BULLETIN OF MARINE SCIENCE, 31(2): 383-398, 1981 CORAL REEF PAPER

SEQUENCE OF COLORATION CHANGES DURING SEX REVERSAL IN THE TROPICAL MARINE ANTHIAS SQUAMIPINNIS (PETERS)

Douglas Y. Shapiro

ABSTRACT The color pattern of females of the protogynous fish Anlhias squamipinnis contrasted strongly with the color pattern of males. Both in the laboratory and in the field, females were induced to change sex by removing a male from their social groups. Color changes of the sex-reversing fish occurred in two phases: a short term phase, in which the basic pattern of male coloration was laid down in black or black-violet ; and a long term phase during which the black-violet coloration became progressively paler and more purely red or red- violet. During the short term phase in the laboratory, color changes appeared first on the pelvic fins or in the head-nape region, next on the dorsal fin or in the superior pectoral region, then on the caudal fin, and finally in the inferior pectoral region. The day-by-day sequence of changes was characterized by an early onset (3-6 days after male removal), a short interim period (2-11 days), and a short total time for completion (7-16 days after male removal). 75% of 44 sex reversals in the lab and in the field followed this typical sequence. Sequences in the field were slightly more prolonged than sequences in the laboratory. Two atypical sequences characterized the remaining sex reversals. Differences in the temporal characteristics of color change sequences appeared to be related primarily to differences in the social circumstances surrounding the initiation of sex reversal. It appeared likely, there- fore, that social factors could influence the sequence of coloration changes during sex re- versal.

Among protogynous marine , i.e., species in which individual females change into males during their adult life, many are sexually dichromatic (Atz, 1964; Reinboth, 1970; Smith, 1975; Robertson and Warner, 1978; Warner and Robertson, 1978). In these species, sex reversal involves an alteration of a fe- male's coloration, gonadal function, and behavior (Shapiro, 1979). Coloration may change approximately in synchrony with gonadal restructuring, as in certain Scaridae, Labridae. and Anthiinae (Choat, 1969; Popper and Fishelson, 1973; Robertson and Warner, 1978; Shapiro and Lubbock, in preparation), or may at least partiaIly precede (Gundermann, 1972; Roede, 1972; 1975) or succeed it (RandaIl and RandaIl, 1963; Quignard, 1966; Buckman and Ogden, 1973). Mor- phological changes in fish coloration, including those accompanying protogynous sex reversal, are generally thought to be hormonaIly controIled (Okada, 1962; Reinboth, 1962b; reviews by Odiorne, 1957; Fujii, 1969). While the color changes foIlowing androgen treatment of females have been briefly described for several protogynous species (Stoll, 1955; Roede, 1972; Fishelson, 1975), the fuIl sequence of color changes in individual fish undergoing natural sex reversal has not received careful scrutiny. In the protogynous, Indo-Pacific serranid, Anthiinae fishes, external differences between males and females are seen generally in body size, fin length, and most strikingly, in coloration (Lubbock and Allen, 1978; RandaIl, 1979; Randall and Lubbock, in press). Anthias squamipinnis is a smaIl, shaIlow water, coral reef species which lives in stable social groups with a median sex ratio of eight adult females per male (Shapiro, 1977b). Sex reversal in a female can be initiated by the removal of a male from her social group (Fishelson, 1970; Shapiro, 1979). This species has served as the subject for a variety of studies on the ecological,

383 384 BULLETIN OF MARINE SCIENCE. VOL. 31, NO.2. 1981

social, and behavioral aspects of sex reversal (Fishelson, 1970; 1975; Gunder- mann, 1972; Popper and Fishelson, 1973; Shapiro, 1977a; b; 1979), but descrip- tions of its coloration remain limited (Peters, 1855; Klunzinger, 1884; Fowler and Bean, 1930; Smith, 1961; Popper and Fishelson, 1973). The aims of this paper are: (I) to describe in detail the coloration of male and female A. squamipinnis; (2) to describe the sequence of color changes accom- panying gonadal and behavioral sex reversal initiated by the removal of a male from a social group; and (3) to comment on changes in size and fin length. The sequence of color changes will be examined both in naturally occurring and in artificially established social groups.

