PERIODICITY OF SEX CHANGE AND REPRODUCTION IN THE RED HIND, EPINEPHELUS GUTTA TUS, A PROTOGYNOUS GROUPER

Transcription

1 BULLETIN OF MARINE SCIENCE. 53(3): CORAL REEF PAPER PERIODICITY OF SEX CHANGE AND REPRODUCTION IN THE RED HIND, EPINEPHELUS GUTTA TUS, A PROTOGYNOUS GROUPER Douglas Y. Shapiro, Yvonne Sadovy and M. Angela McGehee ABSTRACT We examined the hypothesis that individual red hinds, Epinephe/us guttatus, evaluate future reproductive success and the best time to change sex from information available only within the annual spawning aggregation. Gonadal histology on individuals obtained from commercial catches biweekly over a 2.5 year period confirmed that the species is protogynous and that ripe individuals are found primarily during the aggregation months of January or February. A gonadal index peaked sharply in January. Thus, fish reproduced largely within the aggregation time. The sex ratio within the aggregation, 4.9 females per male, differed significantly from the sex ratio of 1.9 in inshore areas outside of the aggregation period. Individuals within the aggregation were significantly larger overall than individuals in inshore areas. Thus, for inshore populations, the information needed in theory to assess when to change sex is only available within the aggregation. However, histologically discernible transitional individuals were found uniformly throughout the year, not primarily soon after the aggregation, as our hypothesis predicted. Additional study of deep, offshore populations and further study of the earliest stages of sexual transition are needed to explain this discrepancy. According to sex allocation theory, individuals in hermaphroditic fishes should change sex whenever net future reproductive success would be higher for the opposite than for the existing sex (Charnov, 1982; Shapiro, 1989). Since the proximate mechanism controlling sex change, in all species for which there is evidence, is triggered by demographic or behavioral alterations within the social system, individuals presumably assess probable future reproductive success by processing information from interindividual interactions (Shapiro, 1988; Ross, 199). We use the term "assess" here in the evolutionary sense that natural selection will favor those individuals changing sex at the most advantageous moment. In many species of sequential hermaphrodites, individuals occupy relatively stable social systems for much of the year (Warner, 1984; Shapiro, 1987a). They thereby have continual access to information concerning, for example, local sex ratio, spawning rates, and dominance relationships, which are likely to be related to future reproductive success. The availability of such information is particularly clear in coral reef fishes that spawn daily or monthly over prolonged periods of the year. In contrast to these fishes, some sex-changing groupers spawn during short periods, sometimes lasting a week or less, that occur once yearly (Smith, 1972; Olsen and LaPlace, 1979; Colin et at, 1987; Shapiro, 1987b). For example, the Nassau grouper, Epinephelus striatus, forms spawning aggregations during 1 or 2 weeks of the year (Colin et at, 1987; Carter et at, in press; Colin 1992). During the remainder of the year aggregation members are widely scattered over many square kilometers of reef. This type of mating system poses the questions of whether individuals assess future reproductive success primarily during the short, aggregation period and how we could test this possibility. We would need to know with certainty that the species is a sequential hermaphrodite, that reproduction occurs predominantly within the aggregation both in time and in space, that relevant information, such as mating sex ratio, is 1151

