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  REPRODUCTIVE BIOLOGY OF THE PANAMA GRAYSBY  Cephalopholis panamensis (TELEOSTEI: EPINEPHELIDAE)        BRAD E. ERISMAN, MATTHEW T. CRAIG AND PHILIP A. HASTINGS 

                      This electronic reprint is provided by the author(s) to be consulted by fellow scientists. It is not to  be used for any purpose other than private study, scholarship, or research.  Further reproduction or distribution of this reprint is restricted by copyright laws. If in doubt about  fair use of reprints for research purposes, the user should review the copyright notice contained in  the original journal from which this electronic reprint was made.   


Journal of Fish Biology (2010) 76, 1312–1328 doi:10.1111/j.1095-8649.2010.02567.x, available online at www.interscience.wiley.com

Reproductive biology of the Panama graysby Cephalopholis panamensis (Teleostei: Epinephelidae) B. E. Erisman*†, M. T. Craig‡ and P. A. Hastings* *Marine Biology Research Division, Scripps Institution of Oceanography, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0208, U.S.A. and ‡Department of Marine Sciences, University of Puerto Rico, Mayaguez, P.O. Box 9000, Mayaguez PR 00681, U.S.A. (Received 3 December 2008, Accepted 1 December 2009) The reproductive biology of the Panama graysby Cephalopholis panamensis was studied from collections and behavioural observations made in the Gulf of California from 2001 to 2006. Histological examinations, particularly the identification of gonads undergoing sexual transition, confirmed a protogynous hermaphroditic sexual pattern. The population structure and mating behaviour provided further support for protogyny. Size and age distributions by sex were bimodal, with males larger and older than females and sex ratios biased towards females. Mating groups consisted of a large male and several smaller females, and courtship occurred in pairs during the evening. Results on spawning periodicity and seasonality were incomplete, but histological data, monthly gonado-somatic indices (IG ) and behavioural observations suggest that adults spawned around the full moon from May to September. Certain aspects of their reproductive biology (e.g. protogyny and low egg production) indicate that C. panamensis is particularly vulnerable to fishing and would benefit from a  2010 The Authors management policy in Mexico. Journal compilation  2010 The Fisheries Society of the British Isles

Key words: groupers; protogynous hermaphroditism; reproduction.

INTRODUCTION Groupers (Teleostei: Epinephelidae: Epinephelini) comprise c. 160 species of carnivorous fishes that occur in all subtropical and tropical seas and show a range of reproductive strategies (Craig & Hastings, 2007; Erisman et al., 2009). They exhibit at least two and possibly three different sexual patterns: protogynous hermaphroditism, gonochorism and bi-directional sex change (Sadovy de Mitcheson & Liu, 2008). Most groupers are protogynous hermaphrodites, in which some (diandry) or all (monandry) individuals within populations reproduce first as females and change sex later in their lifetime to reproduce as males. Some may be capable of bi-directional sex change, as male to female sex change has been induced under experimental conditions in two species, but there is no evidence that this pattern occurs in wild populations (Sadovy de Mitcheson & Liu, 2008). Finally, a few species of groupers follow †Author to whom correspondence should be addressed. Tel.: +1 858 534 4841; fax: +1 858 822 1267; email: berisman@ucsd.edu

