynchus contained the aragonite, calcite, and vaterite morphs of calcium carbonate in varying proportions. Aragonitic otoliths of Allocyttus niger, Hoplostethus atlanticus, and Pagrus major contained sequences of calcite-like crystals. The surface of the vateritic otolith ofAcipenser brevirostrum is shown in detail. Three classes of information are stored in the crystalline structure of the otolith: shape conservation, coexisting crystal morphs, and consecutive changes in crystal morph. Analysis of this crystalline information supports the hypothesis of control of growth of the otolith by proteins from the sensory epithelium or macula. Protein variation involved may be genetic in origin, or non-genetic arising from "stuttering" of the translation process. Proteins extracted from vateric and aragonitic morphs of the otolith of Macruronus novaezelandiae showed differences in infrared absorption spectra that were consistent with two different amino acid sequences. !c 1993 Wiley-Liss, Inc

Otoliths of teleosts occur in the endolymphatic sac of the inner ear (Dale, '76) and are composed of crystals of calcium carbonate, generally in the form of aragonite, that are deposited on an organic matrix (Degens et al., '69; Degens, '76; Gauldie et al., '90; Zhang, '92). Unlike the crystalline material in the bones of fish that is continuously redissolved and reprecipitated, the crystals of the otolith, once deposited, are metabolically inert except under extreme stress (Mugiya and Uch- imura, '89). Thus the otolith has the potential to store information in the sense of retain- ing, unchanged, t he variations in the crystalline structure of the otolith that reflect variations in the organic matrix, or the crystallization environment of the otolith.

Four crystalline forms, called morphs, of calcium carbonate occur in fish otoliths: calcite, aragonite, vaterite, and calcium carbonate monohydrate. Calcium carbonate monohydrate has been found in sharks (Carlstrom, '63; Mulligan and Gauldie, '89) and has been grown in vitro on artificial substrates (Dalas et al., '88). Aragonite is the most common crystal morph of calcium carbonate found in

otoliths (Carlstrom, '63; Degens et al., '69), but calcite (Morales-Nin, '85; Strong et al., '86) and vaterite (Gauldie, '90a, '86) have been reported in teleost otoliths, and calcitic otoliths may have occurred in the fossil acanthodian fish Utahacanthus guntheri (Sch- ultze, '90).

Calcium carbonate polymorphisms have complex behaviors at very high temperatures and pressures (Carlson, '83). However, ions in the solution from which calcium carbonate crystals grow (particularly the amino acids glutamic and aspartic acid) can also determine the morph of the calcium carbonate crystals (Kitano and Hood 19651, and vaterite has been grown in vitro on synthetic fatty acids (Mann et al., '88). The organic matrix upan which the crystallization of biogenic calcium carbonate occurs has also been shown to determine the morph o f the crystal (Wilbur and Saleuddin, '83; Weiner, '86). It has also been shown in molluscs that under certain conditions the morphs calcite, aragonite, and vaterite may co-exist (Wilbur and Watabe, '63), but under other conditions calcite and aragonite may be present in separate layers.

2 R.W. GAULDIE

This paper examines the hypothesis that the chemistry of the otolith parallels that of mol- luscan shells and that the signals provided by changes in the morph of calcium carbonate crystals in fish otoliths may have the potential to provide information about changes in either circulating amino acids, or in the amino acid residues of the matrix of the otolith, although the latter seems much more likely (Weiner et al., '83).

Calcite, vaterite, and aragonite polymorphs of calcium carbonate in fish otoliths have been described in several species (Palmork et al., '63; Jonsson, '66; Campana, '83; Morales- Nin, '85; Gauldie, '86; Strong et al., '86). The variation in crystalline morph described for both teleost otoliths and mollusc shells in- volves the apparent coexistence of different morphs. Co-existing calcite and aragonite morphs have also been described in the otolith of Neoceratodus forsteri (Gauldie et al., '86a), and co-existing aragonite and calcium carbonate monohydrate morphs have been described in the otoconia of some sharks (Carlstrom, '63; Mulligan and Gauldie, '89). Co-existence of morph implies either the expression of more than one gene at the same time, or that different genes are being switched on and off. Multiple genes with vari- able expression have quite different implica- tions for the significance of variation in crystal morph than does a polymorphism of a single locus. Ontogenetic, or environmen- tally induced, gene switching, for example, would prevent the use of crystal morph as a tool for discriminating populations (Strong et al., '86) but would give potentially more important information about the processes of otolith deposition and growth. In addition, the question of either ontogenetic or environ- mentally induced switching of crystal morph genes could have an important influence on the phylogenetic significance assigned to the variation in crystal morph (e.g., Maisey, '87; Schultze, '88).

MATERIALS AND METHODS

Otoliths of the species Hoplostethus atlanticus Collett, 1889 (Trachichthyidae), Macru- ronus novaezelandiae Hector, 1871 (Merluci- idae), Pagrus auratus Bloch and Schneider, 1801 (Sparidae), and Allocyttus niger James, Inada and Nakamura, 1988 (Oreosomatidae) were drawn from collections at the Fisheries Research Centre, Wellington. Otoliths of the species Merluccius australis Hutton, 1872 (Merluciidae), Congiopodus coriaceus Paulin and Moreland, 1974 (Congiopodidae), Kathe-

tostoma giganteum Haast, 1873 (Uranoscop- idae), Argentina elongata Hutton, 1879 (Ar- gentinidae), Rhombosolea tapirina Gunther, 1862 (Pleuronectidae), Neophrynichthys latus Hutton, 1875 (Psychrolutidae), Coelorin- chus aspercephalus Waite, 1911 (Macrouri- dae), Paranotothenia microlepidota Hutton, 1876 (Nototheniidae), and Gonorhynchus gonorhynchus Linnaeus, 1766 (Gonorhynch- idae) were provided from collections made by Dr. Chris Lalas. Two small specimens (25 cm fork length) of Acipenser brevirostrum Le Sueur, 1818 (Acipenseridae) were obtained from a fish farm and were anesthetized with MS222 (Sandoz) and killed by spinal section following the approved methods. Otoliths were collected at sea or from freshly killed animals and stored in paper pockets in a cool, dry environment. Vaterite otoliths stored this way were stable for periods of a t least 22 years based on the time of storage of material held in the general otolith collection at MAFF- ish, New Zealand. Endolymphatic sacs were dissected and fixed at sea for Macruronus novaezelandiae following the procedure described in Gauldie ('93).

The standard nomenclature for orientation of otoliths (Pannella, '80) is made difficult by the angles at which otoliths lie in situ, especially in flatfish. In this study I have foIlowed the simplifying convention of refer- ring to the otolith as if it were laying on a flat surface, sulcus down (Gauldie et al., '91). In this nomenclature there are sulcal and anti- sulcal surfaces [inward and outward faces in Pannella (198011 and dorsal, ventral, inte- rior, and posterior orientations to which broken sections can be referred. Where applica- ble, left and right hand follows from the stance of the fish facing forward.

The standard nomenclature of crystals (McKie and McKie, '86) makes a distinction between crystal morph and crystal habit. Crystal morph refers to the packing orientation of anions and cations that gives rise to different X-ray diffraction patterns. How- ever, a particular crystal morph may adopt many crystal habits involving a variety of crystalline shapes that may even mimic other, different, morphs, but always retain their characteristic X-ray diffraction patterns. Crystal morphology and crystal habit are dis- cussed in more detail in Bloss ('71; pp. 325- 341). Variation in habit can produce many forms that are apparently stable and may even re-appear in different species of fishes. Where possible I have followed the nomencla-

FISH OTOLITHS' POLYMORPHIC CRYSTALLINE STRUCTURE 3

ture of Carriker et al. (1980) in dealing with habit, but in some cases particular habits have acquired specific names. These names are adhered to in the text. Scanning electron micrographs were made with a Philips 505 SEM following the conventional preparation procedures.

All samples for X-ray diffraction were lightly crushed in an agate mortar and pestle and examined with a Philips PW 1279/PW 1710. Errors attached to the proportions of aragonite, calcite, and vaterite were *5%. The proportion of crystal morphs of otoliths was established by X-ray diffraction after SEM or optical pictures had been made.

Maculae (sensory epithelia) were prepared for electron microscopy by critical point dry- ing and sputter coated with gold before examination in a Philips 505 SEM.