2 1152 BULLETIN OF MARINE SCIENCE, VOL. 53, NO.3, 1993 available to individuals within the aggregation but not otherwise, and that sex change occurs in temporal proximity to the aggregation period. If all of these things were true and individuals returned to the same aggregation site each year, then it would be likely that individuals assessed the relative advantage of changing sex from information in the aggregation. The red hind, Epinephelus guttatus, is a reasonable candidate for examining how future reproductive success is evaluated in species spawning during restricted periods. Previous evidence indicates that the species is a female-to-male sex changer, i.e., a protogynous hermaphrodite (Smith, 1959; Burnett-Herkes, 1975; Sadovy et a1., 1992). In southwestern Puerto Rico, aggregations form during approximately 1 week each January (and, occasionally, 1 month later; Sadovy, Rosario and Roman, unpub1. data) at the edge of the insular shelf and contain clear internal structure, with individuals clustering spatially into small groups, many of which consist of one male and a handful offemales (Shapiro et ai., 1993). Spawning is likely to occur within these clusters (Colin et al., 1987). The purposes of this paper are to confirm that the red hind is protogynous, to describe simple aspects of the population in the area producing annual aggregations, e.g., size distribution of individuals and local sex ratios, to describe annual periodicity of reproduction and the temporal distribution of sex-changing individuals with respect to the occurrence of annual aggregations, and to discuss the relevance of these results for the issue of how individuals might assess the correct time to change sex. Since much of the information needed to discuss this issue is based on gonadal histology, e.g., to diagnose protogyny and to specify the periodicity of sex change and gonadal ripening, we include a description of gonad microstructure. METHODS A total of 69 specimens of E. gutta/us was obtained from commercial fishermen, who fished with hook-and-iine and traps in waters less than 25 m deep on the insular shelf in the vicinity of La Parguera, Puerto Rico, between November 1982 and March 1985, inclusive. Specimens were purchased for 29 consecutive months, bimonthly in 22 and monthly in seven. An additional 75 specimens were speared on three shallow reefs in inshore waters. All specimens were returned to the laboratory, weighed without gonads but with viscera intact to the nearest.1 g, and measured (standard length SL) to the nearest mm. Gonads were blotted dry and weighed to the nearest.1 mg. Histological preparations were made of all gonads (N = 65) which had not undergone substantial post-mortem autolysis by the time they arrived in the laboratory. Gonads were preserved in Davidson's fixative, either entire or in 5-mm-thick sections, embedded in paraffin, sectioned at 7 ~m, and stained with Harris' hematoxylin and eosin. Preliminary histological evaluation indicated that gonads were at the same stage of maturation throughout their length. Subsequently, only a single, central transverse section was prepared for each gonad. Specimens obtained too long post-mortem for histology were sexed and staged by macroscopic examination of tissue pressed firmly between two microscope slides. Gonads were evaluated histologically mainly on the basis of the presence or absence of the following germ cell stages (Moe, 1969): gonia; stage 1 and 2 oocytes (previtellogenic and differing from each other primarily by size, shape, and number of surrounding follicular cells); stage 3 oocytes (early vitellogenic); stage 4 oocytes (large, filled with yolk); stage 5 oocytes (rare, hydrated, irregularly shaped); primary and secondary spermatocytes (differing from each other in size, staining characteristics, anrl number of cells per crypt); and sperm (spermatids and spermatozoa in crypts). Periodicity of reproduction was examined, in part, by monthly changes in a relative gonadal index (RGI). The commonly used gonadosomatic index (GSI) could not be used (DeVlaming et al., 1982) because the slopes of regressions relating gonad weight to body weight were not homogeneous among four stages of ovarian maturation (F'(3.) = 53, P <. I). An alternative index, RGI = W/Sbi, where W is gonadal weight, S is standard length, and b, is a parameter fitted to the data using a least squares regression for standard length on gonadal weight, was employed (Erickson et al., 1985). Using our data results, slopes of the regression proved homogeneous among the four stages of gonadal maturation (F~3,52) = 2.588, NS). Hence, a pooled regression was used, with b i estimated by b = 3.456, and differences among the four intercepts were tested with ANCOV A. Because the slopes of the log-