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a gonochoric sexual pattern: individuals function either as male or female without post-maturational sex change (Erisman et al., 2008). Mating behaviour also varies among species. Some spawn in pairs within territories defended by males, others spawn in large groups within massive spawning aggregations, and still others show various combinations of these behaviours (Erisman et al., 2009). Groupers are important to both commercial and recreational fisheries worldwide, accounting for the major portion of the live reef-fish food trade in Asia and the most valuable commercial fishes in fresh-fish markets throughout the tropics (Polidoro et al., 2008). As with most other predatory fishes, grouper populations have been depleted by overfishing, destruction of both juvenile and adult habitats, ineffective management plans for their fisheries or the lack of any management policies at all (Morris et al., 2000; Levin & Grimes, 2002). Understanding factors related to reproduction and development are particularly important for the management and conservation of groupers (Sadovy, 1996). The slow growth rates, restricted spawning periods, sporadic annual recruitment patterns, protogynous sexuality and older age and large size at sexual maturity make most species particularly vulnerable to high levels of fishing pressure and have contributed to their worldwide population and fisheries declines (Vincent & Sadovy, 1998; Sadovy, 2007). Therefore, acquiring data on variables such as size and age of sexual maturation, sexual pattern, growth rate, life span and spawning seasonality are necessary to determine the vulnerability of each species to exploitation or create even the simplest management regulations (e.g. minimum size restrictions or daily catch limits; Young et al., 2006). Thirteen species of groupers occur in the Gulf of California, Mexico, and all but one (the Pacific mutton hamlet Alphestes immaculatus Breder) are targeted by commercial, recreational or artisanal fisheries (Thomson et al., 2000; AburtoOropeza et al., 2008). Increased fishing effort in the Gulf of California over the past three decades is correlated with significant declines in stock sizes and landings of most groupers, and fisheries for larger species such as the Pacific goliath grouper Epinephelus quinquefasciatus (Bocourt) and the gulf grouper Mycteroperca jordani (Jenkins & Evermann) have collapsed and have been replaced by fisheries for smaller, less valuable species (Sala et al., 2004; S´aenz-Arroyo et al., 2005; Aburto-Oropeza et al., 2008). No fisheries regulations exist in the Gulf of California to restrict or manage fishing effort on any species of grouper; however, the Carta Nacional Pesquera (Mexican fisheries regulations) separated groupers and sea basses from all other species of finfish in 2006 as a first effort to design specific management regulations for these fishes. Unfortunately, current efforts to create sustainable fisheries for groupers of the Gulf of California are hindered by an absence of biological information for most species (Aburto-Oropeza et al., 2008). The Panama graysby Cephalopholis panamensis (Steindachner) occurs on inshore rock and coral reefs throughout the tropical eastern Pacific Ocean and is the most abundant grouper in the Gulf of California (Thomson et al., 2000). It feeds mainly on small fishes and invertebrates, grows to a maximum total length (LT ) of c. 400 mm and can live at least 14 years (Craig et al., 1999). Cephalopholis panamensis was long considered a species of low commercial value with catches limited primarily to sport fisheries and incidental catches in artisanal fisheries; however, their relative contribution to fisheries has increased in recent years because populations of larger groupers have declined (Sala et al., 2004; Aburto-Oropeza et al., 2008). The reproductive biology of C. panamensis was investigated to provide a baseline of  2010 The Authors Journal compilation  2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312–1328


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information useful for creating management regulations for its fishery in Mexico. The specific goals of this study were to determine the sexual pattern, size and age of sexual maturity and sex change (if hermaphroditic), population size and age structure, spawning season and mating system of this species.

MATERIALS AND METHODS COLLECTIONS OF SPECIMENS A total of 268 C. panamensis were captured by hook and line (n = 103) or by pole-spear (n = 165) from Loreto, Baja California Sur, Mexico 26◦ 00" N: 111◦ 20" W, from September 2001 to November 2006. The handling, collection and killing of all individuals followed the guidelines of the American Fisheries Society (AFS, 2004). All fish were placed in an ice-slurry immediately after capture and pithed. Standard length (LS ; to the nearest mm) and total body mass (MT ; to the nearest g) were recorded for each fish. Gonads were removed, weighed to the nearest 0·1 g (gonad mass, MG ) and preserved in a 10% formalin–seawater solution. Sagittal otoliths from 107 fish were removed, cleaned in water, dried and stored in manila envelopes. A G E D E T E R M I N AT I O N The right otolith of each fish was mounted on a small wood block with cyanoacrylate. Otoliths were cut with a Buehler low-speed isomet saw (www.buehler.com) equipped with a diamond wafer blade (series 15 hc diamond) to obtain a 0·8 mm transverse section through the focus of the structure. Sections were then polished with 3M lapping film (360-A; www.3m.com), cleaned, immersed in water and observed against a black background by reflected light under a dissecting microscope. Methods of age determination followed those by Craig et al. (1999) for C. panamensis. Two different observers recorded the number of alternating opaque and translucent zones for each section. Each observer performed two separate counts of each section, and if counts differed, a third count was made. R E P RO D U C T I V E D E V E L O P M E N T Sex and reproductive classification were assigned from microscopic evaluations of gonads and were based primarily on the presence and relative abundance of different germ-cell stages. The size and shape of gonads assisted in the distinction between sexually mature and immature individuals, and the presence of thickened muscle bundles in ovaries was used to distinguish mature females that were reproductively dormant from immature females that had never spawned (Shapiro et al., 1993). Classes of ovarian and testicular development were adapted from Chan & Sadovy (2002) for Cephalopholis boenak (Bloch), and stages of gametogenesis followed the definitions of Wallace & Selman (1981). For histological examinations, pieces of gonads were embedded in paraffin, sectioned at 6 µm, mounted on slides, stained with Mayer’s haemotoxylin–eosin and observed under a compound microscope (Humason, 1972). Tissue was taken from the central, posterior and anterior portions of one lobe and sectioned transversely in larger gonads, but smaller gonads were sectioned longitudinally in their entirety. Diagnosis of sexual pattern was based primarily on histological evidence and followed criteria outlined for fishes in general by Sadovy & Shapiro (1987) and for epinephelid fishes in particular by Sadovy de Mitcheson & Liu (2008). Fish undergoing sexual transition from female to male (i.e. transitionals) were identified by the presence of degenerating, vitellogenic oocytes or atretic follicles coincident with proliferating spermatogenic tissue. Size and agefrequency distributions, sex ratios and mating behaviour of mature females and males were used as additional evidence of sexual pattern (Warner, 1984; Sadovy & Shapiro, 1987; Erisman et al., 2008). Comparisons of the mean LS and age of mature females and mature males were analysed with unpaired t-tests, as data conformed to the assumptions of parametric statistics. The ratio of mature females to mature males was determined from both collections and from  2010 The Authors Journal compilation  2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312–1328