Unless otherwise stated the otoliths examined were the sagittae, the largest of the three pairs of otoliths found in the endolymphatic sac.

Infrared adsorption spectra of the organic matrix of partially dissolved vateritic and aragonitic otoliths of Macruronus nouaezelandiae were recovered using a Perkin-Elmer 1720 IR spectrophotometer following the standard methods. The mineral part of the otoliths used in infrared spectra observations was removed using 0.01 m HCl.

The general biology of the fishes whose otoliths are described here can be found in Ayling and Cox ('82) with the exception of Acipenser breuirostrum whose general biology is summarized in Scott and Crossman ('73).

The frequencies of polymorphisms are summarized by species in Table 1, which also includes sample sizes. Crystal polymorphisms in otoliths present a confusing range of morphs and habits. The species with the widest range of crystalline polymorphisms is Hoplostethus atlanticus. This species is described first to provide the reader with some sense of orientation, and the remainin, ,,Z;pe- cies follow in order of complexity of pol$morphism rather than in any taxonomically sig- nificant sequence.

This paper provides a survey of the ultrastructure of those otoliths from a number of teleost species available from the commercial fishery in New Zealand that show mixed crystal polymorphisms. The presence of different calcium carbonate morphs in the fish inner ear reveals itself only after otoliths have been removed. As a result, this survey was limited to those species in which different morphs of calcium carbonate were discovered. In the many species in which only aragonitic otoliths were found it may be possible that with continued sampling other morphs will be found. These considerations made it difficult to organize sampling along the more rational lines of survey by taxa.

RESULTS

The sizes of layers, lamellae, and crystal growth units for all species are summarized by species and crystal morph in Table 2. The distribution of crystal morphs and habits of otoliths described here and in the literature is shown in Table 3. Crystalline growth units, lamellae and layers are described for each

TABLE 1. The occurrence of vaterite and calcite as well as abnormal aragonite is listed by species'

Mixed Mixed calcite,

Partially Totally Partially Totally calcite vaterite, Polymorphic Species Total vateritic vateritic calcitic calcitic vaterite aragonite aragonite

Hoplostethus atlanticus 4,000 20 (0.4%) 2 (0.1%) 2 (0.1%) - 1(0.05%) - - - - - - 7 (0.35%) -

Macruronus novaezelandiae 2,200 - - - - - l(0.04656) -

Allocyttus niger 10 - Kathetostomata giganteum 44 - Coelorinchus aspercephalus 54 2 (3.68) - - - - - -

Pagrus auratus 4,000 -

Merluccius australis 140 3 (2.1R.) - Acipenser brevirostrum 6 - 6(100'%) -

- - - - - - - - - - - - 4 (40%) - - - 1 (2.3%>)

- - - -

- - - - ~ Congiopodus coriaceus 46 l(2.176) - Neophrynichthys latus 18 1(5.5%) - Argentina elongata 174 1(0.5%) -

- - - - - - - - - -

Gonorhynchus gonorhynchus 28 4 (14%)) - - - - - - Paranotothenia microlepidota 66 3 (4.5%) - - - - - - Rhombosolea tapirina 22 - 2 (9%) - - - - -

'The total sample size is the number of otoliths collected for each species. The number and percent frequency of otolith types are shown as a number followed by a percent in parentheses.

4 R.W. GAULDIE

TABLE 2. Crystals'

Diameter of Crystal morph and habit Species Layer width Lamella width growth units

- 1. Vaterite botryoidal H. atlanticus 81-210 -

C. aspercephalus (1) 32 0.5-1 - C. aspercephalus (2) 15 0.8-1.7 - C. coriaceous 100 - - I?. Zatus - 0.1-0.5 - A. elongata 22 P. microlepidotu (1) 16 P. microlepidota (1) 10 H. atlanticus - - ROPY R. tupirina

Tablet A. breuirostrum - -

Stolkowski H. atlanticus 76 2 3

granular H. atlanticus - -

cu boidal M. novaezelandiae -

~ - - - ~ -

2-3 0.5 1.3

__ -

- 2. Aragonite pseudo-mimetic twin K. giganteum - ~

3. Calcite 57 - 12

'Crystal morphs and habits, of fish otoliths, with dimensions of layers, lamellae, and growth units. All units are in micrometres.

otolith type by species. Growth units refer to crystalline nucleation sites, not to the dish- and-ball type of residues described by Watabe ('83) as the incipient formation of the miner- alizing matrix.

Otoliths of Hoplostethus atlanticus Aragonitic otoliths of Hoplostethus atlanti-

cus have been described in terms of whole otoliths and internal crystal morphology else- where in the literature (Gauldie, '87, '88a, '90b).

Vateritic otoliths Completely vateritic otoliths from Hoploste-

thus atlanticus had a characteristic spiny appearance with projections growing from a central axis (Fig. 1A) and showed a strong similarity to the vaterite crystal described by Stolkowski ('62). Although there were radi- cal differences in ultrastructure between the vateritic and aragonitic otoliths of H. atlanticus, the conformation of the normal otolith (i.e., length, height, and covered area) was conserved so that both types of otoliths had a similar general shape, and a similar length and width (Fig. 1B). The growth axis in the posterior part of the otolith of H. atlanticus (Fig. 1A,B) was conserved in the sense that the deep indentations characteristic of the vateritic otolith did not interrupt the continuity and orientation of the growth axes that determine the overall shape of the otolith.

Otoliths with partial replacement of aragonite of the posterior otolith area by vaterite showed a characteristic difference between the morphology of the vateritic and aragonitic parts of the otolith (Fig. 1C). Indenta- tions similar to the entirely vateritic otolith

occurred in these otoliths and continuity of the growth axis was also conserved (Fig. 1C).

The anti-sulcal surface of the completely vateritic otolith (Fig. 1A) showed a series of concentric layers that appeared as a series of steps or checks paralleling the process of growth in aragonitic otoliths of H. atlanticus but with much greater frequency. SEM studies of otoliths composed entirely of vaterite showed further detail of the anti-sulcal surface layering that had a smooth appearance (Fig. ID) formed by small, botryoidal crystals about 1 mm in average diameter. SEM pictures of broken sections of vateritic otoliths showed concentric internal layering (Fig. 1E). The layers were about 76 mm wide on average (n = 20)' both internally and externally upon the anti-sulcal surface of vateritic otoliths. The average width between surface steps on the anti-sulcal surface of the aragonitic otolith of H. atlanticus (Gauldie, '87) was 320 km. At higher magnifications broken sections of the vateritic otolith showed microscopic growth increments with an average width (n = 16) of about 2 p,m (Fig. 2A).

Sections broken along the growth axis of the vateritic layer showed the epitaxial growth common to otoliths (Degens, '76), which resulted in radial and medial growth (Fig. 2B). The vateritic otolith did not show the distinct anti-sulcus and sulcus section separated by a discontinuity in the crystal structure of the otolith that has been described in the aragonitic otolith from Hoplostethus atlanticus (Gauldie, '90b).

The inner sulcal surface of the vateritic otolith, which was exposed to the macula, showed a different crystalline appearance to that of the outer anti-sulcal surface. The


TABLE 3. Descriotion and distribution of crvstal morwhs and habits of fish otoliths'

A. Calcite Cuboidal

Small crystals

Largc crystals Hexagonal

Amorphous

B. Aragonite Hexagonal

Laths

Blocks

Spherules

Pseudo-mimetic twins Pseudo-hexagons

Spindles

Rough spindles

Ornate spindles

Round spindles

C. Vaterite Stolkowski

Botryoidal

Fibrous

Tablet

D. Calcium carbonate

Family Squalidae: Etmopterus bmteri,' Squalus acanthias,1.2 Somniosus sp.,' Cen- troscymnus owstoni, I Centroscymnus crepidator, Centroscymnus plunketi, Dalatias licha, Scymnodalatias sherwood,' Family Oxynotiadae: Oxynotus bruniensis'

Family Gadidae: Macruronus novaezelandiae Family Neoceratidae: Neoceratodus forsteri" Family Gempylidae: Genypterus blacodes4 Family Acanthodidae: Utahacanthus guntheri5 Family Trachichthyidae: Hoplostethus atlanticus Family Gadidae: Pollachius uirens6

Lath-like crystals ranging from simple to complex hexagons are the most common aragonitic crystals of otoliths and have been described from a wide range of species."'