3 SHAPIRO ET AL.: PERIODICITY OF RED HIND REPRODUCTION AND SEX CHANGE I 12 (/) u:: u.. 1 a: w ld 8 :2 ::> z 6 14 FEMALE o MALE [l]) TRANSITIONAL 4 2 a STANDARD LENGTH (mm) Figure 1. Size-frequency distributions for male, female, and transitional Epinephe/us guttatus from inshore waters of southwestern Puerto Rico between November 1982 and March Specimens from different years were pooled by month. transformed model were homogeneous among gonadal stages, the intercepts should differ. Similar slopes and homogeneous intercepts would indicate either a constant reproductive condition or erroneous staging of gonadal development (Erickson et ~1., 1985). The intercepts differed significantly (F,;{'.524)= , P <.1). Thus, the RGI used in our results was RGI = W/Sb, with b = Frequency distributions of standard lengths did not differ significantly from normal (r q =.99, NS, according to Q-Q correlations), and variances in SL of males, females, and transitionals were homogeneous (Bartlett's test, x 2 = 1.76, NS). Thus, lengths of sexes were compared with ANOV A. RESULTS Population Characteristics. -Of the 69 specimens from commercial fishermen, 531 were females, 61 were males, and 17 were transitionals in the process of changing sex (see below). Excluding transitionals, the overall sex ratio was 8.7 females per male. Sex ratios differed significantly, however, between reproductive and non-reproductive periods. During the 3 January aggregation months ( ), when specimens were captured primarily at aggregation sites at the edge of the insular shelf, the sex ratio from all 3 years combined was 4.7 females per male, whereas during the remainder of the year, when specimens were fished primarily in more inshore areas, it was 1.8 females per male (x2 = 8.1, P <.1). The 75 specimens speared from three shallow, inshore patch reefs outside of the aggregation period represented exhaustive samples from the speared areas. All 75 specimens were female. Overall, standard lengths ranged from mm for females, mm for transitionals, and mm for males (Fig. 1). Specimens in the three sexual categories were not of equal length (ANOV A, F = 73.42, P <.1) with females (217 ± 3 mm, mean ± SD) significantly smaller than transitionals (243 ± 33 mm) and males (265 ± 33 mm), and transitionals significantly smaller than males (Scheffe F-test, P <.5 for each for the three comparisons). Females were significantly smaller (t = 3.51, df= 529, P <.1) during the

4 1154 BULLETIN OF MARINE SCIENCE, VOL. 53, NO.3, 1993 non-breeding months (215 ± 3 mm, mean ± SD) than in January (226 ± 26 mm), primarily because the aggregation did not contain females smaller than 182 mm SL, while females as small as 123 mm SL were found at other times. Males did not differ in size between breeding and non-breeding months (t = 1.7, df = 59, NS). Description of Gonads. - Outside of the spawning season, gonads of both sexes occupy a small volume, are pale cream in color, and are compact with rounded margins. Immediately before, and during the spawning season, gonads are much larger. Ovaries, which are then cream or pale yellow in color and contain oocytes that are clearly visible through the gonad wall, can readily be distinguished from ripe testes, which are oval in shape, white, and exude milt when cut. During the reproductive period, the genital papillae of females were distended and vascularized, and could be readily distinguished from the papillae of males. Anteriorly, ovaries and testes form two lobes that unite posteriorly anteriad to the genital pore. In cross-section, gonadal tissue of both sexes form lamellae that surround a central lumen, with an alamellar region ventrally (Fig. 2a, b). Gonads are enclosed by muscle layers and a tunica albuginea, with blood vessels entering dorsally via the mesorchium. In males, sperm sinuses may be extensive and surround both the lamellar and alamellar areas. Females were categorized into four classes, FI-F4, according to the degree of maturation of their ovaries. The gonads of inactive females, Fl, were compact with tightly aligned lamellae and stage 1-2 oocytes deeply embedded in connective tissue. This class included immature females and late, post-spawning females whose gonads had markedly regressed cytologically between annual spawning periods. The gonads of these two female