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observations of mating groups, because sex ratios generated solely from collections may not reflect the true population sex ratio. Mating group size was determined by counting the number of males and females within 2 m of each other during a courtship event.

S I Z E A N D A G E AT S E X U A L M AT U R I T Y A N D S E X C H A N G E The mean size at sexual maturity (Lm50 ) was estimated for females by fitting a logistic function to the proportion (Po ) of mature fish in 20 mm (LS ) size categories: Po = {1 + [−r(LSmid − Lm50 )]}, where LSmid is the midpoint of the LS class, Lm50 is the mean LS at sexual maturity and r is a constant that increases in value with the steepness of the maturation schedule (Grandcourt et al., 2009). The same procedure was used to estimate the mean age at sexual maturity (Am50 ) using the proportion of mature fish in each age class. The size at sex change was estimated from the size ranges of transitional fish and the size range in which females overlapped with males (Shapiro, 1984). Variations in size at sex change were also estimated from two ratios defined by Shapiro (1987): 1) size range of transitional fish divided by maximum size of fish in samples and 2) range of overlap in size of males and females divided by maximum size of fish in samples. S PAW N I N G S E A S O N Gonado-somatic indices (IG ) were calculated for mature fish by: IG = 100 MG MT−1 . The timing and duration of the spawning season were estimated by plotting the proportion of fish by maturity class and IG against sample months for both females and males. Both maturity class and IG data were grouped by month across the entire sampling period. Females collected with hydrated oocytes and direct observations of courtship were used to identify specific dates of spawning and the diel spawning period. M AT I N G S Y S T E M Observations were made by scuba diving from June to August 2004 and from July to October 2006 on shallow reefs at Loreto to determine the mating system. Dives were made from 1000 to 2100 hours and ranged from 30 to 120 min in duration and 3 to 20 m in depth. Identification of specific behaviours related to spawning followed those described for other species of Cephalopholis (Donaldson, 1989, 1995a, b; Shpigel & Fishelson, 1991a, b). The sex of individuals involved in courtship and other social interactions was determined by colour pattern for C. panamensis. The IG of mature active males provided further evidence of the mating system. In broadcast-spawning fishes, IG is indicative of adult investment in gamete production and is used to infer the mating system and intensity of sperm competition in males (Petersen & Warner, 1998; Erisman et al., 2009). The mean IG of mature active male C. panamensis was compared to known values of group-spawning and pair-spawning groupers, which was then used to determine the mating system and estimate the intensity of sperm competition in C. panamensis.