Block-like crystals of aragonite occur in some Ostariophsyian fishes" that have the superficial appearance of calcitic cubes, but retain the basic hexagonal structure of twinned aragonitic.

Spherulitic crystals of aragonite occur in sharks and rays,'J chimera^,'^,'^ teleosts,15J6 and lungfish.'" Sphcrules can be subdivided into a crenellated type,15 and an apparently smoothly spherical typeI3 that is composed of very many small spindles

Large (> 1 mm) aragonitic crystals in some catfish speciesL2 have the bowed lateral faces

A very common form of free aragonitic crystal is thc spindle-shaped crystal with flat-

Aragonitic spindles from some shark species' have a rough surface consisting of many

Some shark species have spindles with multiple lateral facets resulting in a more-

The most extreme form of the multi-facet spindle' has many lateral facets that result in

typical of pseudo-mimetic twins.

tened sides that has a superficial appearance similar to a grain of ~ h e a t . ' . ~ J ~

small crystals.

rounded, lemon-shaped crystal.

an almost spherical, melon-shaped crystal.

Family Trachichthyidae: Hoplostethus atlanticus Family Lampricidae: Lampris imrnacul~tus '~ Family Sparidae: Pagrus auratus Family Gadidae: Merluccius australis Family Salmonidae: Oncorhynchus tshawytscha,'8Jg Argentina elongata, Oncorhynchus

Family Gadidae: Macruronus novaezelandiae, Coelorinchus aspercephalus Family Congopodidae: Congiopodus coriaceus Family Neophrynichthydiae: Neophrynichthys latus Family Gonorhynchidae: Gonorhynchus gonorhynchus Family Notothcnidae: Paranotothenia microlepidota Family Pleuronectidae: Rhombosolea tapirina Family Trachichyidae: Hoplostethus atlanticus Family Pleuronectidae: Rhombosolea tapirina Family Squalidae: Somniosus SP.~' Family Acipenseridae: Acipenser sturio,2 A. brevirostrum, A. guldenstadi,2 A. ruthenus2 Family Salmonidae: Argentina elongata Family Molidae: Mola mola, M. ramsayi Family Chondrostidae: Polypterus ornatipinnis,2 P. delhezi2 Family Holosteidae: Amia Lepisosteus osseusZ Familv SDhvrinidae: Galeocerdo cuuier.2 Swhvrna zvpaena'

kisutchZ0

. _ " "- monohydrate Fami6 do&dae: Alopias vulpinus'

*The table is organized in descending order of polymorphic stability from calcite, through aragonite, vaterite, to calcium carbonate monohydrate. Within each morph crystal habits are listed to include subdivisions hy size where size is an appropriate consideratfon. (1) Mulligan and Gauldie, '89; (2) Carlstrom, '63; (3) Gauldie et al., '868; (4) Morales-Nin, '85; ( 5 ) Schultze, '90; (6) Strong el al., 86; (7) Gauldie, '90b; (8) Dunkelberger et al., '80; (9) Gauldie et al., '91; (10) Gauldie, '88a; (11) Gauldie et al., '91; (12) Gauldie et al., '93; (13) Gauldieet al., '87; (14) Mulligan et al., '89; (15) Dale, '7% (16) Gauldie et al., '86b; (17) Gauldie, '90a; (18) Gauldie, '86 (19) Gauldie, '91; (20) Carnpana, '83; (21) Lowenstarn and Weiner, '89.

inner sulcal surface had crystals with a fibrous or rope-like character (Fig. 2C) which was formed by aggregations of small, 2-3 km diameter crystals. Fibrous vaterite has also been-described in the otoconia of Somniosus sp. (Lowenstam and Weiner, '89).

The internal surface of the broken vateritic otolith of Hoplostethus atlanticus had a fine-grained appearance with small granular crystals (0.5 to 1.0 p m diameter) similar to those of the external sulcal and anti-sulcal surfaces of the vateritic otolith (Fig. 2D). In

6 R.W. GAULDIE

Fig. 1. Hoplostethus atlanticus. A The anti-sulcal surface of the vateritic otolith has a spiny appearance, but with a concentric pattern of layers (arrow) radiating from the nucleus (n) at the apex of the otolith. The principal growth axes are marked from the nucleus (n) to the posterior (p) and anterior (a) edge. B: The vateritic otolith (upper) retains the general conformation of its aragonitic (lower) pair. C: Mixed vateriticiaragonitic oto-

lith of H. atlantzcus has a vateritic posterior segment (arrows) with indentations in the continuous growth axis. D: The anti-sulcul surface of the vateriticH. atlanticus otolith has a smooth, appearance composed of small, granular crystals (arrow). E: Broken sections of vateritic otoliths from H. atlantscus show concentric internal layering (arrow).

FISH OTOLITHS’ POLYMORPHIC CRYSTALLINE STRUCTURE 7

Fig. 2. Hoplostethus atlantzcus. A Microscopic growth increments can be seen in broken cross sections of the vateritic otolith (arrows) B: Broken sections of vateritic otoliths show radial (r) and medial (m) epitaxial crystal growth (arrows). C: The sulcal surface of the vateritic

otolith, which is exposed to the macula, shows a crystal habit composed of fibrous aggregations formed from small granular crystals (arrow). D: The brokcn otolith has small granular crystals (solid arrows) and amorphous shear planes (open arrow).

8 R.W. G

places, the internal surface of the broken vateritic otolith showed amorphous shear planes (Fig. 2D), not the characteristic termi- nating screw dislocations of aragonite.

Calcitic otoliths The partially calcitic otolith of Hoploste-

thus atlanticus showed the development of a glassy amorphous nodule (Fig. 3A). X-ray diffraction studies of two such otoliths showed

AULDIE

them to be composed of aragonite and calcite in the ratios 80:20 and 89:11, respectively.

A single otolith was found that consisted of the vateritic and calcitic morphs in the ratio 60:40 (Fig. 3B). The mixed vaterite/calcite otolith had a granular appearance in the calcitic component which was formed from small crystals about 57 ym in diameter (Fig. 3B). The vateritic part of the otolith had layers about 211 ym wide at the central part of the

Fig. 3. Hoplostethus atlanticus. A: Calcitic nodules (arrow) occur rarely on aragonitic otoliths. B: One otolith consists of a calcitic component that is outlined with dotted lines and is formed of granular crystals (c ) and a layered vaterite component (v). The lapillus (1) in this specimen is abnormally large and is also vaterite.


otolith, decreasing in width to about 80 p,m at edges of the otolith. Otoliths composed of calcite and vaterite, as well as wholly calcitic otoliths, have been described for the cod (Pd- mork et al., '63).

A thin layer of hexagonal crystals was observed on the anti-sulcal (external) surface of the normal aragonite otolith of Hoplostethus atlanticus. The crystals were oriented at almost 90" to the plane of epitaxial growth of the aragonitic crystals within the otolith itself (Fig. 4). These crystals had a similar appearance to crystals described from the same location on the anti-sulcal surface (external apex) of otoliths of Genypterus capen- sis that were described as calcitic (Morales- Nin, '85). Although X-ray diffraction of aragonitic otoliths of H. atlanticus having such a calcite-like layer did not reveal traces of calcite, the amount of calcite potentially present may have been below the resolving power of the available equipment.

Mixed aragonitelcalcitelvaterite otoliths of Pagrus auratus

The aragonitic otolith of Pagrus auratus (formerly Chrysophrys auratus) is illustrated

in Gauldie and Nelson ('90) and Gauldie ('88b).

Polymorphic otoliths of Pagrus auratus (Fig. 5) consistently had the appearance of aragonitic otoliths that had been partially replaced by crystals with a similar appearance to that of the vateritic Hoplostethus atlanticus otolith. However, unlike the otoliths of H. atlanticus, X-ray diffraction showed all of the polymorphic otoliths of P. auratus to consist of a mixture of aragonite, calcite, and vaterite [the ratios in the illustrated otolith (Fig. 5) were 50:25:25, respectively]. Despite their filamentous appearance, the mixed-morph otolith of P. auratus still maintained the integrity of the growth axes equiv- alent to the growth axes of the otolith of Hoplostethus atlanticus.

