otolith microstructure and the demography of coral reef fishes

3
TREE vol. 3, no. 3, March 1988 Otolith Microstructure and the Demography of Coral Reef Fishes Secondly, because otolith growth is generally isometric, patterns of growth in the otolith can be used to measure retrospectively patterns of growth in the fish themselves. The link between growth rate and incre- ment width has significance well beyond the obvious potential for, as an example, correlating changes in growth of herbivores with variations in algal production. Wherever growth rates differ consistently be- tween life history stages, the dura- tion of these stages (and dates of transition) can be determined for all individuals in a population. Prelim- inary work indicates that diverse factors (food ration, temperature, activity levels) can affect the spacing and composition of growth incre- ments4r5; if the interactions of these factors can be resolved into discrete signals, it may well be possible to reconstruct the environmental and developmental history of a fish, from egg to senility, by examination of its otoliths. R.E. Thresher ( The ecology of planktonic larvae - particularly the extent to which lar- vae are transported between habitat patches-is a poorly known aspect of the life history of tropical reef- associated fishes. The logistic diffi- culties of studying larvae in the field and of relating their behavior in cap- tivity to field conditions have greatly impeded the progress of research. However, a recently developed tech- nique, based on analysis of the microstructural growth elements in otoliths (‘ear stones’ ), may prove a powerful means of assessing the early life history of reef fishes and relating it to population and com- munity dynamics. As in most marine organisms, the life history of coral reef fishes typi- cally consists of two wholly different stages: juveniles and adults are relatively sedentary and live on or close to the reef; their larvae are, with few exceptions, planktonic. The duration of the planktonic stage can be as long as a year; during it, the larvae are carried by currents over distances at which we can only guess. The unknown extent of larval transport is the biggest single barrier to our understanding of the de- mography of coral reef fishes. We have virtually no idea of the direction or frequency of exchange of offspring between habitat patches for any species at any location. In- deed, our knowledge of any aspect of the ecology of larval reef fishes is fragmentary. Without more detailed information on this stage of their life history, the prospects of understand- ing the dynamics of reef fish com- munities are slim. Attempts to examine the dispersal and general ecology of larval reef fishes have had little success. The small size of the larvae, their fragility and their relative rarity in a large ocean preclude conventional tagging or direct study in the field. The spatial scale of events critical to the survival of a 5 mm-long larva in the water column is orders of magnitude less than we can routinely sample; larvae operate at a scale where it becomes difficult for oceanog- raphers to discriminate between sampling error and real environmen- R.E. Thresheris at the CSIRO Marine Laborator- ies, GPO Box 1538, Hobart 7001, Tasmania, Australia. 78 tal patchiness. Studies in captivity are seldom technologically feasible, due to difficulties in keeping larvae alive, and are always difficult to interpret. Confinement inevitably affects behavior, distorting the rela- tive importance of factors critical to survival’ . Otolithic aging A new analytic technique, based on the examination of the fine struc- ture of otoliths, offers dramatic promise for investigations of larval and juvenile ecology. Otoliths (‘ear stones’ ) are small calcareous struc- tures that float freely in the semi- circular canals of fishes, where they are important in hearing and the de- tection of orientation and accelera- tion. Otoliths grow more or ‘ less isometrically by accretion of calcium carbonate in a proteinaceous matrix. It has long been known that annual cycles of slow and fast growth pro- duce coupled zones of protein-rich and protein-poor material in otoliths, known as ‘annuli’ . Annuli, once de- posited, are a permanent feature in an otolith and can be counted, like the rings in trees, to determine the age of a fish in years. Recently, it was discovered that there are much finer, microstructural growth increments in otoliths, which can also be used to age fishes*. These fine increments, rarely more than 20 pm wide, also consist of paired zones differing in relative pro- tein content. However, they often form daily, apparently as a result of circadian changes in metabolic activity3. Consequently, in theory, any fish can be aged to the day. In practice, the technique works best for larvae and juveniles. Increment width is proportional to growth rate, which declines as fish age. Conse- quently, there is an upper limit, at about 200 days, beyond which fish cannot usually be aged easily using light microscopy. The possibility of age determina- tion with a resolution in days, even if restricted to larvae and juveniles, has immense implications for the study of the demography of reef- associated fishes. Three types of data can be produced. First, an exact growth rate and date of birth can be determined for each individual. De- tailed comparisons of rates of growth and survival and of recruit- ment patterns between cohorts, reefs, years or species are suddenly not only possible, but even easy. Finally, these developmental data can be recovered for even the oldest individuals in a population. Otoliths are not extensively remineralized in fishes, except under exceptional cir- cumstances The growth increments that were laid down during its larval stage are still present in the otoliths of a 20 year old grouper. It should be possible to reconstruct, for example, inter-annual variations in rates of lar- val growth for periods into the past up to the age of the oldest fishes in a population. Not only could such data be used to study the natural variabil- ity of early life history events, but they also permit interspecific and regional comparisons over a long time span and can be combined with historical data on tropical oceano- graphy to test the effects of environ- mental factors on larval ecology. Despite such possibilities and re- cent enthusiasm for the technique, scientists were slow to apply analy- sis of otolith microstructure to reef fishes. The seminal study was that by Brothers and MacFarland6 on the western Atlantic haemulid, Haemu- Ion flavolineatum. Although pre- vious studies had used daily in- crements to establish growth trajectories for tropical fishes, Brothers and MacFarland’s detailed, quantitative analysis of the rela- tionship between increment spacing and the ecology and development of juvenile fishes in the field led to a widespread realization of the poten- tial of the technique. In H. flavo- lineatum, increment spacing varies with developmental stage, from planktonic larva through to school- ing benthic juvenile. Conspicuous