RESULTS R E P RO D U C T I V E D E V E L O P M E N T Gross morphology of gonads

Gonads were bilobed, fused posteriorly near the urogenital opening and suspended in the body cavity by a network of mesenteries. All gonads contained a central lumen surrounded by lamellae that projected medially from the gonadal walls. Both male and female germinal tissues developed within the lamellae, which were called lobules in males.  2010 The Authors Journal compilation  2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312–1328


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(a)

(b)

w

o1

db o1

mb

(c)

(d) ca

ho

yg yg

o1

(f )

(e) ao

ho

o1 ao

l

l sc

o1 sc

Fig. 1. Gonad development classes for female and bisexual Cephalopholis panamensis. (a) Immature female 166 mm standard length (LS ), (b) mature inactive female 240 mm LS , (c) mature developing female 208 mm LS , (d) mature active female 255 mm LS , (e) mature regressing female 215 mm LS and (f) bisexual 168 mm LS . Scale bar = 100 µm. ao, atretic vitellogenic oocyte; ca, cortical alveoli oocyte; db, dorsal blood vessel; l, gonadal lumen; mb, muscle bundle; o1, primary growth oocyte; sc, spermatogenic cysts; w, gonad wall; yg, yolk globule oocyte.

Immature females [n = 44, Fig. 1(a)]

Ovaries were small, and the gonadal wall was thin. They were composed almost entirely of primary growth stage oocytes (i.e. oogonium, chromatin nucleolar and perinucleolar) packed tightly within rows of lamellae. These gonads contained no evidence of prior spawning activity, because they were small in transverse diameter and thickened muscle bundles were absent. Spermatogenic cysts were present in low numbers in six immature females. Mature inactive female [n = 63, Fig. 1(b)]

The lamellae were dominated by primary growth stage oocytes; however, the ovaries were larger in length, volume and transverse diameter than those of immature females and the gonadal wall was thicker. There was evidence of prior spawning by the presence of thickened muscle bundles and enlarged blood vessels within lamellae. Spermatogenic cysts were present in low numbers in four mature inactive females. Mature developing female [n = 12, Fig. 1(c)]

The gonadal wall was thin, and both cortical alveolar stage and primary growth stage oocytes were abundant within lamellae. Yolk globule stage oocytes were often  2010 The Authors Journal compilation  2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312–1328


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present but few in number, and some were undergoing atresia. Spermatogenic cysts were present in low numbers in one mature developing female. Mature active female [n = 73, Fig. 1(d)]

The transverse diameter of the ovaries was enlarged and the gonadal wall was thin. Lamellae were filled with yolk globule stage oocytes tightly packed throughout the gonad, but oocytes of all stages were present, and primary growth stage oocytes were scattered along the edges of lamellae. When spawning was imminent, the ovaries contained oocytes in the migratory nucleus or hydrated stages, and there was evidence of recent spawning in some individuals via postovulatory follicles. Cortical alveoli and yolk globule oocytes in early to late stages of atresia were also common. Spermatogenic cysts were present in one mature active female. Mature postspawning or regressing female [n = 11, Fig. 1(e)]

The gonadal wall was thickened, and lamellae were filled loosely with cortical alveoli and yolk globule oocytes in early to late stages of atresia. Primary growth stage oocytes were also present, and cortical alveolar were sometimes present but few in number. The large amount of intralamellar debris and cytoplasmic strands gave the gonads a disrupted appearance. Muscle bundles and enlarged blood vessels were present in varied amounts. Spermatogenic cysts were present in one mature regressing female. Bisexual [n = 2, Fig. 1(f)]

The transverse diameter of the gonad was small, and the gonadal wall was thin. Lamellae were filled with relatively equal amounts of primary growth stage oocytes and cysts of spermatogonia and spermatocytes. There was no morphological evidence of prior sexual maturation as a female (muscle bundles or atretic vitellogenic oocytes) or as a male (developed peripheral sperm sinuses, spermatids or spermatozoa). Therefore, these gonads were not classified as female, male or transitional. Transitional [n = 3, Fig. 2(a)]

The gonadal wall was thickened, and the tissues appeared disorganized. Widespread atresia of yolk globule oocytes (demonstrating prior female function) and proliferating spermatogenic cysts and sperm sinuses (demonstrating development towards male function) were abundant throughout the gonad. Muscle bundles, enlarged blood vessels and connective tissue were present in some individuals. Immature male [n = 2, Fig. 2(b)]

The testes were small and thin in appearance, the transverse diameter was also small and the gonadal wall was thick. Lobules were composed mainly of spermatogenic cysts with spermatogonia and spermatocytes. Peripheral sperm sinuses were present in some individuals but were not fully developed and lacked spermatids or spermatozoa. Primary growth stage oocytes were present in one immature male.  2010 The Authors Journal compilation  2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312–1328