Otoliths of Macruronus novaezelandiae Aragonitic otoliths of Macruronus novaez-

elandiae are figured in Gauldie ('93) and Kenchington and Augustine ('88).

Polymorphic otoliths Polymorphic otoliths of Macruronus no-

vaezelandiae were very uncommon; only one

Fig. 4. Hoplostethus atlanticus. The upper anti-sulcas surface of the aragonitic otoliths has a thin layer about 100 mm wide (arrow) of hexagonal calcite-like crystals.

10 R.W. GAULDIE

Fig. 5. Pugrus uurutus. Polymorphic otoliths composed of aragonite, calcite, and vaterite (50:25:25) have the layered appearance and spiny habit similar to that observed in some vateritic otoliths ofHoplosteethus atlun- ticus.

astericus (a), and the polymorphic sagitta are shown. The

sagitta (s) has the normal appearance of an aragonitic otolith at one end and a granular translucent appearance at the other end. Free crystalline granules occur at the edges of the sagitta and the astericus (arrows). Melano- cytes (open arrow) appear on parts of the endolymphatic

Fig. 6. Macruron.us nouuezelandiae. The lapillus (11, sac,

polymorphic otolith was observed in 1,200 individuals. The left otolith was a normal aragonitic otolith, the right otolith was used in this study.

Optical photographs of the endolymphatic sac (Fig. 6 ) of Macruronus novaezelandiae showed a polymorphic sagitta with an apparently normal lapillus, but with a polymorphic astericus. Part of the edge of the sagitta was composed of a number of free particles close to a complex of similar particles that were

fused together to form part of the otolith itself. Other parts of the otolith had the appearance of the normal aragonitic otolith of M. nouaezelandiae. The astericus (Fig. 6) showed a small number of free particles and signs of fusion. The surface of the endolymphatic sac of M. novaezelandiae often showed (for both polymorphic and normal otoliths) groups of melanocytes (Fig. 6).

X-ray diffraction of the powdered sagitta in Figure 6 showed that it was composed of the


Fig. 7. Macruronus novuezelundiae. A The mixture of crystal morphs found in the polymorphic otolith are marked as aragonite (a), calcite (c) , and vaterite (v). The fibrous material on the upper part of the otolith is organic detritus. The vateritic section of the otolith has the botryoidal habit with vateritic particles fused to the surface. Ridges (arrow) in the aragonitic part of thc otolith continue in the constricted part of the otolith. Stacked

calcitic crystals (open arrow) lie in the sulcus. B: The ridges in the aragonitic part of the otolith appear to extend along the sulcus forming ridges (r) in the vateritic part of the otolith. The section outlined by the white box has been magnified in the lower part of B to show ridges r in more detail. C: Calcitic crystals in the sulcus had clearly evident lamellae (arrow).

three morphs, aragonite, calcite, and vaterite, in the proportions 50:25:25, respectively. The proportions compare well with the ap- proximate distribution of morphs identified in the SEM picture of the whole otolith (Fig. 7A).

Scanning electron microscope studies revealed the complex nature of the polymor-

phic sagitta of Macruronus nouaezelandiae. It had four unusual features: a vateric zone, an apparently aragonitic zone, a calcitic zone, and a normally shaped sulcus passing through all of the zones (Fig. 7A). The vateritic zone showed another crystal habit of vaterite, the botryoidal habit (Brunson and Chaback, '791, as well as vateritic particles fused onto the

12 R.W. GAULDIE

surface of the otolith (Fig, 7A). One end of the otolith showed an apparently smooth aragonitic surface, with a series of regular ridges (Fig. 7A) separated by a sharp discontinuity from the calcitic part of the otolith. Another area of regular ridges appeared at the point of constriction of the sulcus (Fig. 7B), apparently a continuation of the ridges in the aragonitic end of the otolith. The other lobe of the sulcus contained a mass of crystals that had the appearance of hexagonal calcite. The lamellae of calcite crystals in the sulcus (Fig. 7C) averaged 5 pm width (n = 25). The polymorphic otolith of M. novaezelandiae conserved the general shape of the polymorphic aragonitic otolith, and the ridges in the aragonitic part of the otolith persisted into the vateritic zone. One of the features of the polymorphic otolith of M. novaezelandiae was the clearly defined boundaries between crystal morphs. The boundaries apparently ex- tended back into the otolith (Fig. 7A) indicating that all the different crystal morphs must have grown continuously and in parallel over the life of the fish.

Examination of the surfaces of a sample of ten otoliths of Macruronus novaezelandiae showed in one case a surface layer of the calcite-like crystals similar to that observed on the otolith of Hoplostethus atlanticus otolith. The remainder showed only the normal surface formed by monoclinical aragonite crystals that grew right to the surface with- out a surface layer of crystals. X-ray diffraction studies showed that the otolith of M. novaezelandiae with calcite-like crystals was aragonite. However, the potential amounts of calcite present may be below the resolving power of the instrument. Otoconia of Macruronus novaezelandiae

The otoconia of the endolymphatic sac of Macruronus novaezelandiae were closely associated with the macula and partially en- cased in a membranous sac. The otoconia were of the crenellated spherulite type (Fig. 8A) described in the endolymphatic sac of Neoceratodus forsteri (Gauldie et al., '86a), some other teleosts (Gauldie et al., '86b; Dale, '76), chimaeras (Gauldie et al., '87; Mulligan et al., '891, and some sharks (Mulligan and Gauldie, '89). Occasionally, the spherulites showed the pock-marked surfaces (Fig. 8A) described for this type of otolith in chimaeras (Gauldie et al., '87). Otoconia of M. novaezelandiae tended to fuse into massive structures (Fig. 8B), which have also been observed in other species (Gauldie et al., '86b).

In addition to otoconia, the endolymphatic sac of the frozen specimen of Macruronus nouaezelandiae (-loco) often contained regular crystalline deposits (Fig. 8B) formed from long, curved orthorhombic crystals. These deposits, which were likely to be preservation artifacts resulting from recrystallization in situ, were firmly embedded in the macula. Ten such frozen sacs were examined. None of the sagittae from these samples showed ab- normalities.

Polymorphic otoliths of Merluccius australis A pair of otoliths of Merluccius australis, a

normal aragonitic otolith and its mixed aragonitic and vateritic pair, are shown in Figure 8C,D.

The vateritic otolith of Macruronus australis (Fig. 8D) showed a progressive replacement of aragonite by vaterite. As with the otolith Macruronus novaezelandiae (Fig. 7A), there was a sharp boundary between the aragonitic and vateritic morphs. However, unlike the otolith of Macruronus novaezelandiae, the polymorphic otolith of M. australis showed vaterite crystals in the Stolkowski form similar to that of Hoplostethus atlanticus and Pagrus auratus, not the botryoidal form of the otolith of Macruronus novaezelandiae otolith. Nonetheless, the polymorphic otolith of M. australis had a similar overall shape to its aragonite pair (Fig. 8C,D).

Vateritic otoliths of Acipenser brevirostrum Carlstrom (1963) showed that the otoliths

of the sturgeons were formed of vateritic otoconia apparently fused into a solid structure. His micrograph (Fig. 2a,b, Carlstrom, 1963) of a section of the otolith of the stur- geon (probably Acipenser sturio) showed a continuous crystalline structure rather like a teleost otolith, with a few surface otoconia. My observations of the otolith of the stur- geon Acipenser brevirostrum provide detail of the otolith surface, particularly the otoconia, that was not available to Carlstrom ('63).

The general form of the sagitta ofAcipenser brevirostrum was similar to those described in general for all sturgeons (Nolf, '85); the sagitta has an irregular surface with a deep sulcus (Fig. 9A). X-ray diffraction showed that the otolith was composed entirely of vaterite. Although the otolith was clearly a solid structure, higher magnification shows that it was formed by the fusion of many tablet-shaped otoconia (Fig. 9B), which indicated yet a fourth habit (see Table 2) of


Fig. 8. Macruronus nouaezelahdiae. A Otoconia are of the crenellated spherulite type, some of which show pock marks (arrow) and a tendency to fuse (open arrow) into a single mass. B: Regular crystal outgrowths occur on the macula of frozen M. nouaezelandiae. They are much larger than otoconia and are formed by rosettes of lath-like crystals embedded in the membrane. The crystals have orthorombic cross-sections (arrow). The area outlined in the lower part of the figure is shown magnified in the upper part of the figure. C: Aragonitic otoliths

of Merluccius australis show the leaf-shape and prisms (arrow) typical of Merluccid otoliths. D The polymorphic pair of the aragonitic otolith (C above) of M. australis shows a distinct boundary between the vateritic and aragonitic parts of the otolith (open arrows). The vaterite in the polymorphic otolith is in the Stolkowski crystal form leading to a spinous development of the prism structure (arrow). The polymorphic otolith is similar in general shape to the normal aragonitic otolith (C above).