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TREE vol. 3, no. 3, March 1988

Otolith Microstructure and the Demography of Coral Reef Fishes Secondly, because otolith growth

is generally isometric, patterns of growth in the otolith can be used to measure retrospectively patterns of growth in the fish themselves. The link between growth rate and incre- ment width has significance well beyond the obvious potential for, as an example, correlating changes in growth of herbivores with variations in algal production. Wherever growth rates differ consistently be- tween life history stages, the dura- tion of these stages (and dates of transition) can be determined for all individuals in a population. Prelim- inary work indicates that diverse factors (food ration, temperature, activity levels) can affect the spacing and composition of growth incre- ments4r5; if the interactions of these factors can be resolved into discrete signals, it may well be possible to reconstruct the environmental and developmental history of a fish, from egg to senility, by examination of its otoliths.

R.E. Thresher (

The ecology of planktonic larvae - particularly the extent to which lar- vae are transported between habitat patches-is a poorly known aspect of the life history of tropical reef- associated fishes. The logistic diffi- culties of studying larvae in the field and of relating their behavior in cap- tivity to field conditions have greatly impeded the progress of research. However, a recently developed tech- nique, based on analysis of the microstructural growth elements in otoliths (‘ear stones’), may prove a powerful means of assessing the early life history of reef fishes and relating it to population and com- munity dynamics.

As in most marine organisms, the life history of coral reef fishes typi- cally consists of two wholly different stages: juveniles and adults are relatively sedentary and live on or close to the reef; their larvae are, with few exceptions, planktonic. The duration of the planktonic stage can be as long as a year; during it, the larvae are carried by currents over distances at which we can only guess. The unknown extent of larval transport is the biggest single barrier to our understanding of the de- mography of coral reef fishes. We have virtually no idea of the direction or frequency of exchange of offspring between habitat patches for any species at any location. In- deed, our knowledge of any aspect of the ecology of larval reef fishes is fragmentary. Without more detailed information on this stage of their life history, the prospects of understand- ing the dynamics of reef fish com- munities are slim.

Attempts to examine the dispersal and general ecology of larval reef fishes have had little success. The small size of the larvae, their fragility and their relative rarity in a large ocean preclude conventional tagging or direct study in the field. The spatial scale of events critical to the survival of a 5 mm-long larva in the water column is orders of magnitude less than we can routinely sample; larvae operate at a scale where it becomes difficult for oceanog- raphers to discriminate between sampling error and real environmen-

R.E. Thresher is at the CSIRO Marine Laborator- ies, GPO Box 1538, Hobart 7001, Tasmania, Australia.