MATERIALS AND METHODS Laboratory Study

The description of male and female coloration was based on approximately 20 males and 120females kept in laboratory tanks over a 3-year period and was modified to include variations from that pattern observed during five months of field study in the Red Sea and Indian Ocean. For a description of the color changes of sex reversal, four social groups, each containing one male and six adult females, were established in separate aquaria. After a 2-month baseline period the male was netted and removed from two of these groups while, in the other two groups, the male was netted and released. Thirty-five days later the male was removed from the groups first used as controls and a control manipulation performed in the originally experimental groups. This procedure was repeated until a total of eight males had been removed from the four groups and, in response, seven females had changed sex (Shapiro, 1977a; 1981). The aquarium size, illumination, water temperature, pH, and specific gravity, feeding routine, general maintenance, detailed methods of experimental proce- dure, and the gonadal histology and behavioral results of these experiments are described elsewhere (Shapiro, 1977a; 1979; 1981). The sequence of color changes shown by six of the seven sex-reversing fish and, in certain instances, by other females that changed sex in similar laboratory experiments, were described in detail. In each group, individual fish were distinguished and identified by peculiarities of size, hue, fin shape, scars, and eye stripe configuration. Prior to male removal all individuals were visually inspected every few days, through the glass wall of the aquarium, for any change in coloration. The day of male removal was counted as day zero. Subsequently, all individuals were inspected daily until coloration changes were noted on one female. Thereafter, this fish was inspected daily while other group mem- bers were inspected approximately on alternate days. For the sex-reversing fish, the day of appearance of the earliest detectable change in coloration was noted for five body regions which, in the male, show characteristic coloration: head-shoulders (the nape and the anterior part of the dorsal body contour to approximately below the sixth dorsal spine will be called the "shoulders" of the fish), pelvic fins, the posterior sixth of the dorsal fin, the superior and inferior regions of the pectoral fins, and the caudal fin. A record was also made of the day when the basic changes in each region were considered complete. For the caudal, pelvic and dorsal fins the criterion for completion was that the new coloration completely covered that portion of the fin which was typically colored in the male. For the superior pectoral region the criterion for completion was that the dark spot, which developed from discrete, thin, black lines (see Results), was fully confluent. For the head-shoulders region, coloration was considered complete when new, dark pigment covered the region smoothly and con- fluently in a typical male pattern. When all of these areas were judged complete the basic coloration change during sex reversal was considered at an end and the date was recorded. Black and white photographs were taken of males, all females considered likely to change sex (on the basis of size and dominance) and at least one female considered not likely to change sex, prior to each experimental male removal and at least once during or after the subsequent sex reversal. Photographs were also taken of specific individuals at various stages of sex reversal. Finally, when a male was removed from his group, he was anesthetized with MS-222 Sandoz (Bell, 1964) and photographed in color, alive, under magnification and standard illumination, for details of fin and body coloration. At the end of the study, several females were photographed in this way as well.

Field Study

The color changes of 26 sex-reversing fish in natural groups were examined on Harvey Reef in the Sudanese Red Sea and at two sites on Aldabra Island in the Indian Ocean (for details of the study sites, see Shapiro, 1977a; 1981). Most of these sex reversals followed experimental male removals SHAPIRO: SEX REVERSAL COLOR CHANGES 385

Figure I. A. squamipinnis (from a painting by W. B. Amos). Male (above), female (below). performed as part of other studies (Shapiro, 1979) and could be dated precisely from the day of male removal. All females in each such group were approached daily after male removal, with a few missed days, to within 2 m and scrutinized for color changes. The day of first appearance of color change was called the onset day. Onset day and completion times were recorded for each sex changing fish. Criteria for judging the completion of color changes were the same as those used in the laboratory study. Several sex reversals followed the natural disappearance of males from social groups and a few sex reversals were either already in progress at the start or were still not complete by the end of the study. For the latter, a minimum time could be specified for the duration of color change, but neitherthe onset day nor the completion time could not be specified. Such cases will be noted specifically in the results. 386 BULLETIN OF MARINE SCIENCE. VOL. 3\, NO.2. 198\

RESULTS Female Coloration The entire head and body of females are uniformly orange-gold except the ventral head and body contours which are pale white-yellow (Fig. 1). The superior part of the cornea is iridescent purple. An orange-red stripe, somewhat narrower than the pupil and faintly edged in violet-lavender, runs from just beneath the eye to the mid-pectoral base. A narrow red stripe runs from the anteriormost part of the isthmus to just anterior to the pelvic base. The fins are pale yellow to trans- parent.

Male Coloration The head is red to brown-violet dorsally, becoming pale red-brown to white ventrally (Fig. 1). A red to orange-red stripe, edged with dark red-brown, and approximately the same width as the pupil, runs from the posterior margin of the orbit to the center of the pectoral fin base. The flanks are pale red to pale yellow- brown, becoming reddish to brown-violet anteriorly and dark red to brown-violet on the caudal peduncle. The dorsal body contour is slightly darker than the flanks posteriorly and becomes red to brown-violet anterior to the base of the sixth dorsal spine. The ventral body contour is pale red to white. Two red-brown stripes run forward from the pelvic-fin bases, merge, and continue as a single stripe to the anterior part of the isthmus. The iris is iridescent green to black and the superior part of the cornea is red-brown. The caudal fin is a deep red or brown-violet color composed of small orange- red circles on a red or blackish background in the membranous parts of the fin, and is margined dorsally and ventrally with iridescent purple and posteriorly with yellow. The dorsal fin is deep red to brown-violet, the anterior third being lighter than the remainder; in addition, the basal one third to one quarter of the fin is lighter than the distal portion. A particularly dark red or brown-violet patch occupies the superior part of the fin approximately posterior to the tenth soft ray. The distal margin of the dorsal fin is iridescent purple to violet. The anal fin is pale yellow. The pelvic fins are deep red or violet, often with light red or orange-red anterior margins. The medial surface contains three or four intensely white stripes occupying the membranous parts between the first and second, the second and third, the third and fourth, and the fourth and fifth soft rays. The pectoral fins are pale pink or yellow with a large round spot of orange-red, red, or violet on the upper part of the fin, usually between the third and the ninth rays. In some males one to seven narrow, ellipsoid areas of red or violet are present on the inferior part of the fins, usually in the membranous areas between rays 12 and 16. In summary, females are bright orange-gold while males are dark red or violet with a pale central flank and red or violet flashes on the pelvic and pectoral fins.