stages could not be distinguished. However, the smallest size class in which 5% of individuals are known to be mature is 175 mm SL (Sadovy, Rosario and Roman, unpubl. data). Since only approximately 4 to 531 females (7.5%) were smaller than this size class (Fig. 1), estimates of reproductive sex ratio will be minimally affected by our procedure of including Fl females in the analyses. The gonads of mature females, F2, were either maturing (Wallace and Selman, 1981), with stage 1-2 oocytes predominating and occasional stage 3 oocytes present, or regressing after the spawning period, with stage 1-2 oocytes present together with either oftwo signs of prior spawning activity. The first sign was degenerating stage 4-5 oocytes, i.e., cells or cell remnants retaining sufficient semblance to oocytes that we could stage them, and occurring soon after the spawning period. We did not consider yellow bodies, which were observed in all females smaller than 17 mm SL, to be reliable indicators of oocyte degeneration (Sadovy and Shapiro, 1987). The second sign of prior spawning were prominent bundles of muscle and connective tissue surrounding blood vessels (Fig. 2c), which appeared characteristically 4-8 days, and generally less than 16 days, after the aggregation period (Fig. 3). These connective tissue bundles became evident following the expansion and subsequent post-spawning collapse of ovarian lamellae. The tunica albuginea of post-spawning ovaries was generally quite thick. The gonads of ripe females, F3, contained stage 1-4 oocytes and an occasional stage 5 oocyte. The ovaries were large and the tunica albuginea thin. Ovaries of immediately post-spawning females, F4, contained predominantly post-ovulatory follicles and early stage degenerating oocytes. Few stage 3-4 oocytes were generally present. F4 gonads were clearly distinguished from late, post-spawning, F2 ovaries by the large number of post-ovulatory follicles in the former and the absence of intact, stage 4 oocytes in the latter. Few gonads were classified as F4, indicating that this stage, as defined, was probably brief.

5 SHAPIRO ET AL.: PERIODICITY OF RED HIND REPRODUCTION AND SEX CHANGE 1155 Figure 2. Cross-sections of gonads of Epinephelus guttatus. a) Testis with membrane-lined central lumen and lamellar structure of an M2 male, 281 mm SL. b) Ovary with central ovarian cavity and lamellae of an FI female, 214 mm SL. c) Ovary with muscle and connective tissue bundles in an F2 female, 332 mm SL. d) Transitional gonad consisting of oocytes, occasional crypts of spermatogenic cells, and muscle and connective tissue bundles in an individual, 193 mm SL. e) Transitional gonad containing about equal amounts of ovarian and testicular tissue in a specimen 275 mm SL. f) Detail of (e) at higher magnification. Symbol key: B: muscle and connective tissue bundle; Bv: blood vessel; CL: central lumen; L; lamella; Ocl-2: stage 1-2 oocytes; Sc: spermatocytes. Scale bar =.25 mm for (a)-(e) and.25 mm for (f). Seventeen specimens had gonads containing both ovarian and testicular tissue in varying proportions. Many were essentially female in configuration, with various stages of oocytes, usually stage 1-2, in lamellae, together with isolated crypts of stage 1-2 spermatocytes and/or sperm (Fig. 2d). Others contained approximately equal proportions of testicular and ovarian tissue, with no sperm sinuses

6 1156 BULLETIN OF MARINE SCIENCE, VOL. 53, NO.3, L (J) w ii: ~ 15 u.. a: w m 1 ::E :J 5 o JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC MONTH Figure 3. Temporal distribution of ovaries containing muscle and connective tissue bundles in Epinephelus guttatus specimens between November 1982 and March Specimens from different years were pooled by month. (Fig. 2e, f). One specimen contained stage 3-4 oocytes, indicating sexual maturation, and crypts of spermatogenic cells. Eight of the 17 specimens, measuring mm SL and captured January-July, contained muscle and connective tissue bundles (Fig. 2d) indicative of prior spawning as a female. The remaining nine specimens, measuring mm SL and caught June-December, lacked such bundles, probably because bundles rarely persisted in post-spawning ovaries beyond June (Fig. 3). We called these 17 specimens transitional gonads. We did not