78

tal patchiness. Studies in captivity are seldom technologically feasible, due to difficulties in keeping larvae alive, and are always difficult to interpret. Confinement inevitably affects behavior, distorting the rela- tive importance of factors critical to survival’.

Otolithic aging A new analytic technique, based

on the examination of the fine struc- ture of otoliths, offers dramatic promise for investigations of larval and juvenile ecology. Otoliths (‘ear stones’) are small calcareous struc- tures that float freely in the semi- circular canals of fishes, where they are important in hearing and the de- tection of orientation and accelera- tion. Otoliths grow more or ‘less isometrically by accretion of calcium carbonate in a proteinaceous matrix. It has long been known that annual cycles of slow and fast growth pro- duce coupled zones of protein-rich and protein-poor material in otoliths, known as ‘annuli’. Annuli, once de- posited, are a permanent feature in an otolith and can be counted, like the rings in trees, to determine the age of a fish in years.

Recently, it was discovered that there are much finer, microstructural growth increments in otoliths, which can also be used to age fishes*. These fine increments, rarely more than 20 pm wide, also consist of paired zones differing in relative pro- tein content. However, they often form daily, apparently as a result of circadian changes in metabolic activity3. Consequently, in theory, any fish can be aged to the day. In practice, the technique works best for larvae and juveniles. Increment width is proportional to growth rate, which declines as fish age. Conse- quently, there is an upper limit, at about 200 days, beyond which fish cannot usually be aged easily using light microscopy.

The possibility of age determina- tion with a resolution in days, even if restricted to larvae and juveniles, has immense implications for the study of the demography of reef- associated fishes. Three types of data can be produced. First, an exact growth rate and date of birth can be determined for each individual. De- tailed comparisons of rates of growth and survival and of recruit- ment patterns between cohorts, reefs, years or species are suddenly not only possible, but even easy.

Finally, these developmental data can be recovered for even the oldest individuals in a population. Otoliths are not extensively remineralized in fishes, except under exceptional cir- cumstances The growth increments that were laid down during its larval stage are still present in the otoliths of a 20 year old grouper. It should be possible to reconstruct, for example, inter-annual variations in rates of lar- val growth for periods into the past up to the age of the oldest fishes in a population. Not only could such data be used to study the natural variabil- ity of early life history events, but they also permit interspecific and regional comparisons over a long time span and can be combined with historical data on tropical oceano- graphy to test the effects of environ- mental factors on larval ecology.

Despite such possibilities and re- cent enthusiasm for the technique, scientists were slow to apply analy- sis of otolith microstructure to reef fishes. The seminal study was that by Brothers and MacFarland6 on the western Atlantic haemulid, Haemu- Ion flavolineatum. Although pre- vious studies had used daily in- crements to establish growth trajectories for tropical fishes, Brothers and MacFarland’s detailed, quantitative analysis of the rela- tionship between increment spacing and the ecology and development of juvenile fishes in the field led to a widespread realization of the poten- tial of the technique. In H. flavo- lineatum, increment spacing varies with developmental stage, from planktonic larva through to school- ing benthic juvenile. Conspicuous

TREE vol. 3, no. 3, March 1988

transition points in increment width appear to reflect changes in physiol- ogy, morphology and behavior dur- ing ontogeny. The correspondence between otolithic features and de- velopmental stages documented by this study, along with subsequent work primarily by Brothers and his CD-workers, has resulted in a general concept of ‘settlement marks’ in oto- liths: an abrupt change in increment width and optical density that, for many taxa, appears to indicate the end of a planktonic larval stage and the onset of a benthic juvenile one7r8. Through the use of such a mark, and by counting pre- and post-transition increments, it has already been possible to establish, in unpre- cedented detail, temporal and spatial patterns of settlement and juvenile survival in a western Atlantic labridg, the relationship between planktonic duration and breadth of distribution for a variety of Indo-west Pacific specieslo, and regional differences in pianktonic larval durations among closely related taxa”,‘*.