The Typical Color Sequence The time course of color changes can be divided into two phases: the short- term phase, during which the basic male coloration pattern was laid down, usually within 19 days of male removal, and the long-term phase, a much longer period during which refinements to the basic male color pattern were made. The short- term phase began when the experimental male was removed from the group and ended when the color changes in all five body regions were judged complete. The changes occurring during this short-term phase were rapid and dramatic. Long- SHAPIRO: SEX REVERSAL COLOR CHANGES 387

Figure 2. (Left) Small, irregular areas of darkening on the head-shoulders regions of a sex-reversing A. squamipinnis 5 days after male removal. Figure 3. (Right) Early darkening (arrow) on the pelvic tips of a sex-reversing A. squamipinnis 5 days after male removal. term changes extended for an indefinite period thereafter and were slow and gradual. Most of the data from this study pertain to short-term changes.

The Short-Term Phase in Laboratory Fish The earliest sign of color change occurred either in the head-shoulders region, where onset ranged from day 3-7, or on the pelvic fins where onset ranged from day 4-6 (Table 1). On the head-shoulders this change consisted of the appearance of several, small black dots irregularly scattered over the inter-orbital area of the forehead, over the postorbital portion of the head above the eye stripe, or oc- casionally on the shoulder. Over the next 2-9 days more of these dots appeared and individual dots enlarged and eventually merged with neighboring dots to form dark splotches (Fig. 2) and, finally, large confluent areas of darkening. By day 16 the head-shoulders changes were complete (range 7-16 days, median day 9; Table I). The first change on the pelvic fins was invariably a darkening of the extreme tips of the fins (Fig. 3). In two cases, this was the first noticeable change on the fish, preceding the first change on the head-shoulders region by one or two days. Generally it occurred on the same day as the first head-shoulders change or 1-2 days later (Table 1). Often the earliest changes could only be seen when the fins were collapsed. On ensuing days, the blackish pigment spread anteriorly over the fin. The anteriormost margin of the dark area was well-defined and formed the shape of an inverted V. As the days progressed this inverted V moved farther anteriorly until the entire pelvic fin was covered with dark pigment. During this time the pigment in the darkened area increased in intensity and began to assume a violet hue. On the medial surface of the fin the four membranous areas between soft rays 2-5 assumed a brilliant white color. On the lateral surface these same areas were dark (Figs. I and 5). The changes of the pelvic fins were complete by the 16th day (range 7-16 days, median day 10). Shortly following the first appearance of new pigment in the head-shoulders region and on the pelvic fins, dark pigment appeared on the posterior sixth of the dorsal fin. The onset day on the dorsal fin ranged from 4-7 (Table 1). Darkening here began as a small area of black spots surrounding tiny circles of yellow pigment. This area gradually expanded in circumference and the yellow circles became overlaid with red. This dark purple area deepened in intensity and spread anteriorly in a narrow band along the dorsal fin (Fig. 4). The darkest part of this band was always the original area of black-violet color on the posterior sixth of 388 BULLETIN OF MARINE SCIENCE. VOL. 31. NO.2. 1981

Table 1. Onset and completion days of A. squamipinnis color changes on five body regions during typical sequences in the laboratory

Group Fish Head- Pelvic Posterior Superior Caudal Identity Identity shoulders Fins Dorsal Pectoral Fin

Onset Day

1975 LT I 5 4 5 5 5 NT] I 3 5 5 5 5 NT2 I 3 4 4 6 6 NT2 2 3 5 5 6 7 LT 2 6 6 7 7 8 MT 2 5 5 6 5 6 1974 MT 5 7 5 7 7 8 Median 5 5 5 6 6 Range 3-7 4-6 4-7 5-7 5-8 Completion Day

