The utricular otoliths , lapilli , of teleosts : their morphology and relevance for species identification and systematics studies *

According to Chaine and Duvergier (1934), the presence of a “stone” in the utriculus of fish, later called the lapillus (plural: lapilli), was first reported by Julius Casserius in 1600. Since then, the knowledge on the morphological characteristics and diversity of utricular otoliths has not increased much, as no detailed morphological study on these otoliths has ever been undertaken (Assis, 2000). During the first half of the 20th century several studies on recent fish otoliths were published. In some of them, the lapilli of a few species were represented (Shepherd, 1910, 1915; Frost, 1925a, b, c, 1926a, b, c, 1927; Sanz Echeverría, 1928, 1929, 1930, 1931, 1932, 1935, 1936, 1941, 1949; Bauzá Rullán, 1956, 1958). Since then, the lapilli of some species, especially those of otophysine fish (which are rather large and robust), have been used in the identification of recent species (Adams, 1940; Tilak, 1963; Mollo, 1981; Martinez and Monasterio de Gonzo, 1991), have been found with relative frequency in the fossil record (Koken, 1884, 1891; Bassoli, 1906; Schubert, 1915; Martin and Weiler, 1954; Frizzell, 1965; Frizzell and Dante, 1965; Frizzell and Koenig, 1973; Nolf, 1976, 1985), and have been collected in shore sediments (Martini and Reichenbacher, 1997). However, no one has yet considered the lapilli of fishes SCI. MAR., 69 (2): 259-273 SCIENTIA MARINA 2005


INTRODUCTION
According to Chaine and Duvergier (1934), the presence of a "stone" in the utriculus of fish, later called the lapillus (plural: lapilli), was first reported by Julius Casserius in 1600.Since then, the knowledge on the morphological characteristics and diversity of utricular otoliths has not increased much, as no detailed morphological study on these otoliths has ever been undertaken (Assis, 2000).During the first half of the 20 th century several studies on recent fish otoliths were published.In some of them, the lapilli of a few species were represented (Shepherd, 1910(Shepherd, , 1915;;Frost, 1925aFrost, , b, c, 1926aFrost, , b, c, 1927;;Sanz Echeverría, 1928, 1929, 1930, 1931, 1932, 1935, 1936, 1941, 1949;Bauzá Rullán, 1956, 1958).Since then, the lapilli of some species, especially those of otophysine fish (which are rather large and robust), have been used in the identification of recent species (Adams, 1940;Tilak, 1963;Mollo, 1981;Martinez and Monasterio de Gonzo, 1991), have been found with relative frequency in the fossil record (Koken, 1884(Koken, , 1891;;Bassoli, 1906;Schubert, 1915;Martin and Weiler, 1954;Frizzell, 1965;Frizzell and Dante, 1965;Frizzell and Koenig, 1973;Nolf, 1976Nolf, , 1985)), and have been collected in shore sediments (Martini and Reichenbacher, 1997).However, no one has yet considered the lapilli of fishes SCI.MAR., 69 (2): [259][260][261][262][263][264][265][266][267][268][269][270][271][272][273] SCIENTIA MARINA to be worthy of a detailed comparative morphological study.The fact that these otoliths are generally very small, not frequently found in geological layers, and rarely identified as fish otoliths in stomach contents or predator food remains, and the belief that their diversity is not significant, may have been the main reasons for their neglect (Assis, 2000).As a result, the morphological diversity of the lapilli is still almost unknown (Frizzell and Koenig, 1973;Nolf, 1976Nolf, , 1985) ) and their real potential as sources of taxonomic information remains unevaluated.In order to help in the description of fossil or recent lapilli, essentially from otophysine fishes, some authors (Tilak, 1963;Frizzell, 1965;Frizzell and Dante, 1965;Frizzell and Koenig, 1973;Nolf, 1976;Mollo, 1981;Martinez and Monasterio de Gonzo, 1991) have arbitrarily assigned provisional terminologies for their parts.Nevertheless, their terminologies are based on a very narrow range of taxa and are too incomplete to be useful.Furthermore, most of the terms proposed are similar to those already in use for features of the sagittae.It is in this context that this account of the lapilli of fishes aims to: (1) introduce the general morphology of the lapilli of teleosts; (2) propose a terminology for their parts; and (3) evaluate and discuss the potential of their use in species identification and their relevance in taxonomic and phylogenetic studies.