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(b)

(a)

sg

ao sg

o1

sc o1 ao

l w

(c)

sp

(d)

sg

l

o1

sc

sc

(e)

(f)

sp ss

sc

rs ss

cl w

w

Fig. 2. Gonad development classes for transitional and male Cephalopholis panamensis. (a) Transitional 260 mm standard length (LS ), (b) immature male 285 mm LS , (c) mature inactive male 314 mm LS , (d) mature developing male 292 mm LS , (e) mature active male 290 mm LS and (f) mature regressing male 280 mm LS . Scale bar = 50 µm. ao, atretic vitellogenic oocyte; cl, coalesced lobule; l, gonadal lumen; o1, primary growth oocyte; rs, residual sperm; sc, spermatogenic cysts; sg, spermatogonia; sp, spermatozoa; ss, sperm sinus; w, gonad wall.

Mature inactive male [n = 2, Fig. 2(c)]

The testes appeared similar but could be distinguished from those of immature males by their relatively large transverse diameter, the presence of fully developed (but constricted) sperm sinuses and small amounts of residual spermatozoa within lobules or sperm sinuses. The gonadal wall was thin, and lobules were filled with cysts of sperm cells at all stages of development but were dominated by cysts of spermatocytes and spermatogonia. Sperm sinuses were fully developed but constricted. Small amounts of residual spermatozoa were sometimes present in peripheral sperm sinuses or in lobules. Mature developing male [n = 10, Fig. 2(d)]

Cysts of spermatogonia, spermatocytes and spermatids were present within lobules. Peripheral sperm sinuses contained some spermatozoa but were not completely enlarged or full, but not all lobules were coalesced and filled with spermatozoa. Primary growth stage oocytes were present in five mature developing males. Mature active male [n = 42, Fig. 2(e)]

Lobules were enlarged, coalesced and filled with spermatozoa, and peripheral sperm sinuses were expanded and also completely full of spermatozoa. Some cysts  2010 The Authors Journal compilation  2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312–1328


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of spermatogonia, spermatocytes and spermatids were also present. Primary growth stage oocytes were present in seven mature active males. Mature postspawning or regressing male [n = 4, Fig. 2(f)]

Lobules were only partially full of residual spermatozoa, but they were more separated and smaller compared with active males. Peripheral sperm sinuses were enlarged or constricted but not completely full of spermatozoa. Large amounts of connective tissue, stromal tissue and spermatogonia were present throughout the gonad. S I Z E A N D A G E AT M AT U R I T Y A N D S E X C H A N G E

The minimum size and age at sexual maturity were 145 mm LS and 1 year for females and 200 mm LS and 3 years for males. The mean size (Lm50 ) and age (Am50 ) at sexual maturity for females were 170 mm LS and 1·5 years (Fig. 3). All females were sexually mature by 220 mm LS (4 years). All males were sexually mature by 200 mm LS (3 years), but one immature male of 230 mm LS and one of 285 mm LS were present. The estimated size range for sex change was 195–260 mm LS from the lengths of transitionals and 200–287 mm LS from the size overlap of males and females, which equates to 59–88% of maximum body size. The only transitional in which age was determined was 245 mm LS and 6 years of age, which equates to 38% of maximum age. The estimated age range for sex change was 3–11 years from the age overlap of males and females or 19–69% of maximum age. The two bisexuals were 230 (4 years) and 240 mm LS (3 years), respectively. Both bisexuals and two transitionals were collected in July 2003, and one transitional was also collected in May 2005. P O P U L AT I O N S T R U C T U R E A N D S E X R AT I O

The size range of mature females (145–287 mm LS ) overlapped with mature males (200–328 mm LS ; Fig. 4), but the mean size of mature females (216·96 mm LS ) and mature males (262·88 mm LS ) was significantly different (unpaired t-test, d.f. = 102, P < 0·01). Mature females (range = 1–11 years) overlapped in age with mature males (range = 3–16 years), but the difference in the mean age between mature females (3·66 years) and mature males (8·04 years) was significant (unpaired t-test, d.f. = 24, P < 0·01). The sex ratio of mature fish was 2·74 females per male from collections (n = 217) and 3·67 females per male from courtship observations (n = 6). S PAW N I N G S E A S O N