include in this category predominantly testicular gonads containing scattered stage 1-2 oocytes, because such oocytes often appear in the testes of non-sexchanging species and are thus not reliable indicators of sexual transition (Sadovy and Shapiro, 1987). Finally, the gonads of males were placed into four classes, MI-M4, according to the stage of maturation of constituent testicular tissue. The testes of inactive males, M1, were distinguished by stage 1-2 spermatocytes, with only occasional clumps of sperm. Spermatogenic tissue was compact and arrayed in discrete crypts. Sperm sinuses, either with or without sperm, were present in the gonadal tunica; gonia were apparent. Testes of mature males, M2, contained all stages of spermatogenesis in approximately equal proportions. Gonia were not apparent. Sperm was often present in sperm sinuses. The testes of ripe males, M3, consisted predominantly of sperm. Lobular walls surrounding spermatogenic crypts were often broken down, creating large lumina filled with sperm. Lumina appeared to form initially in central regions of the testis and to expand centripetally. The testes of post-spawning males, M4, were large and flaccid, and contained numerous empty lumina. Internal structure was disorganized, contained clear proliferations of gonial cells, and generally lacked later stages of spermatogenesis. Sperm sinuses were discernible but contained little or no sperm.

7 SHAPIRO ET AL.: PERIODICITY OF RED HIND REPRODUCTION AND SEX CHANGE lis X W ~ 15...J < < C) W > w 75 a: z ~ 5 w ::E DATE Figure 4. Monthly medians of the relative gonadal index for female Epinephelus guttatus. Numbers at and around peak values are sample sizes. Solid lines and dotted lines connect bimonthly and monthly samples, respectively. Periodicity of Spawning and Sex Change. - The relative gonadal index (RGI) for ovaries (Fig. 4) was not homogeneous throughout all months ofthe year (Kruskal- Wallis analysis of variance, H = 262.5, df = 11, P <.1, when data from all years were pooled by month), with sharp peaks in January of each year and, in 1985, a second peak in February. On Dunn's multiple range comparison test, the RGI for December-January were significantly larger than RGI values during the remainder of the year. Similarly, the RGI values for testes were not homogeneous over all months of the year (H = 48.8, df= 1, P <.1), with values of from March-November, in December and February, and a peak of 19.3 in January. The relative proportion of ovaries, and testes, falling into maturation classes F1-F4, and M1-M4, respectively, was not constant throughout all months of the year (x2 = 37.7, df = 33, P <.1 for ovaries; x 2 = 17.5, df = 3, P <.1 for testes). For example, of the 59 ripe (F3) ovaries, 33 of them (56%) fell in January and 19 (32%) in December, with one in November, five in February, and one in March. Of 29 ripe testes (M3), 21 were in January (72%), three in December and five in February. The degree of maturation, as indicated by gonadal stages F1-F3 and M1-M3, correlated significantly positively with RGI both for females (Fig. 5; Spearman r =.58, P <.1) and for males (r =.68, P <.1). Thus, the RGI was an accurate indicator of gonadal ripeness, as judged histologically, and both measures indicated that spawning occurred almost entirely during the aggregation period in January. Transitional individuals were found uniformly throughout the year, with no

8 1158 BULLETIN OF MARINE SCIENCE, VOL. 53, NO.3, o 4 >< 35 W 3...J «25 «CJ 2 w > 15 ~ W 1 a:. 5 o ~ o 8 o I8 i GONADAL MATURATION CLASS 2 3 Figure 5. The relative gonadal index of female Epinephe/us gutta/us in three gonadal maturation classes (N = 284, 114, and 59 for classes 1,2, and 3, respectively). tendency to cluster in the months immediately after the aggregation period in January (Fig. 6). The amount of spermatogenic proliferation in these individuals was not related to time elapsed after spawning. DISCUSSION Gonadal histology demonstrates clearly that the red hind is protogynous. All testes contained a membrane-lined central lumen that was not used in the transport of sperm, and testicular tissue was often arrayed in lamellae. Transitional gonads displayed a wide range of relative proportions of ovarian and testicular tissue in various stages