MATERIALS AND METHODS
This study is based on the analysis of a collection of utricular otoliths from 4735 positively identified teleost fish, representing 183 species from 23 orders and 62 families.All specimens were collected in Portuguese coastal waters, estuaries and rivers.
Before dissection, the total length (L T ) of each specimen was recorded.The otoliths were extracted through ventral dissection of the brain case, cleaned, and stored dry.All drawings were made with a camera lucida, using the most suitable magnification in each case.

The morphology of the utricular otoliths, the lapilli
The lapilli are normally very small otoliths, more or less equivalent to the asterisci in size.However, due to their generally rounded shape and thickness, they are often less fragile than the other two pairs of otoliths.
While the sagittae and the asterisci have their largest development on a vertical plane, are laterally compressed and have their medial side in contact with the respective maculae, the lapilli have their largest development in a horizontal plane, are dorsoventrally flattened and rest on the macula utriculi, with their ventral face contacting it.
Among the three pairs of otoliths the lapilli are the ones that have the most regular shape, the most homogeneous constitution in all fish taxa and the fewest number of usable diagnostic features.Nevertheless, they are still considerably diverse in terms of morphology (Figs. 1 and 2).
Basically, the lapilli are composed of a wedgeshaped dorsal body, narrowing from the lateral to the medial side, below which occurs a more or less voluminous protuberance (Fig. 3), the gibbus maculae (macular hump).This hump corresponds to the "mond" of Tilak (1963) or the "protubérance dorsale" of Nolf (1976).It develops on the ventral face of the otoliths, may have a diversely shaped outline, tilted antero-laterally with an angulous or globose apex (Fig. 4), and its surface has a texture similar to that of the collicula that pave the sulcus acusticus and the fossa acustica (sensu Assis, 2000Assis, , 2003) ) of the sagittae and asterisci respectively.
As in the sagittae and asterisci, the anti-macular side may have a plane, regularly curved, or sinuous surface, and is frequently devoid of useful features.For this reason, the macular side (the ventral face in the lapilli) is considered preferential for the observation of these otoliths.
When the lapilli are viewed from their macular side, in a position equivalent to the one occupied in the fish skull observed from bellow, the characteristics of several aspects of the outline can be used in their description (Fig. 5).
The extremum anterior and the extremum posterior (anterior and posterior extremes) are the anterior-most and posterior-most points in the outline of the otolith.Sometimes they are located at the tip of more or less pointed prominences, but often they are the apical points of regular curves.
The prominentia marginalis (marginal swelling) is a more or less conspicuous, normally round swelling of the lateral margin of the dorsal body, along the curvature of the apical region of the gibbus maculae.It frequently corresponds to the thickest zone in the dorsal body of the otolith.When the gibbus maculae is well developed it may intercept the lateral outline of the dorsal body and totally or partially cover the prominentia marginalis, which becomes only visible from the dorsal face.In such cases, a band of the dorsal surface of the gibbus maculae, the regio apicale gibbi maculae (apical region of the macular hump), is visible on the outside of the lateral outline of the dorsal body.
The structure that several authors (Adams, 1940;Tilak, 1963;Frizzell, 1965;Frizzell and Dante, 1965;Frizzell and Koenig, 1973;Mollo, 1981) have called the sulcus in the lapilli rarely looks like a furrow and is not associated with the sensitive epithelium of the macula in the same way that the sulcus acusticus or the fossa acustica (sensu Assis, 2000Assis, , 2003) ) are in the other pairs of otoliths.Instead, it simply represents the region of contact between the lateral margins of the gibbus maculae and the dorsal body of the otolith.Confluentia gibbi maculae (confluence of the macular hump) is the proposed designation for this feature, which probably corresponds to the zone of the otolith adjacent to the striola, a thicker region of the utricular epithelium with larger cells that occurs around the dorso-lateral border of the macula utriculi (Popper and Platt, 1993).
The linea basalis (basal line) is not equally clear in all otoliths, especially its medial portion, which may be inconspicuous or interrupted.This line corresponds to a discontinuity in texture, sometimes associated with a small slope or step, and seems to represent the limits of the zone of contact between the otolith and the macula utriculi, or the limits of the collicular region of the gibbus maculae.Frequently, the linea basalis displays a more or less pronounced, diversely shaped indentation directed to the middle of the surface of the gibbus maculae, the incisura lineae basalis (indentation of the basal line) (Fig. 6).