The spawning season was broadly identified as May to September. The values IG in females and males increased in the spring (April to June), peaked in the summer (July to August) and decreased to minimum levels in the autumn (September to November; Fig. 5). Mature active females were collected from May to September, and mature active males were collected from May to November (Fig. 6). Females with hydrated oocytes (n = 13) were collected 3 days before to 8 days after the full moon in July 2003 (n = 10), 9 days after the full moon in July 2004 (n = 1) and 1–5 days after the full moon in September 2006 (n = 2). The IG of mature active females ranged from 0·31 to 7·24% and averaged 2·01%, whereas the IG of mature active males ranged from 0·04 to 1·02% and averaged 0·26%.  2010 The Authors Journal compilation  2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312–1328


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100

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Age (years) Fig. 3. Logistic maturation curves for female Cephalopholis panamensis showing (a) the mean standard length (LS ) at first sexual maturity and (b) the mean age at first sexual maturity, and the mean size (Lm50 ) and age (Am50 ) at 50% sexual maturity.

M AT I N G S Y S T E M A N D C O L O U R PAT T E R N S

Courtship in C. panamensis was observed on 4 July (n = 2) and 5 July (n = 3) 2004, or 2–3 days after full moon, where it occurred between 1855 and 1930 hours and ranged from 35 to 140 s in duration. Courtship was also observed once on 13 September 2006 (6 days after full moon) at 1725 hours and lasted for 45 s. All courtship events involved a male–female pair and consisted of a combination of one  2010 The Authors Journal compilation  2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312–1328


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Fig. 4. (a) Standard length (LS ) and (b) age-frequency distributions for immature female ( ), mature female ( ), bisexual ( ), transitional ( ), immature male ( ) and mature male ( ) Cephalopholis panamensis in the central Gulf of California.

or more of the following behaviours: dance, parallel swim, follow, rise, parallel rest, shake and bump. During courtship periods, adults were clustered on the reef in groups comprised of a single male and two to six females. Both males and females frequently engaged in aggressive interactions with conspecifics during this period. Some interactions were brief, in which an individual would erect its dorsal fin, flare its gills or chase an approaching individual for a short distance. Others were more intense, where ď&#x203A;&#x2122; 2010 The Authors Journal compilation ď&#x203A;&#x2122; 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312â&#x20AC;&#x201C;1328


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Fig. 5. Mean ± s.e. monthly gonado-osomatic indices (IG ) for female ( ) and male ( ) Cephalopholis panamensis in the central Gulf of California. Data were pooled by month across sample years from 2001 to 2006.

participants would face each with their mouths wide open, occasionally biting one another. Interactions usually involved two neighbouring males or two females within the same mating group. Courtship began when a male approached a female within the group. When this happened, the female would rise up c. 0·5 m above the reef, oriented in a head-up posture. She would then shake her head and body back and forth at a rapid pace (dance). The male would then swim up alongside and parallel to the female and perform the same behaviour. The two would then swim side by side along the reef for several seconds (parallel swim), or the male would follow close behind the female as she swam (follow). While swimming, the female would change directions several times, and the male would respond by making contact with the opercular area or the abdomen of the female with his snout (bump). During two courtship events, parallel swim and follow behaviours were followed by parallel rise, in which the pair rose up slowly into the water column for 1–2 m while maintaining body contact and oriented parallel. Next, the pair would separate and swim away from each other, or they would settle down onto the reef next to each other and facing the same direction (parallel rest). Once both settled onto the reef, the male would shake his head rapidly for 1–2 s, while remaining next to the female (shake). After one or more shake behaviours, the female either responded by swimming up into the water column and performing dance behaviour or swam away from the male. The release of gametes was not observed during any courtship events. When solitary, males and females were similar in colour (Fig. 7). During interspecific interactions, females were black, with high-contrast white bars on their heads and bodies, whereas in males the black vertical bars on the body were faded on  2010 The Authors Journal compilation  2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312–1328


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Fig. 6. Monthly percentage of gonad development stages for Cephalopholis panamensis (a) females: immature ( ), mature inactive ( ), mature developing ( ), mature active ( ) and mature regressing ( ), and (b) males: immature ( ), mature inactive ( ), mature developing ( ), mature active ( ) and mature regressing ( ) in the central Gulf of California. Numbers above bars show the monthly sample sizes. Data were pooled by month across sample years from 2001 to 2006.