1975 LT I 9 10 10 10 10 NT! ] 9 12 10 12 12 NT2 I 8 9 7 9 9 NT2 2 9 9 9 9 9 LT 2 II 11 II 10 II MT 2 7 7 7 7 7 1974 MT 5 ]6 16 16 16 16 Median 9 10 10 10 10 Range 7-16 7-16 7-16 7-16 7-16 the fin. The more anterior along the fin the color spread, the paler the band became. It looked as if the dark pigment were migrating anteriorly from the posterior part, becoming less intense as it migrated (cf. melanophore migration in Brachydanio, Goodrich et aI., 1954). In some cases a narrow, second band of pigment appeared beneath the first band. By the 16th day (range 7-16 days, median day 10) most of the dorsal fin had been darkened by the expanding band of black-violet pigment, and the basic changes were considered to be complete (Table 1). On the same or the following day as the onset of color changes on the dorsal fin, the earliest sign of development of a superior pectoral spot appeared. In the male, the superior pectoral spot is a prominent, round area of uniform, red-violet color. But this circle of color began its development not as a circle but as 5-6 thin, parallel lines of melanic pigment which gradually broadened, until they united in a confluent, circular area of color. The first sign of this development was the appearance of 1-3 dark lines, each in the membranous region between two adjacent rays (Fig. 5). This occurred on day 5-7 (Table 1). The first dark line was often quite faint and barely visible on the free-swimming fish. With the fin compressed these early lines became more visible. Within 12-24 hours the initial line became broader and darker and readily apparent. On subsequent days the number of lines increased (Fig. 6) and finally became confluent. The number of separate, dark lines which merged to form the superior pectoral spot on the right and left fins was recorded for nine fish that changed sex in these experiments. The number of lines ranged from 4-6 on the right fin and 4- 8 on the left fin, with a median of 6 lines for both. The first lines to appear were those forming the central diameter of the spot. They were thus the longest lines in the spot. Subsequently, lines appeared above and below the central lines. This SHAPIRO: SEX REVERSAL COLOR CHANGES 389

Figure 4. (Left) A narrow band of dark pigment (arrows) spreading anteriorly along the dorsal fin of a sex-changing A. squamipinnis. Figure 5. (Right) A single dark line in the superior region of the right pectoral fin of a sex-changing A. squamipinnis 7 days after male removal. Note white lines on medial surface of left pelvic fin not visible on lateral surface of right pelvic fin. tendency for the spot to form from the center outward, on the vertical axis, is revealed in Table 2 which lists the order of appearance of dark lines in the superior pectoral region of all fish showing six or more lines prior to the formation of a confluent spot. The lines were numbered 1-6 starting with the superior line. If a fish showed more than 6 lines, the 7th or 8th lines were eliminated from the analysis. Both the median rank list and the sum of ranks, Rj, suggest that on the right and the left fin central lines appeared first, followed successively by increas- ingly peripheral lines. The order of appearance of these lines was fairly uniform for all fish (Kendall coefficient of concordance, W = 0.563, P < 0.01, on the right fin; W = 0.603, P < 0.01, on the left fin) except fish No.1, group NT2, in which the spot formed from the top downward. The superior pectoral spot was confluent and, hence, complete by day 16 in all seven animals for which there were adequate data (Table 1).

Figure 6. Same fish (A. squamipinnis) as in Fig. 5 but 4 days later (i.e., II days after male removal). The fish now has five dark discrete lines on the right pectoral fin. The lines have begun to broaden and the outline of a round spot is becoming clear. Note the absence of any line in the inferior pectoral region. 390 BULLETIN OF MARINE SCIENCE, VOL. 31. NO.2, 1981

Table 2. Order of appearance of superior pectoral lines in sex changing A. squamipinnis in the laboratory

Right Fin Line Identity Left Fin Line Identity

Group Fish Top Bottom Top Bottom Identity Identity 1 6 1 6

1975 LT I 5.5 2 4 1 3 5.5 5 4 2 I 3 6 MT I 5 2 I 3 4 6 5 2 I 3 4 6 NT1 1 5 3 2 I 6 4 5 2 I 3 4 6 NT2 I 2 1 3 5 4 6 1 2 3 5 4 6 LT 2 4 3 I 2 6 5 5 3 I 2 4 6 NT2 2 3 6 I 2 4 5 Median 5 2 2 2 4 5.3 5 3 I 2 4 6