The two morphological types of lapilli
In morphological terms, two very distinct types of lapilli can be defined: the clupeiform and the nonclupeiform types.
The clupeiform type of lapilli is a highly modified otolith, found only among the clupeiform fishes.It is characterised by a tetrahedrical shaped dorsal body, an extremely thin and sometimes inconspicuous gibbus maculae, with a very thin and fragile extension projecting beyond the anterior border of the dorsal body in an antero-lateral and ventral direction, and an almost inconspicuous confluentia gibbi maculae (Figs. 5 B and 7).
The non-clupeiform type of lapilli, which occurs in most fish taxa, is characterised by a diversely shaped dorsal body (although never tetrahedrical), a normally voluminous and conspicuous gibbus maculae in the ventral surface of the dorsal body, and a marked confluentia gibbi maculae (Figs. 5 A and 8).

The potential of the lapilli in fish species identification
The characteristics of the lapilli, like those of any other anatomical part of a fish, are subjected to a considerable diversity and can be used as an aid in species identification, provided that their vari-THE LAPILLI OF TELEOSTS 263 ability is lower than the diversity, i.e. that the otoliths from co-specific fish are more alike than those of different species, even if the species are closely related.
The examples from the rather unrelated families Berycidae, Mugilidae and Sparidae, presented as an appendix to the present work, show that, although with different degrees of precision, and at least in some taxa, the morphology of the lapilli is sufficiently specific to allow them to be used as a tool in fish species identification.
Although the diversity of those examples and the variability illustrated in each of them are rather limited, the three cases presented point out the specific nature of some combinations of morphological characteristics of the lapilli, which may enable them, at least in some cases, to be of use in the discrimination between species.Among the characteristics that enter the combination, the general shape of the otolith and the characteristics related to the gibbus maculae, the prominentia marginalis, the linea basalis, and its incisura, seem to be the most relevant in terms of discriminative power.

The potential of the lapilli in studies of fish phylogeny and systematics
In contrast to the sagittae and asterisci, the lapilli show a noticeable structural homogeneity in almost every taxon studied.This indicates that they are anatomical parts with an extremely conservative general morphology.
Only among the clupeiforms do the lapilli assume a completely modified morphology, a fact that is probably related to the particular otophysic connection developed between the gas bladder and the utriculi in this group (Grassé, 1958;Popper and Platt, 1979;Nelson, 1994;Bond, 1996).Different from the otoliths of other taxa, the clupeiform lapilli have a tetrahedric shaped dorsal body with a narrow lateral margin, a broad medial margin, a concave curvature in the ventral face, and an extremely thin gibbus maculae with an anterior extension that projects beyond the dorsal body (Figs.5b and 7).
The specificity and the probable universality of this type of lapillus among the Clupeiformes allow its peculiarities to be added to the synapomorphies that define the group, and to the arguments that support its holophyly.
With the exception of the clupeiforms, however, no morphological characteristic of the known diver-sity of the lapilli seems to display a clear tendency among the Teleostei, or to be specific of one higher level taxon in particular.This suggests that the lapilli may be of limited utility in studies of fish macroevolution and macrosystematics.
Nevertheless, the fact that some morphological features of the lapilli can be used to discriminate and identify with reasonable precision some fish taxa points to the possibility of their use, along with other characteristics, in the establishment of phylogenetic/systematic relationships between closely related species.
In this respect, each of the morphological features of the otolith, per se, seems to be of little diagnostic value at a high taxonomic level, but increases in discriminative power below the family level.In fact, the occurrence of otoliths with similar characteristics, for instance outline, is common in distantly related taxa (Fig. 9), as well as with different corresponding characteristics within the same lower ranked taxon (Fig. 10).The same is true for all the other relevant features displayed by these otoliths (the gibbus maculae, the prominentia marginalis, the linea basalis, the incisura lineae basalis, etc.).This causes the diversity among higher level taxonomic groups to overlap considerably, and makes it necessary to combine all available characters when using the morphology of the lapilli for purposes of taxon characterisation or identification.For this reason, the information given by the characteristics of the lapilli, in terms of the study of phylogenetic relations, might be limited in relevance to the relationships between taxa positioned at the lower levels of the taxonomic hierarchy.
Above all, the low number of usable characteristics is probably related to the lower complexity of the macula utriculi in comparison with the maculae sacculi and lagenae, which causes the associated parts of the respective otoliths, i.e. the sulcus acusticus, the fossa acustica and the gibbus maculae, to differ significantly in the degree of differentiation and, consequently, in the number of usable diagnostic features.