(a)

(b)

(c)

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Fig. 7. Colour patterns of adult Cephalopholis panamensis in the central Gulf of California, organized by circumstances under which they were observed: (a) solitary adult, (b) female, agonistic and courtship events, (c) male, agonistic events and (d) male, courtship events.

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the upper body. Differences in colour pattern by sex were verified by collections of individuals (12 males and 17 females). During courtship, the male was a pale grey colour, and the female was black with high-contrast white and black bars on her head and body.

DISCUSSION S E X U A L PAT T E R N

Definitive evidence of protogynous hermaphroditism was found in C. panamensis, as transitional gonads containing atretic vitellogenic oocytes coexistent with proliferating spermatogenic crypts and sperm sinuses were present in two individuals. The presence of a gonad undergoing sexual transition is the strongest evidence of sex change in fishes, and this characteristic has been used to diagnose sex change in other groupers (Mackie, 2006; Sadovy de Mitcheson & Liu, 2008). Bisexual gonads were also found in C. panamensis, in a size range similar to that of transitional individuals. It is possible that bisexual fish were also undergoing sex change, although this could not be confirmed, because bisexual gonads did not show clear evidence of prior female function or development towards male function. Moreover, bisexual gonads are known to occur in both gonochoric and protogynous groupers, and so they cannot be used to determine sexual pattern (Liu & Sadovy, 2004; Erisman et al., 2008). The population structure of C. panamensis provided further support of protogyny. The size and age distributions of adults were bimodal, in which males were larger and older than females, and adult populations had female-biased sex ratios. These patterns are common in protogynous fishes, where males develop from sex-changed females, and social groups often consist of a large male and a group of smaller females (Warner, 1984; Munday et al., 2006). Differences in mortality rates, growth rates and habitat preferences between the sexes may also produce bimodal distributions or female-biased sex ratios, and thus do not provide unequivocal evidence of hermaphroditism (Sadovy & Shapiro, 1987; Devlin & Nagahama, 2002). When present in combination with histological evidence of sex change, however, these characteristics support this diagnosis. All males were larger and older than immature females, indicating that all males develop from sex-changed females (i.e. monandry). Monandry is the most common form of protogyny reported in groupers, although diandry has been reported in species of Cephalopholis, Plectropomus and Epinephelus, and bi-directional sex change has been reported in Cephalopholis boenack (Bloch) and Epinephelus akaara (Temminck & Schlegel) under experimental conditions (Sadovy de Mitcheson & Liu, 2008). The broad range of overlap in the size and age ranges of females, transitionals and males is common among protogynous groupers and consistent with the hypothesis that sex change is socially mediated (Shapiro, 1987; Munday et al., 2006). S PAW N I N G S E A S O N A N D T I M I N G O F S E X C H A N G E

Results from this study were not sufficient to determine precisely the seasonal timings of spawning or sex change, because data were collected over a 5 year period and all months of the year were not sampled; however, some patterns did ď&#x203A;&#x2122; 2010 The Authors Journal compilation ď&#x203A;&#x2122; 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312â&#x20AC;&#x201C;1328


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emerge. First, a broad spawning season of May to September was identified based on the presence of mature active females and males and elevated IG values during those months. Next, there was evidence that spawning occurs in rhythm with the lunar cycle in C. panamensis; collections of hydrated females and observations of courtship were made 3 days before to 9 days after the full moon. Spawning during specific lunar phases is common in groupers (Levin & Grimes, 2002; Soyano et al., 2003; Frisch et al., 2007), but more information is needed to confirm this pattern for C. panamensis. Finally, the collection of transitionals in both May and July demonstrates that adults are capable of sex change during the spawning season. Fish were not sampled during most of the non-breeding season (i.e. December to March); so it is unknown if sex change occurs during this time, although many groupers are known to change sex in the months that follow the spawning season (Sadovy, 1996; Frisch et al., 2007). M AT I N G S Y S T E M