14 R.W. GAULDIE

Fig. 9. Acipenser brevirostrum. A The sagitta has a deep sulcus (arrow). B The vateritic otolith of A. brevirostrum is composed of many tablet-shaped otoconia that have the granular crystal (arrow) surface typical of vaterite. C: The aragonitic otolith of Allocyttus nzger shows occasional interruptions formed by what appear to be irregular deposits of large hexagonal calcite-like crystals (arrow). The white box in the left hand part of the figure is magnified in the right hand part.


vateritic crystals. In places, the otoconia on the surface of the otolith were incompletely fused, resulting in a layer of easily dislodged individual otoconia overlying the mass of the otolith (Fig. 9B, and Fig. 2 in Carlstrom, '63). The tablet-shaped otoconia have a clearly defined edge (Fig. 9B). The otoconia of A. brevirostrum have the same granular surface characteristic of vaterite in otoliths of Hoplo- stethus at2anticus and other species described above with crystal grains of about 1.3 pm average diameter.

Otoliths of Allocyttus niger Although the crystal structure of the prin-

cipal growth axes of the otolith of Allocyttus niger is unusual (Davies et al., '881, X-ray diffraction studies confirmed that the typical otolith was aragonitic and no polymorphism was indicated. However, SEM studies of the internal structure of the otolith frequently (four out of ten otoliths) encountered narrow layers of calcite-like crystals (Fig. 9 0 , which were laid down in an apparently disorganized fashion. The amount of calcite involved may have been below the resolving power of the instrument.

Polymorphic otoliths of Kathetostomata giganteum

The polymorphism of the otolith of Kathe- tostoma giganteum was very unusual in that the replacement for aragonite was aragonite in another, distinctive habit. In this case polymorphic is a misnomer, the difference being in habit, not morph. The normal and replacement aragonitic otoliths are shown in Figure 1OA. The replacement aragonitic otolith was composed of an aggregation of many small, otoconia-like aragonitic crystals and was brittle and broken into a number of pieces, two of which are shown (presumed sulcal side up) in Figure 10A. At higher magnifications the replacement aragonitic crystals varied widely in form and degree of fusion. For example, in some parts of the otolith, crystals were formed from readily observable lamellae (about 3 km average width) but the crystals varied from hexagonal to almost circular in cross section (Fig. 1 0 0 . Most hexagonal crystals had the bowed sides typical of aragonitic pseudomimetic twins (Fig. 10D). Otoconia-like crystals similar to those described in the endolymphatic sac of some shark species (Mulligan and Gauldie, '89) occurred on the edge of the otoconial mass where fusion had resulted in a more solid,

but still coarsely crystalline otolithic mass (Fig. 10E). At high magnifications the fused crystalline otolithic mass was composed of lamellae between 0.5 and 1 pm wide (Fig. 10F). The approximately 64" twinning angle typical of aragonite was observed in both free and fused crystals (Figs. 10D,F).

Polymorphic otoliths of Coelorinchus aspercep halus

Polymorphic otoliths of Coelorinchus aspercephalus were composed of aragonite and vaterite. About 30% of the first of the polymorphic otoliths of C. aspercephalus was vateritic (Fig. 11A). The vaterite component of the first otolith grew from the centre of the otolith from a point near, or at, the nucleus (Fig. 11A). The vaterite component was in the botryoidal form (Fig. l lB) , sharply delineated from the aragonitic part of the otolith and in places had the appearance of layers with an average width of about 32 km (Fig. 11B). The surface of the botryoidal vaterite had a laminated appearance in which individual lamellae varied between 0.5 and 1 pm in width (Fig. 1lC). About 10% of the second polymorphic C. aspercephalus otolith was vateritic (Fig. l lD) , but the vaterite component did not grow from the centre of the otolith. The vaterite component of the second otolith had the appearance of botryoidal vaterite, with a layered surface (Fig. 11E) similar in appearance to that of the vateritic otolith of Hoplostethus atlanticus but the average width of layers was about 15 pm. At higher magnifications the surface of the botryoidal vaterite of the second otolith had a laminated appearance in which individual lamellae varied between 0.8 and 1.7 fim in width.

Polymorphic otoliths of Congiopodus coriaceus

About 30% of the anterior end of the otolith of Congiopodus coriaceus was replaced by vaterite (Fig. 12A). Vaterite may have grown from the nucleus of the C. coriaceus, but the nucleus is obscured in both the aragonitic and vateritic otoliths by an unusual arrangement in which the central part of the sulcus was overgrown by a pitted crystalline structure (Fig. 12A). The vaterite component of the polymorphic otolith of C. coriaceus was in the botryoidal habit with layers about 100 pm wide (Fig. 12A).

16 R.W. GAULDIE

Fig. 10. Kuthetostorna giganteurn. A Two broken pieces of the replacement aragonitic otolith shown with a normal aragonitic otolith. B: The replacement aragonite otolith of K. giganteurn is composed of free crystals of aragonite with different forms and sizes. C: Free aragonitic crystal shape varies from almost circular to hexagonal, but most are formed by lamellae (arrow). D: Crystals sometimes have the approximately 64" twinning angle

(bars) and the bowed sides (arrow) typical of pseudomimetic aragonitic twin crystals. E: At the edge of the otolith mass there are single crystals (arrow) fused onto a coarsely crystalline mass. F: At high magnifications the coarsely crystalline mass is composed of interpenetrant lamellae (open arrow) oriented at the approximately 64" twinning angle (bars).

Polymorphic otoliths of Neophrynichthys latus

Normal aragonitic otoliths of other species of Neophrynzchthys have been described in Gauldie et al. (1991). The polymorphic otoliths of N . latus consisted of a normal, aragonite central core surrounded by a corona of vaterite (Fig. 12B). The vaterite had the botryoidal habit with the added complication of

apparent twinning (Fig. E C ) . In places the botryoidal vaterite assumed a complex arrangement of faces in individual crystals with lamellae crossing not only at an angle close to go", but also in places forming interpenetrant planes between the crystal lamellae (Fig. 12D). The complex interpenetrant planes lead to apparent discontinuities within the crystal (Fig. 12E). Lamellae in the botry-

FISH OTOLITHS' POLYMORPHIC CRYSTALLINE STRUC'NJRE 17

Fig. 11. Coelorinchus aspercephalus. A The vateritic component of the first otolith (sulcus up) forms about 3U% of the otolith mass and grows from a point (arrow) at or near the nucleus of the otolith. B: The vateritic component of the first otolith of C. aspercephalus is in the botryoidal habit sharply delineated from the aragonitic surface. The surface between the arrows can be inter- preted as layers of vaterite. C: Thevateritic component of

oidal crystals in Figure 12E had widths between 0.1 and 0.5 Frn wide.

Polymorphic otoliths of Argentina elongata The normal aragonitic otolith and the poly-

morphic otolith of Argentina elongata are shown in Figure 13A. The anterior end of the polymorphic otolith was replaced by vaterite in layers with an average width of about 22 km (Fig. 13B). The sulcus of the otoliths

the first otolith of C. aspercephalus is formed of lamellae (arrow). D The vateritic component of the second otolith (sulcus up) of C. aspercephalus forms about 10% of the otolith mass and does not grow at the nucleus (arrow) of the otolith. E: The surface of the vateritic component of the second otolith of C. aspercephalus is layered (arrow). F: The vateritic component of the second otolith of C. aspercephalus is formed of lamellae (arrow).

continues across the boundary between aragonite and vaterite (Fig. 13B). Vateritic replacement at the posterior end of the polymorphic otolith of Argentina elongata was more difficult to define structurally because of the less clearly defined boundary (Fig. 1 3 0 , but, remarkably, the vaterite surface at the anterior end of the otolith was decorated with the characteristically tablet-shaped otoconia of vaterite (Fig. 13D). Replacement of aragonite

18 R.W. GAULDIE

Fig. 12. CongLopodus coriaceus. A: The normal (left hand side) and polymorphic otoliths (right hand side) show layered (open arrow) vateritic replacement of the anterior (a) third of the right hand otolith and fusing of the central sulcal groove (arrow). B: The polymorphic otolith (sulcus side up) of Neophrynichthys latus has an aragonite central area (a) fringed with botryoidal vaterite crystals (v). C: The vateritic component of the otolith of N. latus is clearly dissimilar in texture from the aragonitic surface of the otolith (a) and shows evidence of symmetric (arrow) and interpenetrant (open arrow) crystal twinning. D: The botryoidal vateritic crystals of the otolith of N. latus show multiple faces with lamellae (open and closed arrows) running in different directions. E: At higher magnification the lamellae (open and closed arrows) of the vateritic crystals of the otolith of N . latus show a complex pattern of interpenetrant lamellae leading to an apparent discontinuity (a,d).

by vaterite did not start a t or near the nucleus of the otolith.