DISCUSSION
In 1868, Higgins described the "superior" otolith, as he then called the lapillus, as an amorphous stone, completely devoid of value in studies of fish classification, phylogeny and palaeontology, and so unspecific that it would not even be usable for purposes of species identification.His arguments were that the utricular otoliths: (1) are normally rather small; (2) are very difficult to extract from the fishes' heads; (3) are not found in the fossil record or in the stomach contents or excrements of piscivorous animals; and (4) are too homogeneous in terms of shape.
Since that period, Higgins's (1868) statement has been reinforced by the inexistence of studies on the comparative morphology of the lapilli, and stressed by some authors who have subscribed to some of his arguments (e.g.Rado, 1968;Stinton, 1968;Jonet, 1972/73;Popper and Coombs, 1982).
Although the lapilli are normally rather small, if the fish skulls are carefully dissected and the relative position of each pair of otoliths is known, their retrieval does not involve any major difficulties.For this purpose, the ventral dissection of the otic region of the neurocranium has proven to be the best method for exposing the membranous labyrinth from underneath and extracting the otoliths (Assis, 2000(Assis, , 2003)).
It is true that the presence of lapilli in the fossil record has not been reported with as much frequency as that of sagittae, and that no published reference is known concerning their occurrence in the stomach contents or excrements of piscivorous animals.Nevertheless, many palaeontologists have reported the occurrence of fossil lapilli in geological strata from Europe (e.g.Koken, 1884Koken, , 1991;;Bassoli, 1906;Priem, 1906;Schubert, 1915;Martin and Weiler, 1954;Paghida, 1962;Weiler, 1963;Martini, 1965a, b;Stinton, 1977;Jonet, 1979;Nolf and Lapierre, 1979;Nolf and Capetta, 1980;Steurbaut, 1984;Reichenbacher and Mödden, 1996), from the Americas (e.g.Frizzell, 1965;Frizzell and Dante, 1965;Frizzell and Koenig, 1973;Nolf, 1976;Nolf and Stringer, 1996;Martini and Reichenbacher, 1997;Nolf and Aguilera, 1998) and from New Zealand (e.g.Schwarzhans, 1980), and the author of the present work has had the opportunity to identify two lapilli from Atherina sp. and Pomatoschistus sp. from stomach contents of green crabs, Carcinus maenas (Linnaeus, 1758), from the Tagus estuary, and two myctophid lapilli from stomach contents of THE LAPILLI OF TELEOSTS 265 blackmouth catsharks, Galeus melastomus Rafinesque, 1810, captured in the eastern north Atlantic.This shows that lapilli may be found both in the fossil record and in stomach contents of marine predators.What remains to be known is the extent to which their occurrence is, in fact, rare or whether they have been neglected or considered as unidentified material in other works.
It is also true that the lapilli are morphologically rather homogeneous among most fish groups, and that the number of taxonomically usable features in these otoliths is fewer than in the other pairs, especially if the sagittae are used for comparison.However, even if there are taxa that cannot be discriminated trough the morphological characteristics of the respective lapilli, as shown before, these otoliths may be used with considerable precision in the identification of at least some other teleost groups (Adams, 1940;Tilak, 1963;Mollo, 1981;Martinez and Monasterio de Gonzo, 1991;Assis, 2000).
An exception to the morphologic homogeneity of the lapilli among the teleosts is the case of the clupeiforms, in which there is a close association between the gas bladder and the utriculus (Allen et al., 1976;Denton and Blaxter, 1976;Best andGray, 1980, 1982;Popper and Coombs, 1982;Popper and Platt, 1993), related to an enhanced involvement of this chamber in sound perception (Popper and Platt, 1979), and justifying the fact that their lapilli have evolved to assume a morphology completely different from that of the otoliths of other teleosts.