of maturation, suggesting a developmental sequence from ovary to testis. All transitional specimens collected between January and June contained muscle and connective tissue bundles, indicating earlier reproductive function as a female. These characteristics indicate that testes have transformed from an earlier, functional ovarian stage (Sadovy and Shapiro, 1987) and confirm previous indications of protogyny in this species (Smith, 1959; Burnett-Herkes, 1975; Sadovy et ai., 1992). The sharp peaks in RGI in January of each year, for both sexes, with a secondary peak in February of one year, strongly suggest that reproduction in the red hind is limited to the aggregation period. This interpretation is supported by the temporal occurrence of ripe gonads, which predominated in January, with smaller numbers in the 1-2 months preceding and following January. Whether spawning itself is confined within aggregations spatially remains an unanswered question. The few instances of spawning that have been observed in

9 SHAPIRO ET AL.: PERIODICITY OF RED HIND REPRODUCTION AND SEX CHANGE en --l < o 2 ~ (j) < a: I- u. o a: ~ 1 :::2: :::l o JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC MONTH Figure 6. Temporal distribution of transitional Epinephelus guttatus between November 1982 and March Specimens from different years were pooled by month. the red hind occurred within an aggregation, but observations of individuals during the aggregation period but spatially outside of any aggregation have been few in number (Colin et al., 1987). In some other groupers in which aggregations have been massive, aggregations are likely to be the prime location of mating (Colin, 1992). We conclude from these data that red hinds spawn almost exclusively during the aggregation period and, inferring from data on the Nassau grouper (Carter et al., in press; Colin, 1992), probably predominantly within the aggregation itself. The relevant values of parameters to assess the best time to change sex, e.g., spawning sex ratios, relative size of males and females, the quality of behavioral interactions, should be those found during the aggregation. For the inshore population, social structure outside of the aggregation period differed markedly from structure within the aggregation. The entire population of three patch reefs, which included adult individuals, was female, with individual home ranges varying widely in size and overlapping irregularly with the home ranges of other individuals (Garcia-Moliner, 1986; Shapiro et al., in press). In contrast, aggregations were characterized by overall sex ratios approximating five females per male and by spatial clusters containing a small number of females or one male and several females (Shapiro et al., 1993). Thus, for individuals living in inshore regions, parameter values we expect them to use to evaluate when to change sex would not be available except within the aggregation. This argument leads us to predict that sex change by individuals living in inshore areas should be initiated within or soon after the dissolution of the annual aggregation. Our prediction assumes that the parameters theoretically playing an evolutionary role in determining the timing of sex change are identical or coterminal with the parameters causally initiating sex change. Because sex change, once initiated, is generally completed within 9-18 days (Sadovy and Shapiro, 1987),

10 116 BULLETIN OF MARINE SCIENCE, VOL. 53, NO.3, 1993 transitional individuals should be found predominantly in the first few months after the aggregation. However, histologically transitional individuals were found without temporal pattern throughout the year. There are five possible interpretations of these results. First, sex change is initiated well before the subsequent, variable appearance of histologically recognizable changes in the gonad in this species. There is no apparent advantage to individuals completing sex change soon after the aggregation ends; they will not spawn again for another year and the absence of social structure in inshore areas indicates no behavioral advantage to becoming a male quickly. Evaluating this possibility would require examination of large numbers of post-spawning females using genetic, immunological, or physiological