Sum of Ranks, Rj 21.5 II 11 12 23 26.5 24 19 9 16 23 35

On the same day that changes appeared on the pectoral fin, or one day later, the earliest color changes were distinguishable on the caudal fin (Table 1). These consisted of general darkening of the membranous portion of the fin. With suc- cessive days, this darkening intensified, but spared small circular areas of yellow which gradually turned orange-red as they became covered with tiny specks of red pigment. Changes in the caudal fin were complete by day 7-16, with a median day 10 (Table 1). The last portion of the fish to change color was the inferior pectoral region. Data available from five fish showed that onset day in this region ranged from day 6-7, with median 6, on the right fin, and ranged from day 6-9, with median 7, on the left fin. This feature was highly variable in appearance. Generally, it consisted of several, non-contiguous, dark lines (Fig. 1). In these laboratory ex- periments, the number of lines ranged from 1-7 on the right fin, with median 2- 3, and from 2-6 on the left fin, with median 3 (Table 3). In the field, males were often found with no inferior pectoral lines at all. Rarely, a male had inferior lines that were broad enough to be contiguous. The data in Table 1 suggest that, although the onset day for a specific body region varied, changes in different regions appeared in a regular order for most fish. This can be confirmed by ranking the five body regions, for each individual fish, according to the order of first appearance of color change. A Kendall test of concordance shows these rank orders to be essentially uniform (W = 0.683, P < 0.01). Changes appeared first on the head-shoulders or pelvic fins, followed

Table 3. Number of lines in the inferior region of the right and left pectoral fins in sex changed A. squamipinnis in the laboratory

Number of Lines Group Identity Fish Identity Right Left

1975 LT 1 4 6 NTI I 7 5 NT2 1 I 3 MT 2 2 2 NT2 2 3 3 MT 3 2 2 Median 2.5 3 Range 1-7 2-6 SHAPIRO: SEX REVERSAL COLOR CHANGES 39\

Table 4. Day of onset, interim time, and day of completion for laboratory and field cases of the typical coloration sequence in A. squamipinnis

Day of Onset Interim Time (days) Day of Completion

Laboratory Cases Median 4 6 10 Range 3-6 2-11 7-16 N = 7 Field Cases Median 5 8 14 Range 4-9 5-11 8-19 N = 18 Mann-Whitney V-test P-values, two-tailed <0.02 NS <0.02 by changes on the dorsal fin and/or on the superior pectoral region, then by changes on the caudal fin, and lastly by changes in the inferior pectoral region. When all the changes of specific body regions are considered together, the onset day for any body area ranged from 3-6 (Table 4), with median day 4. Completion of short-term changes occurred on day 7-16, with median day 10. The interim time between onset day and final completion of color changes ranged from 2-11 days, with median 6 days. The general temporal features of short-term coloration changes, then, are three: early onset, a short interim period, and a short total time for completion.

The Long-Term Phase in Laboratory Fish During the short-term phase the basic coloration pattern of the male was com- pletely laid down. This required, in the laboratory cases, 16days or less. By this time, all male color features were present, but the color itself often differed from the final color seen in most males in the field. Generally, the dark areas which appeared during the short-term phase were black, red-brown-black, or violet. In older males, these areas were either rich red or red-violet. Slowly, over a period of months, the black hue to these areas became more violet or reddish. This was particularly noticeable in the superior pectoral spot, where the central portion of the spot turned violet before the circumferential margins. The spot was then seen to have a violaceous interior and a dark, black outline. Similar changes of hue proceeded on all body areas which were initially dark. These slow changes constituted the long-term phase.

The Typical Sequence in Natural Groups Twenty sex reversals occurring in the field conformed to the basic features of the typical color sequence seen in laboratory groups: early onset, a short interim period, and a short total completion time. In 18 of these cases there were suffi- ciently good data for a quantitative comparison with laboratory sex reversal (Ta- ble 4). Although these field sequences were similar to, they were not identical with the laboratory sequences. A Mann-Whitney V-test shows the day of onset for the 18 field cases to be significantly later than the onset day for the seven laboratory cases (V = 24, P < 0.02, two-tailed). The interim time was the same for both (V = 32, P > 0.05 NS, two-tailed). As a result of the later onset day, 392 BULLETIN OF MARINE SCIENCE, VOL. 31, NO.2. 1981

Table 5. Day of onset, interim time, and day of completion for the atypical coloration sequence, Type I, in laboratory and field cases of sex changing A. squamipillllis

Interim Time Day of Group Identity Fish Identity Day of Onset (days) Completion

Laboratory Cases 1974 LT 4 4 30 34 1974 LT 2 19 34 53 ]975 MT 3 28 6 34 Field Cases Aldabra, site 2 20 6 26 Red Sea 29A 20 17 37 29A 25 ]2 37 22 ? >18 ? Median 20 17 35.5 Range 4-28 6-34 26-53

the field sequences had a slightly longer completion time (U = 21, P < 0.02, two- tailed).