At least in the case of the sagittae, it is known that otolith morphology is not only related to common ancestry (Schwarzhans, 1978;Gaemers, 1984;Nolf, 1985Nolf, , 1993;;Lombarte and Castellón, 1991), but also to the habitat in which the fish live (Wilson, 1985;Volpedo and Echeverría, 2003) and to the anatomical specialisations connected with sound perception, such as the structure of the entire inner ear (Nolf, 1985(Nolf, , 1993;;Popper and Platt, 1993;Lychakov and Rebane, 2000;Popper and Lu, 2000;Lombarte and Popper, 2004).They are thus subjected to different degrees of morphologic convergence between evolutionary distant taxa, and divergence between evolutionary close ones (Nolf, 1985(Nolf, , 1993)).The same probably happens with the asterisci (Assis, 2003) as both the sacculus and the lagena are especially engaged in sound detection (Popper, 1980;Rogers et al., 1988) and seem to have evolved in close association with specialisations of the sense of hearing (Popper and Coombs, 1982).On the con-trary, the utriculus and the remaining pars superior appear to be more engaged in the sense of posture and have a very homogeneous structure among all vertebrates (Cordier and Dalcq, 1954;Popper and Coombs, 1982).It is therefore not surprising that the lapilli have such a homogeneous morphology.
Although it ought to be kept in mind that otolith morphology alone should not be used to define taxonomic or phylogenetic affinities (Schwarzhans, 1978;Nolf, 1985Nolf, , 1993Nolf, , 1995;;Smale et al., 1995), there is no objective reason for the characteristics presented by the otoliths, when available, not to be used along with other characters of the fish body to increase the amount of information available in studies of fish systematics and phylogeny, as already proposed by Adams (1940).
In this respect, it is important to note that, as in the other two pairs of otoliths, some of the most relevant discriminative characters of the lapilli are those associated with the otolith outline and with the region that contacts the sensitive macula, which most certainly reflect its shape.Among them, the shapes of the gibbus maculae, of the linea basalis, and of the incisura lineae basalis assume a particular importance.
However, due to the morphological homogeneity of the utriculus among fish, and probably also to the little differentiation of the macula utriculi, most of the diversity of the lapilli is related to details less evident than those that characterise the other pairs of otoliths.
While it is true that the lapilli have a limited number of usable morphological features, and that no clear tendency in character distribution has yet been noticed among the teleosts, it is evident that these otoliths can be used for purposes of identification, in some cases to the species level.
For the above reasons, it is natural that the knowledge on the morphologic diversity of the utricular otoliths, which is still very poorly known and of limited use in the deduction of phylogenetic relationships and for taxonomic studies at the higher levels of classification, may prove to be a valuable tool in fish species identification and as a source of additional phylogenetic and taxonomic information at the family, genus and species levels.To deal with these limitations, future work directed towards an increase in the range of taxa studied and the evaluation of the amount of intra-and interspecific variation associated with the features of these otoliths is necessary before their real potential as taxonomic tools can be properly evaluated.