The problem of validation, and other limits to the technique

Such studies make one common, critical assumption: that the in- crements being counted form on a daily basis, Unfortunately, there are two reasons to question the validity 0’: this assumption. First, at least one widely cited study, on a temperate species, reports that the number of increments deposited each day varies with growth rate in larval fiches13. This disturbing result has not been widely substantiated, and nay be the result of methodological problems specific to that study14. Nonetheless, it may be dangerous to assume that increments always form daily, even where their structure is unambiguous. The second reason fcr caution is that otolith microstruc- ture is often far from unambiguous. A ong with the presumed daily incre- ments, there are often ‘sub-daily’ elements, ‘growth hesitations’, in- tersecting growth planes, and optical interference. Indeed, otolith micro- structure is often sufficiently com- plex that without being able to see far oneself the structural elements being counted, it is difficult to evalu- ate the results of published studies.

The obvious and essential solution is to validate the aging technique each time it is used. In practical terms, there are four ways to test directly the assumption of daily in- crement formation: (I 1 examination of larvae of known age reared in captivity; (2) back-calculation to a known spawning date on the basis of estimated age and the date of cap-

Otoliths of larval and ven/ small juvenile fishes can be examined microscopically without special preparation, by focusing on the appropriate growth plane. Otoliths from larger fishes, however, have to be cut and polished in order to expose the structure deposited during early development. In transmitted light microscopy, growth increments are conspicuous as paired light and dark elements, each pair forming a complete shell around the otolith (or a ring when the otolith is sectioned). Increments formed prior to settlement to the reef (Pre-s) are often, as in this species of lizardfish (Sawida sp,), distinctly wider than those that form after settlement (Post-s). Between the two growth regions there is a ‘settlement mark’, which in this species consists of a zone of confused growth and poorly defined structure

ture; (3) measurement of hourly changes in the width of the out- ermost increment, which should vary consistently with time of day; and (4) tag and recapture studies, in which the otolith is marked.

All these approaches, however, have procedural weaknesses. Growth of larvae in captivity doubt- less incorporates effects of numer- ous artifacts, and increments observed in laboratory-reared speci- mens may not often match those of wild-caught material, or form under similar conditions. Back-calculation to a spawning date requires strongly episodic and discrete spawning events, ideally over limited time spans. This seldom occurs with reef fishes. Even where species have dis- crete lunar or semi-lunar spawning cycles, these cycles can be out of phase for populations within short distances of each other (R.C. May, MSc thesis, University of Hawaii, 1967). Analysis of diurnal changes in the width of the marginal increment, although reported successful in the Iiterature15, is, in my opinion, nearly impossible for most species. Curva- ture along the edge of the otolith makes it difficult to measure any width confidently unless the margin- al increment is exceptionally wide. Of the four direct tests listed, that involving tagging and recapture of marked fishes is potentially the most powerful. Recaptures may not be an unbiased sample of the tagged population, however, and otolith

marking may itself produce effects on growth and otolith formation.

Beyond these logistic problems, validation studies reported to date are not, in my opinion, sufficiently rigorous. Two conceptual weak- nesses seem to be pervasive. A typical validation study for larvae, for example, would proceed as follows. Larvae are reared in the laboratory; they are sacrificed at various known ages; their otoliths are extracted and the increments in these otoliths counted. Increment number is re- gressed against known age. If things have gone well, a high value of r* is obtained, the intercept is around 0 and, most importantly, the slope of the least squares line is not signifi- cantly different from 1.0. In sloppy studies, the last ‘proves’ that in- crements form daily; in more careful studies, it is only consistent with the hypothesis of daily increment forma- tion. In most studies, it proves only that the data obtained are not suffi- cient to discriminate between the observed trend line and the null hypothesisle. Most studies thus far published do not specify the power of a data set to falsify the null hypothesis. Given the typically small sample sizes used to validate aging studies, type II errors (the error of accepting a false null hypothesis) are doubtless common in the literature. These errors are overlooked because the conclusions are consistent with expectations and, to put it bluntly, with those desired.