Atypical Color Sequences Two types of color change sequences were observed which were atypical. Only a limited number of these cases have been seen, so description will be limited to characterizing their differences from the typical sequence. The first atypical sequence, called Type 1, differed from the typical in either or both of two ways: the day of onset of color change was considerably delayed and/or the interim period of change was considerably prolonged. In either case, the total time for completion ofthe short term phase was increased. This occurred in three instances in the laboratory and four instances in the field (Table 5). In each of these cases the day of onset was at least seven days later than the latest day of onset in the typical sequences and/or the interim period was seven or more days longer than the longest interim period in the typical sequences. Of these seven cases four occurred in circumstances which differed noticeably from the circumstances of all typical sequences. The unusual circumstances were of two types: (I) the sex reversal occurred in an initially all-female group (three cases); (2) a male was removed from a group during the first week of the sex reversal of another group member, i.e., before the sex reversal was completed (one case). Three fish were observed to have a color pattern which resembled male col- oration but which could have been formed neither via the typical sequence nor via the atypical sequence described above. Each of these fish retained the orange- gold color of the female on its head, shoulders, body and on its dorsal and caudal fins. Each had a faint, red superior spot on the pectoral fins and red coloration on the central portion of the pelvic fins. The pelvic fin tips were spared, remaining a transparent yellow. In the typical color sequence, pelvic coloration began with a darkening of the pelvic tips. By the time the central portions of the pelvic fins were dark the tips were densely black-violet. The pelvic color seen in the three atypical fish, with a red central area but transparent tips, could not have occurred by the typical sequence. Similarly, in the typical sequences a confluent superior pectoral spot SHAPIRO: SEX REVERSAL COLOR CHANGES 393

Table 6. Prevalence of three types of coloration sequence in sex-changing A. squamipinnis in the laboratory and in the field

Atypical Sequences

Typical Sequence Type I Type 2 Total

Laboratory Cases 13 3 2 18 Field Cases 20 5 I 26 Total 33 8 3 44 Percent 75 18 7 100

was never observed without well-advanced changes on the head-shoulders and on the dorsal and caudal fins. The total absence of dark pigment on the head- shoulders, dorsal, and caudal regions of these fish in the presence of a superior pectoral spot represents a color pattern which could not have developed accord- ing to the typical sequence or the delayed form of it represented by the atypical sequence, Type I. Two of these three fish had developed this color pattern in very small laboratory groups containing two or three females after the single male in the group was removed. In each case, one female gradually assumed this color pattern over a three month period, but no detailed observations on the sequence itself are avail- able. It is noteworthy, however, that both instances involved groups with very few females. In three other groups of this size, removal of the male resulted in no sex reversal or color change at all. These results suggested that very small groups may be inadequate for the normal production of a male. A third fish with this peculiar color pattern was observed in a group at site I, Aldabra. This group contained, in addition to the aberrantly colored fish, a full male and nine adult females. When the full male was removed from the group, the aberrantly colored fish developed all of the coloration changes normally shown by a sex-reversing female. These subsequent color changes followed the typical sequence in its entirety. Gonadal histology was not available for any of these three fish. It is not certain what relation this aberrant color pattern had to the development of testes or to the performance of male behavior. What is certain is that the emergence of this color pattern must follow a temporal sequence entirely distinct from the typical color sequence. This atypical sequence will be called Type 2. Several additional individuals showing Type 2 coloration sequence have since been observed in the Philippines. A total of 44 instances of coloration change have been observed, in the labo- ratory and in the field, in sufficient detail to be characterized according to col- oration sequence. Thirty-three of these, or 75%, conformed closely to the typical color sequence (Table 6). The remaining II cases followed one of the two atypical sequences.

Body Size and Fin Length Two additional features by which males and females differed externally are body size and fin length. It has been shown elsewhere (Shapiro, 1977a; 1981) that within each social group and generally within each study site, males were longer than females. In addition, the body height of males was greater than that of females and, as is true for several Anthias species (Randall, 1979), the third dorsal spine and the caudal fin streamers were often greatly elongate in males and 394 BULLETIN OF MARINE SCIENCE. VOL. 31. NO.2. 1981 only occasionally so in females. When females with a short third dorsal spine changed sex, the third dorsal spine elongated. It generally required at least 2 weeks before the elongation of the third dorsal spine was noticeable, and much longer before elongation was complete. Although measurements of standard length and height were not taken before and after sex reversal, it was obvious in some individuals that a growth spurt had accompanied sex reversal. This was particularly noticeable in a case where the two largest females in the group were of about equal size and one changed sex. The sex reversed fish ended by being larger than the remaining female. My impression generally was that an increase in body size was part of sex reversal.