The lapilli of the genus Beryx (Berycidae)
The utricular otoliths of the two european Beryx species (Fig. 11) are characterised by an irregular shape, with a lobed medial margin.They have a long and triangular anterior region, with a pointed extremum anterior; a longitudinal axis closer to the medial side than to the lateral one; a large prominentia marginalis individualised from the rest of the dorsal body by a conspicuous strangulation in its base; a rather large gibbus maculae, with concave anterior and posterior outlines, narrower in the base than in the lateral margin; and the absence of an incisura lineae basalis.The gibbus maculae covers the prominentia marginalis completely on the ventral face, and forms a continuous regio apicale gibbi maculae on the dorsal one.However, the lapilli of each of the species can be identified by means of the

. Beryx decadactylus
The lapilli of the Mugilidae The lapilli of four mugilid species (Figs.[12][13][14][15]) are characterised by being oblong, almost semielliptical, with round and prominent anterior and posterior extremes.They have a small, premedian prominentia marginalis, perpendicular to the otoliths' longitudinal axis; and a rather asymmetrical gibbus maculae, with a premedian apex tilted towards the anterior direction.
Nevertheless, all species can be discriminated through the morphological characteristics of their otoliths.For this purpose, the following key can be used as a guide:  Apart from that, some characteristics of the gibbus maculae, such as its thickness and elevation in relation to the dorsal body; and of the linea basalis, namely its clarity and the depth of its incisura, become more noticeable with the increase in fish length.Although only 3 species are represented by otoliths from larger sized fish, it seems that the increase in size brings about a reduction in the anterior region of the otolith (the extremum anteri-or becomes progressively less conspicuous) and a relative increase in the longitudinal axis of the otolith.
Although the differences between the otoliths of the various Diplodus species are not as clear as in the examples presented before, which may lead to a larger percentage of misidentified specimens, especially for specimens smaller than 70 mm, as they have not yet developed the most important differentiating characters, the following key can be used to discriminate between the larger specimens of the Diplodus species represented: FIG. 2. -Ventral face of the lapilli of several teleost fish species.The shaded area corresponds to the gibbus maculae.The species in the upper four lines are represented by their right otoliths, while the ones in the bottom line are represented by both right (R) and left (L) otoliths.All specimens with lateral (distal) margin to the top of the page; anterior region of the right otoliths to the right and of the left otoliths to the left.

FIG. 3
FIG. 3. -Different views of the right lapillus of Sarpa salpa, showing the dorsal body of the otolith (white) and the gibbus maculae (black).D, V, A, P, L and M represent the dorsal, ventral, anterior, posterior, lateral (distal) and medial (proximal) directions respectively.

FIG. 5
FIG. 5. -Proposed terminology for the features displayed by teleost lapilli.Two types of utricular otoliths are represented: A -a nonclupeiform type otolith, the right lapillus of Epigonus telescopus; B -a clupeiform type otolith, the right lapillus of Alosa alosa.D, V, A, P, L and M represent the dorsal, ventral, anterior, posterior, lateral (distal) and medial (proximal) directions respectively.

FIG. 6
FIG. 6. -Ventral face of the lapilli of four species, illustrating differently shaped incisurae lineae basalis (indicated by the arrows).The shaded areas correspond to the gibbi maculae.L. cavillone, C. rhonchus and M. barbatus are represented by right otoliths, while S. solea is represented by both right (R) and left (L) otoliths.All specimens with lateral (distal) margin to the top of the page; anterior region of the right otoliths to the right, and of the left otolith to the left.

FIG. 10
FIG. 10. -Outline of the right lapilli of several cyprinid species, illustrating the occurrence of otoliths with considerably different appearance within the same fish taxon.All outlines are represented with lateral margin to the top of the page and anterior region to the right.
FIG. 11. -Ventral and dorsal faces of right (R) and left (L) lapilli of two species of genus Beryx (Berycidae) allowing a comparison between them.The shaded area corresponds to the gibbus maculae in the ventral face images, and to the regio apicale gibbi maculae in those of the dorsal face.All otoliths are represented with the lateral margin to the top of the page, and posterior region to the middle of each pair of columns (ventral face views), or to the outside of each pair of columns (dorsal face views).

1
FIG. 12. -Three aspects of right lapilli from Chelon labrosus (Mugilidae) of four size classes.The shaded area corresponds to the gibbus maculae in the ventral face and lateral view images, and to the regio apicale gibbi maculae in those of the dorsal face.The otoliths are represented with the lateral margin to the top of the page and the anterior region to the middle of the first two columns (ventral and dorsal faces), or with the dorsal face to the top of the page and the anterior region to the right (lateral view).

FIG. 13
FIG. 13. -Three aspects of a right lapillus from Liza aurata (Mugilidae) of three size classes.The shaded area corresponds to the gibbus maculae in the ventral face and lateral view images and to the regio apicale gibbi maculae in those of the dorsal face.The otoliths are represented with the lateral margin to the top of the page and the anterior region to the middle of the first two columns (ventral and dorsal faces), or with the dorsal face to the top of the page and the anterior region to the right (lateral view).