Polymorphic otoliths of Gonorhynchus gonorhynchus

The normal aragonitic and polymorphic otoliths of Gonorhynchus gonorhynchus are shown in Figure 13E. Vaterite replacement occurs at both the anterior and posterior parts of the otolith. At the anterior end the sulcus is maintained, but at the posterior end there was only growth on the ventral side of the sulcus, mimicking the form of the normal otolith (Fig. 13E). The posterior vaterite re-

placement showed signs of vestigial prisms (Fig. 13E). All four polymorphic otoliths showed similar patterns of vaterite replacement. The vaterite of the otolith G. gonorhynchus was layered, but the layering pattern was obscured by a coarsely crystalline surface unlike any other vaterite surfaces in this study (Fig. 13F).

Polymorphic otoliths of Paranotothenia microlepidota

Polymorphic otoliths of Paranotothenia microlepidota were composed of vaterite and


Fig. 13. Argentina elongata. A The aragonitic (right hand side) and polymorphic otoliths show similar size and shape. White and black segments of the scale bar are both the size indicated. Vateritic replacement occurs at both the anterior (a) and posterior (p) ends of the polymorphic otolith and the sulcus (arrow) is maintained in the vateritic replacement. B The vateritic component of the polymorphic otolith of A. elonguta is layered (arrow) and the sulcus (open arrow) is retained in the vateritic section. C: At the posterior end of the polymorphic otolith of A. elongata showing vateritic replacement (arrow) is obscured by the aragonitic component of the otolith

(open arrow). D: In places the vateritic surface of the polymorphic otolith ofA. elongata is covered with tablet- shaped vateritic otoconia. E: The aragonitic (left hand side) and polymorphic otoliths of Gonorhynchus gonorhynchus are about the same length, and the sulcus (s) and posterior ramus (r) of the otolith are conserved in the vateritic component, which also shows some traces of prismatic growth in the posterior ramus (arrows). F: The vateritic component of the polymorphic otolith of G. gonorhynchus has traces of layers (arrow) and continues the sulcus (open arrow) which are partly obscured by the coarse crystallinity of the vateritic surface.

aragonite, but the vaterite was in a different habit in each polymorphic otolith.

The first polymorphic otolith of Paranoto- thenia microlepidota is shown with its aragonitic pair in Figure 14A. The polymorphic

otolith in this pair did not retain the shape of the aragonite otolith and was wholly composed of vaterite in a crystal habit similar to the vaterite in the polymorphic otolith of Gonorhynchus gonorhynchus (Fig. 14B).

20 R.W. GAULDIE

Fig. 14. Paranotothenia microlepidota. A The first vateritic otolith (left hand side) does not retain the overall shape and dimensions of the normal aragonitic otolith. B: The crystal habit of the vateritic otolith of P. microlepidota generates a complex surface. C: The second wholly vateritic otolith of P. microlepidota retains the overall shape and dimensions of the aragonitic otolith (right hand side) but has crystalline nodules (arrow) along the ventral edge. D: The second vateritic otolith of

The second polymorphic otolith of Parano- tothenia microlepidota was wholly vateritic but retained the overall shape and dimensions of its aragonite pair (Fig. 14C). The second vateritic otolith had a crystal habit similar to the first vateritic otolith of P. microlepidota (Fig. 14D) but had, in addition, layered structures along the ventral edge that

P. microlepidota has a complex crystalline surface similar to B above (arrow), but the structure of the layered nodules along the ventral edge (open arrow) is similar to those of the mixed vateritic and calcitic otolith of Hoplo- stethus atlanticus in Figure 3B. E: The third vateritic otolith of P. microlepidota retains the overall shape and dimensions of the normal aragonitic otolith. F: Detail of the third vateritic otolith of P. microlepidota shows a layered surface.

were similar in shape and crystal texture to those of the polymorphic otolith of Hoploste- thus atlanticus shown in Figure 3B. The layers in these structures in the otolith of P. microlepidota were on average about 10 p,m wide.

The third polymorphic otolith of Paranoto- thenia microlepidota was wholly vateritic but


retained the overall shape and dimensions of its aragonitic pair (Fig. 14E). Detail of the third polymorphic otolith revealed a layered structure with layers of about 16 km average (n = 21) width (Fig. 14F).

Polymorphic otoliths of Rhombosolea tapirina

Polymorphic otoliths of Rhombosolea tapirina were wholly vateritic and similar in form to each other. The vateritic otolith of R. tapirina and the normal aragonitic otolith are shown in Figure 15A. The sulcal surface on the dorsal and ventral sides of the sulcus are deeply recessed giving the otolith a shell- like appearance (Fig. 15A). Botryoidal nodules of vaterite grew at the dorsal edge of the otolith (Fig. 15A). The sulcus was layered (Fig. 15A) in the vateritic otolith of R. tapirina, but most of the surface had either a smooth or fibrous (Fig. 15B) crystal texture. The fibrous surface in Fig. 15B showed small growth units about 0.5 pm in diameter.

Spectra ofproteins associated with vaterite and aragonite

Vateritic and aragonitic otoliths of Macru- ronus novaezelandiae were partially dissolved with weak (0.01 M) hydrochloric acid and the exposed organic matrix was scanned by infrared spectrometry. Although there were similarities in the resulting spectra (Fig. 16), there were differences in the spectra indicating differences between proteins in both the amounts and kinds of the bonds that produce the infrared absorption effect. In particular, the shifts in intensity of absorbance in the 1,200 to 1,600 wavenumber

range indicate conformation changes in the amide-I and amide-I1 absorbance parts of the proteins (Gendreau et al., '82).

DISCUSSION

The teleost otolith grows as a single large twinned crystal (Gauldie and Nelson, '88) in the fluid of the endolymphatic sac. All of the calcium required by the otolith enters through the macula, as has been demon- strated by experiments with isolated endolymphatic sacs (Mugiya, '87). Some species (for example, Macruronus novaezelandiae) have melanocytes on the external surface of the endolymphatic sac that may have some as yet unknown role in maintaining the integrity of the sac and its fluids. Higher vertabrates have 'dark cells' associated with the ampul- lae of the endolymphatic sac that are known to be involved in maintaining the chemistry of the endolymph (Kimura, '69; Bernard et al., '86). The melanocytes of M. novaezelandiae have a similar appearance to the 'dark cells' illustrated in Kimura ('69). The macula area is nearly always much smaller than that of the otolith (Gauldie, '88c) so that the most rapidly growing edge of the otolith may be millimeters away from its source of calcium. The result is a most extraordinary situation in which an unknown physiological mechanism maintains differences in growth rate in different parts of the otolith, even though all of the otolith is bathed in the same endolymphatic fluid. This unknown mechanism is even more remarkable in that i t can maintain differences in size and shape between sagitta, astericus, and lapillus otoliths which all grow in the same reservoir of endolym-

Fig. 15. Rhombosolea tupirzna. A: The vateritic otolith (left hand side) is shown with its aragonitic pair. The vateritic otolith has a deep sulcus ( s ) that is layered (arrow) with deep recesses (r) on the dorsal and ventral sides of the sulcus and nodules growing on the dorsal edge of the otolith. B: Parts of the surface of the vateritic otolith of R. tupirirzu have the fibrous habit of vaterite.