DISCUSSION Three types of color sequence were observed. In the most common, typical sequence the onset of coloration change was rapid, the interim period was short, and completion occurred within 19 days of male removal. In the laboratory, typical sequences were quite consistent but showed a slightly earlier onset than typical sequences in the field. Five field groups accounted for the statistical difference in onset time between the laboratory and field cases. Three of these were on Aldabra where the lower water temperature (22-24°C) than that of laboratory groups (25-27°C) could have retarded onset time. But two of the five field groups were in the Red Sea where water temperatures were high (25-28°C). Illumination intensity and background contrast have been claimed to affect the rapidity of appearance of melanophores and xanthophores in scale transplants of Thalassoma bifasciatum and Ha/i- choeres bivittatus (Goodrich et aI., 1950). The greater the light intensity, the more rapidly the appeared. The lighting conditions of the laboratory groups were certainly different from the five field groups, but the intensity of illumination was much less than that in the field. If illumination were a factor, then the field groups should have shown an earlier rather than a later onset time than the laboratory groups. Furthermore, while there were clear ecological differences (e.g., in water tem- perature, water pH, aquarium size and illumination) between the four laboratory groups which were consistent in onset time, the water conditions within each Aldabra site were likely to have been nearly identical for all groups. But within each site the individuals of some groups showed early onset times while members of other groups showed later onset times. Ecological conditions differed widely between the study sites on Aldabra and in the Red Sea, but there were no con- sistent differences in onset times between sites. It seems unlikely, therefore, that the longer onset times of typical sequences in the field can be explained primarily by ecological factors. A second possibility is that the onset time was influenced by the nature of the social groups involved. Three of the five field groups in which individuals showed delayed onset times during the typical color sequence were socially exceptional. Two of them contained three or fewer females. The third was a Red Sea group from which four fish had disappeared within 39 days: two males and two fish midway through a sex reversal (the first male was removed experimentally; the others disappeared naturally). Atypical sequences also tended to occur in socially unusual groups. Six of the II cases of atypical sequences involved all-female groups, groups with three females or fewer, or groups in which a male was removed during the first week of the sex reversal of another group member. It would appear that the coloration sequence of a sex-reversing fish may be influ- SHAPIRO: SEX REVERSAL COLOR CHANGES 395 enced by the composition of its social group and by the timing of the male removal which initiated the sequence. Recent evidence from the Philippines suggests a similar conclusion (Shapiro, 1980). When one male was removed from each of 15 multi-male, bisexual social groups, the onset day for coloration change ranged from 1-7 with median 3. When 3-9 males were removed simultaneously from each of 11multi-male groups, 3-9 fe- males changed sex in each group. The onset times for sex change in the fish of each group were evenly spaced, with 1-2 days separating successive onsets. For example, the first female to change sex had onset a mean of2.3 days after all males were removed; the third female to initiate changes had onset 6.0 days after remov- als; and the seventh female had onset 11.3 days after male removals. The range of onset times for the first two females to initiate sex change closely resembled the range for sex reversals in the 15 groups from which only one male was removed. The range of onset times for the last four females to begin sex reversal in the mul- tiple removal groups showed no overlap with the range of times for the single removal groups (Shapiro, 1980). The temporal sequence for coloration change is therefore unlikely to be completely physiologically predetermined within the individual but can be influenced by the social circumstances surrounding the initiation of sex reversal. In the typical color sequence the onset time of 3-9 days corresponded to the time lag after male removal for the first performance, by the sex-reversing fish, of a characteristically male behavior, V-swims, and for an increase in third dorsal erections, a movement performed at very high rates by males (Shapiro, 1977a; 1979). This correspondence suggests that the development of male coloration and the appearance of these behaviors share a common cause, which is likely to be hormonal (see review by Fujii, 1969). The addition of testosterone to aquaria water led female A. squamipinnis to assume male coloration (Fishelson, 1975). With a testosterone concentration of 5.0 mg/l, fin and body pigment appeared in two and four days, respectively. With a lower concentration, the onset was delayed until day 6. When these fish were placed in fresh aquaria containing no testosterone, male coloration only persisted in individuals treated with 5.0 mg/1. The social composition of the tanks into which treated females were placed was not described in this study. This omission could be important since normal males placed in social isolation or together only with one or two females tended to lose the male features of their coloration (personal observation). In other, sexually dimorphic, protogynous species, treatment of females with testosterone has often resulted in the appearance of male coloration. This has been found in Corisjulis (Reinboth, 1962b), Thalassoma bifasciatum (Stoll, 1955; Reinboth, 1962a; Roede, 1972) and Halichoeres poecilopterus (Okada, 1962), but was not found in H. bivittatus or H. garnoti (Roede, 1972), the difference perhaps being related to dose (Okada, 1962, and Okada, 1964). Dose-related phenomena may occur in natural populations. Primary males of several labrid and scarid species, although possessing larger testes than those of brightly colored secondary males (Roede, 1966; 1972; Choat, 1969), retain a drab coloration, but become bright and gaudy when injected with testosterone (Stoll, 1955; Reinboth, 1962b). The restriction of fin growth during sex reversal to the third dorsal spine is not inconsistent with there being a hormonal basis for third dorsal spine elongation. Differential sensitivity of fins, or of portions of fins, to androgens have been reported in other fish (Yamamoto, 1969). The early appearance of dark pigment in the prospective male-colored regions of sex-reversing individuals, followed in the long-term phase by a change to violet and red, is similar to the sequence of appearance during larval 396 BULLETIN OF MARINE SCIENCE. VOL. 31. NO.2. 