Courtship and social behaviours observed in C. panamensis were similar to those described in other members of the genus. Cephalopholis argus Bloch & Schneider, C. boenak, Cephalopholis miniata (Forssk˚al), Cephalopholis spiloparaea (Valenciennes) and Cephalopholis urodeta (Forster) also engage in paired courtship during the afternoon and evening, and mating groups consist of single male, multiple female groups (Donaldson, 1989, 1995a; Shpigel & Fishelson, 1991b). In addition, behaviours such as dance, parallel swim, follow, rise, parallel rest, shake and bump have all been observed in these species (Donaldson, 1989, 1995a). Male C. panamensis engaged in paired courtship with females, displayed aggressive behaviours towards other males and invested little energy in sperm production; mean IG of mature active males was <0·5% of MT . Such traits are common in protogynous groupers, in which males attempt to maximize their mating success through female defence behaviour rather than through sperm competition with other males (Donaldson, 1995a, b; Erisman et al., 2009). In contrast, spawning in gonochoric species occurs in groups with several males and one or more females, male territorial behaviour is rare, and males have relatively large testes (e.g. IG > 4) indicative of high levels of sperm competition (Sadovy & Colin, 1995; Erisman et al., 2009). M A N A G E M E N T C O N S I D E R AT I O N S

There is evidence that small groupers like C. panamensis and its congeners are not resilient to fishing. A study on three species of Cephalopholis from the Red Sea found that adults and juveniles failed to recolonize the area 3 years after experimental removal (Shpigel & Fishelson, 1991a). This failure was attributed to competition by other resident predators and limited larval recruitment. Recruitment patterns in C. panamensis have not been investigated, although the low mean IG (2) of mature active females in comparison with other groupers suggests that egg production is limited, which could result in limited larval recruitment (Sadovy, 1996). Other aspects of their reproductive biology also suggest that C. panamensis is susceptible to overfishing. Cephalopholis panamensis are protogynous hermaphrodites with population structures in which males are larger and less abundant than females. Since traditional fisheries tend to remove the largest fishes, they select for the removal  2010 The Authors Journal compilation  2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1312–1328


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of males and highly fecund females from populations (Coleman et al., 1996; Beets & Friedlander, 1999). Such size-selective mortality may drastically alter the population structure and reduce reproductive output through decreases in sperm production, fecundity, life span, mate encounter rates, size or age at first maturity and the timing of sex change (Vincent & Sadovy, 1998; Hamilton et al., 2007). Changes in population structure and reproductive output due to fishing have resulted in the collapse of grouper fisheries in several regions in the western Atlantic Ocean (Coleman et al., 1996; Nemeth, 2005). Commercial fisheries for most large groupers have collapsed throughout the Gulf of California and Pacific Ocean coasts of Mexico (Sala et al., 2004; S´aenz-Arroyo et al., 2005; Aburto-Oropeza et al., 2008), and efforts have shifted towards the exploitation of smaller species such as C. panamensis. The life history of this small, slow-growing, protogynous species indicates the required caution in considering commercial fishing and points to the need for a management policy. A minimum size restriction of 25 cm LT would allow fish to spawn at least once as males and contribute to the population before they are captured; however, size-based regulations may be unreasonable, given the small size of C. panamensis. At the Great Barrier Reef in Australia, all groupers are managed by a minimum size restriction of 38 cm LT , which protects small species of Cephalopholis and Epinephelus from being taken (State of Queensland, Fisheries Regulation 2008; www2.dpi.qld.gov.au/fishweb/ 18778.html). Size-based restrictions or any other form of management policy do not exist in the Gulf of California for groupers, but they are urgently needed to hasten the current trends of declining populations and fisheries of most species. The authors would like to thank J. Hyde, H. J. Walker, M. Buckhorn, J. Rosales-Casi´an, J. Sanchez, E. Presutti, L. Allen, R. Hauserman, B. Lamoureux, M. Adriani and A. Layman for their assistance in the field, H. Bassett, M. McCormick and B. Chlebowski for their assistance in the laboratory and B. Macewicz and N. Holland for help with histological preparations and analyses. J. Graham, R. Rosenblatt, J. Moore, N. Holland and R. Warner provided valuable suggestions and comments on this research. This study was supported by the PADI Foundation (Proposal #48), David and Lucile Packard Foundation, SIO Center for Marine Biodiversity and Conservation (CMBC) in cooperation with the National Science Foundation (Grant #0333444), Maxwell Fenmore Fellowship, Carl Hubbs Memorial Fellowship, California Seagrant, UC MEXUS, SIO Graduate Department and the SIO Marine Vertebrate Collection.

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Electronic Reference Sadovy, Y. (2007). Workshop for Global Red List Assessments of Groupers, Family Serranidae; Subfamily Epinephelinae: Final report. Available at http://www.hku.hk/ ecology/GroupersWrasses/iucnsg/Docs/Final Report Workshop 2007.pdf

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