22 R.W. GAULDIE

4000 3000 2000 1000 Wavenumbers (cm- 1)

Fig. 16. Infrared absorption spectra are shown for matrices associated with aragonitic otoliths (continuous line) and vateritic otoliths (dotted line) as absorbance plotted against wave number. The wavenumbers of major peaks are indicated.

phatic fluid. Furthermore, the differences in growth rates of different parts of the gross structure of the otolith are so finely modu- lated that not only is the otolith species- specific, but individual variation, and even variation by region (Parrish and Sharman, ’59; Smith, ’92) and by depth (Wilson, ’85), still results in what is usually a remarkably faithful mirror-image between pairs of otoliths from what are, within the individual physically isolated endolymphatic sacs.

Some of the shape constraints of the otolith are provided by the physical restrictions, particularly along the ventral edge, of the otolith caused by occlusion within the bony otic cleft (Gauldie and Nelson, ’90; Smith and Kostlan, ’91). However, most of the constraints on gross structure must be gener- ated by the matrix components, proteins, and perhaps polysaccharides (Weiner, ’86) of the otolith, simply because there is no other source of genetically explicit information that could be made available simultaneously to both endolymphatic sacs and still result in differently shaped otoliths within the same sac. Soluble proteins and other organic chem- icals would also provide the chemical messen-

gers needed for the process of growth-at-a- distance characteristic of otoliths. Recent studies of the developing otolith have shown that some, a t least, of the proteins produced by the macula are incorporated into the otolith of Oreochromis niloticus (Zhang, ’92). There may also be transient ambient events such as temperature shock or disease that may, for example, change the viscosity of the endolymphatic fluid, which is known to effect the induction of vaterite (Pach et al., ’90).

How are we to gain experimental access to this complex process of control of crystal morph, habit, and growth rate? Unfortu- nately otoliths grow out of a chemical gradi- ent within a solution of calcium and carbonate, rather than a transfer of mineral by exocytosis. We are therefore denied a conve- nient anatomical experimental “karyotic mineralization window” (sensu Simkiss, ’84) on the growth of the otolith. The alternative is to examine the otolith itself for the record it maintains of its own growth. Proteins and organics can be extracted and analyzed, and a good deal has been learned in this way about the biochemistry and physiology of biogenic calcium carbonates. However, characteriza-


tion of the organic matrix cannot by itself explain the conformation of biogenic calcium carbonate crystals and the otoliths that result from these crystals. The crystal structure of the otolith must also be examined in terms of both calcium carbonate crystal polymorphism, variations in crystal habit within those morphs, and variations in matrix material.

Chemistry ofpolymorphism The different morphs of calcium carbonate

are produced by variations in the geometry of the crystal that result from the differences in the packing of calcium and carbonate ions in the crystal lattice (Mckie and Mckie, '86). Repulsion between ions in the crystal lattice determines the stability of the morphs (Evjen, '32).

The chemistry of calcium carbonate polymorphisms is well known (Dalas et al., '88). The driving force for the general process of calcium carbonate crystallization can be described quantitatively as the change in Gibbs free energy, AGO, for the transition from the supersaturated solution of calcium and carbonate to the equilibrium between crystal and free salt. The Gibbs free energy for each calcium carbonate polymorph, x, is estimated by

IP AG; = -RT In -,

qx where IP is the ion activity product, (Ca2+)(C032-), in which the parentheses de- note activities rather than molar quantities, and K& is the thermodynamic solubility product of the polymorph x, and R is the gas constant and T the Kelvin temperature. For a standard set of solute conditions the critical determinant of AG: is the solubility product, KZx. The following values for &Ox have been calculated from the literature by Dalas et al. (1988):

calcite K& = 3.311 x (1) aragonite K& = 4.613 x (0.718)

vaterite K& = 1.222 x (0.271)

monohydrate K&,, = 1.279 x (0.269) calcium carbonate

There is a decrease in Gibbs free energy from calcite to calcium carbonate monohydrate resulting in decreasing thermodynamic stability. The proportional decrease can be made more obvious by giving calcite a value of 1 and proportional values to the other morphs. These are shown in parentheses in

the list of solubility coefficients above. The differences in free energy indicate substan- tial differences in the stability of morphs. For example, inorganic vaterite occurs a t temperatures higher than 400°C (Brunson and Cha- back, '79) and tautomerises to calcite and aragonite at various temperature and pressure conditions (Albright, '71). The persis- tence of morphs other than calcite at more-or- less standard temperatures and pressures implies a large source of energy in the matrix necessary to counteract thermodynamic equilibrium.

Matrix effects on polymorphism Calcite is the only morph thermodynami-

cally stable at biological temperatures and pressures, but the expected tautomeric changes of aragonite, vaterite, and calcium carbonate monohydrate morphs to calcite are slowed to the order of millions of years (Wein- er et al., '76) by the organic matrices stabiliz- ing the crystal. This slow tautomerism means that the information contained in the variation in crystal morph is for practical purposes permanently recorded in the otolith.

Calcium carbonate crystals have been shown to crystallize into different morphs depending on the presence and the concentra- tion of certain amino acids (Kitano and Hood, '65). The amino acid effect on crystallization extends to the amino acid residues of the matrix proteins (Weiner et al., '83) as well as to free amino acids. Viscosity has also been shown to be important in vaterite crystallization (Pach et al., '90). The role of the amino acid as either a nucleation site or simply as a charge (or charge conformation) aspect of the ionic environment is not clear (Degens, '76). Amino acid metabolism, including changes in the sequence of amino acid residues in proteins, is normally determined genetically. Consequently, the determination of a particular morph on the basis of amino acid metabolism may be regarded as a signal of the expression of a particular gene. This ap- proach to the genetics of shell morph via the analysis of the amino acids of the organic matrix aragonite has been used by Weiner and Lowenstam ('80) and Weiner et al. ('76) on well-preserved fossil mollusc shells and by Jope ( '80) on brachiopods. The differences observed in the infrared spectra between the protein matrices of vateritic and aragonitic otoliths lend weight to the hypotheses that difference between vateritic and aragonitic otoliths may be genetically determined. Dif- ferences in the amide conformation detected

24 R.W. GAULDIE

by absorbance indicate either different amino acids, or changes in the conformation (i.e., the tertiary structure) of the protein. The bond structures of proteins that generate many of the observed infrared absorption peaks (including those in the 1,200-1,600 wavenumber region) are also activated by the bond-specific Raman technique (Sharma, '89). Other studies of the Raman spectra of complex organics (Saito et al., '87) and proteins (Spiro and Burke, '76; Champion et al., '88) have shown that even single amino acid sub- stitutions cause detectable changes in spectra, including the spectra associated with absorbance in the amide-I and amide-I1 region.

Crystal habit While the morph of the crystal reflects the

geometry of the lattice (the angle of orientation of calcium and carbonate ions), the habit of the crystal depends on the size of the crystal faces, which in turn depend on the velocity of crystallization, temperature, and the viscosity of the surrounding medium (De Jong, '59; Li et al., '91). There may be consid- erable variation (fibrous, flat, chalky, etc.) in crystal habit of the same morph within the same tissue. For example, Carriker et al. ('80) showed a large range of habits for aragonite crystals in the dissoconch valve of the oyster Crassostrea virginica. As with the vir- tual stability of the different morphs of calcium carbonate, the different habits within calcium carbonate morphs (once crystallized) are stable at biological temperatures and pressures. There are a number of different crystal habits within different calcium carbonate morphs reported in the literature that deals with fish otoconia and otoliths. These variations in habit within morphs are listed in Table 3. Otoconia in vertebrates other than fishes are mostly calcitic in the hexagonal form and have been described in Lim ('80). In addition to the species listed in Table 2, Stronget al. ('86) found calcite in the otoliths of 18 species of fish from the Atlantic, in one of which, Pollachius virens, the calcite was identified by X-ray diffraction, but they do not say how calcite was recognized in the other 17 species. Vaterite was the most commonly observed non-aragonite polymorph of calcium carbonate observed in the otoliths of fishes from New Zealand waters. Calcite was relatively rare in otoliths of New Zealand fish, but in Strong et al. ('86) it is the only non-aragonite polymorph of calcium carbonate described for Atlantic species. In a study of the otoliths of Limanda limanda from

Iceland, Jonsson ('66) refers to "crystalline" otoliths with insufficient description to iden- tify a possible morph. However Weiler ('59) describes otolith with apparent calcium carbonate polymorphisims form ten species of recent Atlantic fishes. Two of those species, Pleuronectes platessa and Lophius piscato- rius are compared by Weiler ('59) with Acipenser sturzo and notes the similarity of crystallinity. It is possible, therefore, that some of Weiler's otoliths may have been vateritic.