probes to detect early stages of sex reversal. Second, grouper juveniles tend to recruit in shallow, inshore waters and move into deeper areas as they grow (Moe, 1969; Keener et ai., 1988; Sadovy, Rosario and Roman, unpubl. data). When the spawning aggregation ends, sex changing individuals may not enter inshore areas, where most of our commercially obtained specimens were caught, but move offshore to deeper, unsampled waters to join larger fish. I[so, then we may have sampled only aberrant, transitional individuals for whom both the timing of sex change and the tendency to resume inshore existence was atypical. The small number of transitional specimens, representing only 2.8% of histologically examined individuals, the smaller size of individuals in inshore areas than in the aggregation itself and the positive correlation between body size and depth in other studies (Sadovy, Rosario and Roman, unpubl. data) support this interpretation. Sampling in deeper, offshore areas would be required to test this possibility. The third interpretation is that individuals do not assess the best time to change sex from information within the aggregation. The only data we have on sex ratios and social structure outside of the aggregation period concerns inshore areas. If the deeper population resides year-round in behavioral clusters whose structure and sex ratio match those found within the aggregation, then information on when to change sex would be available to them throughout the year. Again, intensive study of the deeper population is needed to address this possibility. The fourth possibility is that sex change is not mediated by behavioral cues available only within the aggregation but by attaining a critical developmental stage that might occur at any time of the year. In a separate study, placing a male and six female E. guttatus together in underwater cages, and then removing the male, failed to induce females to change sex (Shapiro, Figuerola, McGehee, Sadovy and Wingfield, unpubl. data). While this result provides no evidence that sex change is developmentally induced, it does raise the possibility that behavioral cues might not control it, at least within the 18-week time frame of the experiment. The fifth possibility is that our data on sex ratios are inadequate to conclude that information on future reproductive success is only available to inshore individuals within the spawning aggregation. Presumably, the sex ratio of interest for a fish to "assess" the pros and cons of changing sex would be the spawning sex ratio in future aggregations. That sex ratio would depend upon the current spawning sex ratio within the aggregation, the proportion of current adults surviving to spawn in future years, and the proportion of current, inshore juveniles surviving to mature and enter successive aggregations. The present study does not provide such predictive, prospective information. In conclusion, the red hind population in our study reproduces primarily during the aggregation period in January of each year, with occasional reproduction during a portion of a second month, probably spatially within the aggregation

11 SHAPIRO ET AL.: PERIODICITY OF RED HIND REPRODUCTION AND SEX CHANGE 1161 itself. The species is protogynous. Overall, sex ratios and size distribution of individuals differed significantly between inshore areas outside of the aggregation period and the aggregation. Hence, information needed evolutionarily for inshore individuals to assess the best time to change sex is probably only available within the aggregation. Our hypothesis that individuals assess the best time to change sex within the spawning aggregation and actually initiate sex change shortly thereafter led to the prediction that transitional individuals should occur predominantly soon after the aggregation. This prediction was not supported by the evidence. Inshore, transitional individuals were rare and found during most months of the year. Until more is known about the sex ratio and social structure of offshore individuals and about early stages of sex change, we can not choose among alternative explanations for this discrepancy. ACKNOWLEDGMENTS We thank G. Garcia-Moliner for help processing and reading gonadal histology. Work was done in the Department