1981 and juvenile development of other fish. In several species of goldfish, carp, and dace, black melanophores appeared on the trunk of the embryo within 24 h of spawning. Yellow or red xanthophores did not appear for 4-5 days. In the larval red-scaled goldfish, the body was colored initially by melanophores which later began to depigment. By 50 days after hatching, melanophores had disappeared and were being replaced by adult xanthophores containing red pigment (Matsumoto, 1965). An increase in orange-red pigment at the ex- pense of pigment laid down earlier in development has been observed in Oryzias latipes (Hama and Hasegawa, 1967). The initial invasion of a fin re- gion by melanophores, followed later by replacement with xanthophores and erythrophores, also characterized the development of color in Brachydanio rerio, normally (Goodrich and Green, 1959) and during regeneration of destroyed fin regions (Goodrich and Nichols, 1931). When yellow and striped regions of the anal fin were frozen, all chromatophores in the area were phagocytized. After 5- 6 days, both areas were invaded by melanophores. In the prospective yellow areas, the invading melanophores subsequently degenerated and were removed by phagocytes, and yellow xanthophores appeared (Goodrich et aI., 1954). The description of the process of melanophore removal and replacement by xanthop- hores closely resembles the color sequence of sex-reversing A. squamipinnis. The rapid, integumentary blackening of the head and dorsal body regions of post-spawning Tasmanian whitebait has been described, but no time scale given for the process (Blackburn, 1950). During normal development of fish fins, color usually develops first on the proximal and later on the distal regions (Matsumoto, 1965). On the pelvic fins of A. squamipinnis this process was reversed. Male color first appeared distally. Similarly, in female Thalassoma bifasciatum injected with testosterone, male color on the pectoral fins appeared first on the distal tips (Stoll, 1955). It is not known whether the chromatophores appearing on sex-reversing A. squamipinnis migrated from other body regions or differentiated in situ (Goodrich et aI., 1954). It is also not known whether localized color features, e.g., the superior pectoral spot, represent specific developmental properties of the local tissue or whether any tissue transplanted to that position would develop the local color pattern. Goodrich and Biesinger (1953) have described the histological basis of the male color pattern of two sex-reversing wrasses, T. bifasciatum and H. bivittatus (see also Kawaguti, 1965), and have shown that in T. bifasciatum, tissue transplanted from a green to a black region, or vice-versa, did not change its colora- tion. In H. bivattatus, however, tissue rapidly assumed the coloration of the region to which it was transplanted (Goodrich et aI., 1950). Each sex of A. squamipinnis has been described here as having a single color pattern. Males, however, occasionally showed a slightly different pattern when doing V-swims. As this pattern appeared and disappeared rapidly, it was probably physiological (i.e., involved the dispersion or aggregation of pigment within chro- matophores) rather than morphological (i.e., involved a change in the number of chromatophores or in the quantity of pigment present within chromatophores; Odiorne, 1957)and was thus not of primary concern in the process of sex reversal. The pattern itself has been described by Popper and Fishelson (1973) who claim that it was shown only by "territorial" males during the evening. While I have seen this color pattern both in the laboratory and in the field, it was often difficult to detect and it occurred so infrequently that no evidence was obtained on the time of day at which it appeared. The male color pattern described in this study contained features of two color patterns, A and C, described by Popper and Fishelson (1973) to distinguish "ter- SHAPIRO: SEX REVERSAL COLOR CHANGES 397 ritorial" and "non-territorial" males, respectively. A study of the location and movements of the five and eight males of two bisexual groups (Shapiro; in prep- aration) showed no such distinction between two male types. Furthermore, al- though males which I have seen did not all have absolutely identical color pat- terns, the variations were subtle and did not fall into distinct categories.

ACKNOWLEDGMENTS

This work was supported by a studentship and a Paton-Taylor Travelling Fellowship from Gonville and Caius College, Cambridge, the Durham Fund, King's College, Cambridge, the Cambridge Phil- osophical Society, a grant to H. W. Lissmann from the British Science Research Council, and in various ways by the University Sub-Department of Animal Behaviour and the Zoology Department of Cambridge University, the Cambridge Coral Starfish Research Group, and the Royal Society. The author is grateful to the following people for help and advice at various stages of this work: the late T. Weis-Fogh, D. A. Parry, H. W. Lissmann, M. J. A. Simpson, R. A. Hinde, P. P. G. Bateson, G. W. Potts, R. Sankey, R. Lubbock, K. Leighton-Boulon, D. Allen, J. Rodford, and M. E. Leighton- Shapiro. I thank P. Colin, Y. Sadovy, and R. Waldner for helpful criticisms of the manuscript.

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DATE ACCEPTED: October 24, 1979.

ADDRESS: Department of Marine Sciences. University of Puerto Rico, Mayaguez, Puerto Rico 00708.