Otolith crystal habit variation It is evident from Table 3 that there is a

wide variation in the different habits that a particular morph of calcium carbonate can assume in the otoliths of fishes. A similar wide range in crystalline habit of calcium carbonate has been observed in the exoskele- ton of invertebrates (Watabe, '81). Much of the basic crystallography of morph and habit is reflected in size of layers, lamellae, and growth units. Although it was not always possible to measure these in the otolith crystal, differences in sizes of layers, lamellae, and growth units by both morph and habit were observed in the otoliths of several species (Table 2). Although the sample size was small and individual variation was high, there was less range in size at the level of the lamella and growth unit levels than at the level of the macroscopic layers. The size and structure of the lamellae and growth units reflect the underlying properties of the crystal (Li et al., '911, so that the macroscopic layering is more likely to be biological than chemical in origin. This would tend to support the idea that habit (i.e., macroscopic variation in what is essentially the same crystalline material) is a species character requir- ing different levels of energy to maintain in different species. It is also evident from Table 3 that certain characteristic habits of a particular morph may appear in a wide range of unrelated species, e.g., the tablet morph of vaterite occurs in Acipenser brevirostrum and Argentina elongata, and the spindle morph of aragonite occurs in some sharks as well as in Kathetostoma giganteum.

Recent theoretical studies of the growth of super-molecular structures in multiphase polymers indicate that secondary nucleation diameters and lamella widths are important indications of the thermodynamics of the phase equilibria associated with habit (Li et al., '91). It is instructive to note that layers in polymorphic otoliths (Table 2) vary over two


orders of magnitude while lamellae and growth units only vary in size by a factor of about two. These observations suggest that the basic crystalline properties are consistent from habit to habit, but that the biological component of the otolith (the dimensional interrelationships implied by layering) varies from species to species.

Causes of otolith crystal polymorphism Direct experimental growth in vitro of var-

ious morphs of calcium carbonate (Mann, ’89) suggests that habit is as much controlled by the matrix (both in terms of conformation and stability) as is the morph of the crystal. Energetic differences between habits may be much lower than those between morphs, but there may be a decreasing series of energy states corresponding to each habit as there is for each morph. Just as energetically incom- patible morphs coexist in vivo and in vitro (Dalas et al., ’881, so energetically heteroge- neous habits may co-exist. For example, studies of shell repair in molluscs have shown existing aragonites to be repaired by aragonites of a different crystal habit (Watabe, ’83). However, just as each morph can be expected to arise from a morph-specific matrix, so one might expect some, at least, of the stable and characteristic habits to arise from a habit-specific matrix. The possibility of specific matrices suggests that there are either a number of morph and habit genes expressed in the fish inner ear or some other control of crystal growth that reappears consistently among species. Examination of Table 3 shows a surprising consistency of crystal habit within particular morphs that crosses many species boundaries. The consistent re-appearance of the same habit at the same morph in widely separated species implies either that same conditions of crystallization, or the same specific genes coding the matrix protein. There are several possible alternative expla- nations.

A genetic basis for polymorphism and habit Detailed SEM studies of the otolith of Ma-

cruronus novaezelandiae showed sharp boundaries between the crystal morphs in the otolith. Such boundaries could have been maintained only if the agent causing the morph had an extremely short effective half- life. Because otoliths are close to the macula, about 10 km (Dunkelberger et al. ’80), it is reasonable to conclude that any causative agent released from the macula would have

to have a short half-life in solution. The macula is known to release proteins that are incorporated into the otolith (Zhang, ’92). The spectral analysis reported here shows differences in the protein matrix of aragonite and vaterite morphs. Differences in proteins imply differences in genotype. Studies of the proteins found in vateritic, calcitic, and aragonitic otoconia from higher vertebrates have shown that each polymorph of calcium carbonate also has a unique, polymorph specific protein (Pote and Ross, ’91). Otoconia are very similar to otoliths in their chemical composition and structure, and also occur in the inner ear of fishes; sometimes simultaneously with the otolith (Gauldie et al., ’86b).

I t is difficult to avoid the conclusion that different proteins are being produced by the macula and that these proteins are largely responsible for the observed variations in morph and perhaps habit. But the co-expression of the different genes needed for the simultaneous production of different proteins needed to produce simultaneous polymorphism presents some problems.

Simultaneous expression of two or more genes does not constitute a genetic polymorphism in the sense of different alleles occurring at the same locus. The simultaneous co-expression of variants of the same gene is common in fish in the form of tissue-specific isozymes. The sharp boundaries between polymorphs of calcium carbonate in the otolith suggest that different genes are being expressed in different parts of the macula. There is evidence to support the division of the macula into functionally separate units (Platt and Popper, ’81). Separate functions in the macula imply different cells and, consequently, the expression of different genes- not genetic polymorphism at a particular lo-

One flaw in the argument for the co- expression of different genes is that calcium carbonate polymorphisms sometimes appear a t the edges of the otolith. It may be possible that ontogenetic changes occurring in the chemistry of the endolymphatic sac that affect the ion activity product term, IP, that can in turn affect the carbonate polymorphism. Otherwise it would be necessary to propose that an otherwise silent gene be switched on during the life of the individual. “Stuttering”

There is a non-genetic protein coding mechanism possibly involved in the co-existence of different morphs in the otolith: stuttering.

cus.

26 R.W. GAULDIE

Stuttering occurs when t-RNAs are being translated into proteins in the absence of certain amino acids. Shortage of amino acids results in another amino acid being substi- tuted, resulting in a novel, and non-genetic, variant protein (Parker et al., '78). Under certain conditions of stress in some fishes some amino acids fall well below their normal metabolic levels (Mommsen et al., '80). Thus the co-existence of crystal morphs may reflect a chronic metabolic disorder in individual fish rather than the expression of a certain gene.

Gliding An alternative non-genetic mechanism for

calcite occurrence would be simply that in some cases the matrix failed to stabilise the aragonite-to-calcite tautomerism. The calcitic otoliths described by Strong et al. ('86) showed calcite as occurring most commonly (about 70%) in the anterior ventral quadrant of the otolith. This is the part of the otolith in which contact with the floor of the bony otic cleft is most likely to occur. Calcite is sensitive to pressure and has a well-known gliding property in which calcite crystals will be as- sembled in an alternative plane at the point of pressure (Bloss, '71). Crystals that have apparently glided occur on the anterior ventral quadrant of the otolith of Pagrus auratus (Gauldie and Nelson, '90). However, glide- plane instability inevitably leads to calcite if a tautomeric change accompanies pressure gliding; therefore these properties cannot explain the widespread occurrence of vaterite.

CONCLUSIONS

The very existence of calcium carbonate polymorphisms in otoliths draws attention to the changes in otolith shape that they cause. Shape is an important component of the basic role of the otolith as a mechanical frequency transducer (Fay, '80; Gauldie, '88c). In some cases there is conservation of the overall shape of the otolith, suggesting mini- mal disturbance to the function of the otolith. In other polymorphic otoliths there are major changes in otolith shape which would indicate effective deafness to the frequencies to which particular otolith shapes are most sensitive. This study was limited in its sample size, but it would be interesting to assess potential selection against otolith polymorphs in terms of departure from the species- specific shape of otoliths that is sensitive to a particular range of frequencies.

ACKNOWLEDGMENTS

The otoliths used in this study were collected while the author was at the Fisheries Research Centre, MAFFish, Wellington, New Zealand. Technical assistance in the preparation of the otoliths was provided by Kevin Mulligan of the Fisheries Research Centre and Robert Thompson of Victoria Univer- sity, and Brent Fry and Ray Soong of the New Zealand Geological Survey. The otoliths of Merluccius australis were generously do- nated by Dr. Chris Lalas. Correct zoological identification was provided by Peter McMil- lan of MAFFish. I am grateful to Dr. Gerald Smith and the anonymous reviewers for their constructive comments. This is SOEST contribution number 3274.

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Polymorphic crystalline structure of fish otoliths