of Marine Sciences, University of Puerto Rico, Mayagiiez, Puerto Rico, and was supported by NOAA National Sea Grant Program Office (NSGCPO), Department of Commerce, under Grant No. UPR-SG 4-F (Project No. R/LR-6-87-DSP2), the NOAA Undersea Research Program, and NIH grant S6RR-813. The U.S. Government is authorized to produce and distribute reprints for governmental purposes, notwithstanding any copyright notation that may appear hereon. LITERATURE CITED Burnett-Herkes, J Contribution to the biology of the red hind, Epinephelus guttatus, a commercially important serranid fish from the tropical western Atlantic. Ph.D Dissertation, University of Miami, Coral Gables. 154 pp. Carter, J., G. J. Marrow and V. Pryor. In press. Aspects of the ecology and reproduction of Nassau grouper, Epinephelus striatus. off the coast of Belize, Central America. Proc. Gulf Carib. Fish. Inst. 43. Charnov, E. L The theory of sex allocation. Princeton University Press, Princeton. 355 pp. Colin, P. L Reproduction of the Nassau grouper, Epinephelus striatus (Pisces: Serranidae), with relationship to environmental conditions. Environ. BioI. Fishes 34: , D. Y. Shapiro and D. Weiler Aspects of the reproduction of two groupers, Epinephelus guttatus and E. striatus in the West Indies. Bull. Mar. Sci. 4: DeVlaming, V., G. Grossman and F. Chapman On the use of the gonosomatic index. Compo Biochem. Physiol. 73A: Erickson, D. L., J. E. Hightower and D. Grossman The relative gonadal index: an alternative index for quantification of reproductive condition. Compo Biochem. Physiol. 81A: Garcia-Moliner, G. E Aspects of the social spacing, reproduction and sex reversal in the red hind, Epinephelus guttatus. M.Sc. Thesis, University of Puerto Rico, Mayagiiez. 14 pp. Keener, P., G. D. Johnson, B. W. Stender, E. B. Brothers and H. R. Beatty Ingress ofpostlarval gag, Mycteroperca microlepis. through a South Carolina barrier island inlet. Bull. Mar. Sci. 42: Moe, M. A., Jr Biology of the red grouper, Epinephelus moria (Valenciennes), from the eastern Gulf of Mexico. Aorida Dept. Nat. Resources Prof. Pap. Ser. 1: Olsen, D. A. and J. A. LaPlace A study of a Virgin Island grouper fishery based on a breeding aggregation. Proc. Gulf Carib. Fish. Inst. 31: Ross, R. M The evolution of sex-change mechanisms in fishes. Environ. BioI. Fishes 29: Sadovy, Y. and D. Y. Shapiro Criteria for the diagnosis of hermaphroditism in fishes. Copeia 1987: , M. Figuerola and A. Roman Age and growth of red hind, Epinephelus guttatus. in Puerto Rico and St. Thomas. Fish. Bull. 9: Shapiro, D. Y. 1987a. Differentiation and evolution of sex change in fishes. BioScience 37: b. Reproduction in groupers. Pages in J. J. Polovina and S. Ralston, eds. Tropical snappers and groupers: biology and fisheries management. Westview Press, Boulder, Colorado.

12 1162 BULLETIN OF MARINE SCIENCE, VOL. 53, NO.3, Behavioural influences on gene structure and other new ideas concerning sex change in fishes. Environ. BioI. Fishes 23: Sex change as an alternative life-history style. Pages in M. N. Bruton, ed. Alternative life-history styles of animals. Kluwer Academic Publishers, Dordrecht. ---, G. Garcia-Moliner and Y. Sadovy. In press. Social system of an inshore population of the red hind grouper, Epinephelus gultatus. Environ. BioI. Fishes. ---, Y. Sadovy and M. A. McGehee Size, composition, and spatial structure ofthe annual spawning aggregation of the red hind, Epinephelus gultatus (Pisces: Serranidae). Copeia 1993: Smith, C. L Hermaphroditism in some serranid fishes from Bermuda. Pap. Michigan Acad. Sci. 44: A spawning aggregation of Nassau grouper Epinephelus striatus (Bloch). Trans. Am. Fish. Soc. 11: Wallace, R. A. and K. Selman Cellular and dynamic aspects of oocyte growth in teleosts. Am. ool. 21: Warner, R. R Mating behavior and hermaphroditism in coral reef fishes. Am. Sci. 72: DATEACCEPTED: January 21, ADDRESSES:(D. Y.s. and M.A.M.) Department of Marine Sciences. University of Puerto Rico. Box 5, MayagUez, Puerto Rico 681; (Y.S.) Fisheries Research Laboratory, Department of Natural Resources, Box 3665, MayagUez, Puerto Rico 681; Present Addresses: (D. Y.S.) Department of Biology, Eastern Michigan University, Ypsilanti, Michigan 48197; (Y.S.) Department of oology, University of Hong Kong, Hong Kong.