79

TREE vol. 3, no. 3, March 7988

Even when the power of statistics is calculated, however, a validation like that outlined above is nonethe- less ‘weak’. It deals with the mean response of the population under some sort of average conditions. Under normal conditions most, if not all, fishes may well deposit growth increments daily. But the norm is not necessarily of interest. Before daily increment formation can be used to unravel elements of larval or juvenile ecology, the limits of the technique must be determined. Beyond what range of daily rations, water temper- atures or salinities does daily aging cease to be usable? How much and in what fashion does the relationship between increment width (optical density, composition, substructure) and environmental variables deviate from linearity, particularly near the upper and lower limits of tolerance? And, crucially, is it possible to tell from the structure of an otolith that estimated ages or growth rates for an individual are no longer accurate?

This is the essence of what I refer to as ‘strong validation’, a delin- eation of those circumstances be- yond which the technique cannot be applied to field specimens. A complete, strong validation is prob- ably impossible; there will always be some unknown variables that could distort otolith microstructure. But it is an approachable goal, particularly for those studies that rely heavily on analysis of otolith microstructure as their primary research tool. Yet de- spite several such studies, it has not yet been attempted for any reef fish species. Stronq validation normally

those done on larvae of some temperate fishes16f17. It also requires a great deal of time and effort. Work- ers, myself included, are enamored with the possibilities of the tech- nique for determining patterns of growth, dispersal and recruitment, and in virtually all cases must - or choose to - validate only weakly, bypassing more rigorous analysis in a rush to apply the technique. Until the limits of the technique are probed, there must remain uncer- tainty over results generated with it.

Epilogue It is easy to criticize a new tech-

nique. In this case, it is not the tech- nique that is at fault, however, but rather its uncritical application. Tech- nical constraints and the often con- fusing patterns of daily and sub-daily incremental structures in otoliths dictate an awareness of the limits of otolithic analysis. Within these limits, analysis of otolith microstruc- ture could well prove to be the most powerful tool yet developed to ex- amine aspects of the early life history of fishes. The easy applications, to studies of the growth and settlement patterns of post-larval and juvenile fishes, merely use otolithic aging as a convenience, one that makes possible synoptic studies without de- manding extensive effort in the field. The real power and promise of oto- lithic studies, however, lie in those applications that no amount of effort could duplicate, such as analysis of historical patterns of larval growth or reconstructing the environmental history of individual fishes. Retro-

life histories, as far back as the egg stage in some fishes, could rev- olutionize our understanding of the life histories of fishes, the links be- tween life history stages and the re- lationships between larval and post- larval ecology.

References 1 de Lafontaine, Y. and Leggett, W.C. (1986) Can. J. Fish. Aquat. Sci. 44, 54-65 2 Panella. G. (1971) Science 173.24-27 3 Mugiya, Y., Watabe, N., Yamanda, J., Dean, J.M., Dunkelberger, D.G. and Shimuzu, M. (1981) Comp. Biochem. Ph ysiol. 68A, 659-662 4 Campana, S.E. (1983) Can. J. Zoo/. 61, 1591-1597 5 Gutierrez, E. and Morales-Nin, B. (1986) J. Exp. Mar. Biol. Ecol. 103, 163-l 79 6 Brothers, E.B. and McFarland, W.N. (1981) Rapp. P-V. Reun. Cons. Int. fxplor. Mer. 178,369-374 7 Victor, B.C. (1982) Mar. Biol. 71, 203-208 8 Brothers, E.B., Williams, D.M. and Sale, P.F. (1983) Mar. Biol. 76, 319-324 9 Victor, B.C. (1986) Ecol. Monogr. 56, 145-160 10 Thresher, R.E. and Brothers, E.B. (I 983) Evolution 39,878-887 11 Thresher, R.E. (1985) Proc. Ecol. Sot. Aust. 14, l-5 12 Victor, B.C. (1986) Mar. Biol. 90, 317-326 13 Geffen, A.J. (1982) Mar. Biol. 71, 317-326 14 Campana, S.E. and Neilson, J.D. (1985) Can. J. fish. Aquat. SC;. 42, 1014-I 032 15 Tanaka, K., Mugiya, Y. and Yamada, J. (1981) fish. Bull. /US) 79,459-466 16 Rice, J.A., Crowder, L.B. and Binkowski, F.P. (1985) Trans. Am. Fish. sot. 114,532-539 17 McGurk. M.D. (1984) Fish Bull. (USI

requires experymental studies, like spectibe, detailed data on individual 82,113-120

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