Reproductive Life-History Traits of Two Aggregating Reef-Associated Groupers (Red Hind and Yellowfin Grouper) in Marine Protected Areas of Southern Gulf of Mexico

Abstract

Overexploitation is the main anthropogenic threat to groupers (Epinephelidae) that aggregate to spawn. Fishing negatively affects their reproductive success and indirectly harms fishery economic yield. In the southern Gulf of Mexico, grouper catches, which include thirteen species, are in decline. A lack of biological information on each exploited species prevents optimising fishery management. Using histological examination of the gonads, the reproductive traits of red hind Epinephelus guttatus and yellowfin grouper Mycteroperca venenosa were studied from January 2008 to October 2009. Collections were made at two reef systems (Alacranes Reef and Bajos del Norte) on the continental shelf of the Yucatan Peninsula, Mexico, where these species form transient spawning aggregations. The results confirmed that previously identified spawning aggregation sites at both reefs constitute productive seasonal and perennial “hotspots” for both groupers; they spawn annually between January and April. Females of these protogynous hermaphroditic species exhibit a reproductive strategy characterised by asynchronous ovarian development organisation and ovulation. Sex ratios and maximum sizes at each reef suggest that populations of both groupers had a good conservation status as of the late 2000s. Both reefs are now marine protected areas, and a discussion is made of the consequent possible benefits to grouper population conservation and sustainability in the southern Gulf of Mexico.

1. Introduction

Groupers (Epinephelidae, Epinephelinae) are high-trophic-level predatory fishes that control energy flows in the ecosystem through their top–down regulatory role. Significant changes in grouper population composition can therefore substantially alter other functional groups in an ecological community [1]. Although groupers do not represent a high percentage of global fish catches, their exploitation is of considerable importance in commercial and recreational fisheries operating in marine coral or rocky reef areas around the world [2,3]. Between 2006 and 2016, worldwide grouper catches increased from 237,000 to 450,000 metric tons (t), most of it (~85%) from the Indo-Pacific region. In the Americas, grouper catches peaked in the 1980s and 1990s and have since been in decline [4,5]. The western central Atlantic hosts the highest concentration of grouper species in critical conservation conditions, with eight to ten threatened species per 100 km² [4]. Overfishing represents the main anthropogenic threat to groupers. Three biological traits increase their vulnerability to this threat: (1) they often develop a K-selected life strategy (species with slow growth and late maturation, high maximum size and longevity, and low natural mortality); (2) they are generally protogynous hermaphrodites, implying the presence of a reduced number of males in their populations; and (3) they frequently form spawning aggregations at predictable sites and during specific periods [6,7,8,9,10].

Fish population productivity and resilience in the face of fishing exploitation and other anthropogenic impacts depend largely on reproductive dynamics [11]. Knowledge of reproductive biology is therefore essential to provide reliable scientific recommendations for the conservation of exploited species and the management of their fisheries. Reproduction research has been conducted on red hind (Epinephelus guttatus, Linnaeus, 1758) and yellowfin grouper (Mycteroperca venenosa, Linnaeus, 1758) in the North Atlantic Ocean (Bermuda [12] and the Bahamas [13]) and throughout the Caribbean Sea (Cuba [14], Jamaica [15], Puerto Rico [16,17,18,19], British Virgin Islands (BVI) [20], United States Virgin Islands (USVI) [21,22], Netherlands Antilles [23], Antigua and Barbuda [24], and Venezuela [25]). However, little reproductive data are available for either species in the Gulf of Mexico [26,27].

Grouper fisheries in the state of Yucatan, in the southern Gulf of Mexico (SGM), account for the largest grouper catches in Mexico [28]. At least thirteen species (from the genera Cephalopholis, Epinephelus, Hyporthodus, Mycteroperca, and Paranthias) are traditionally exploited by the grouper fisheries operating in the northern region of the continental platform of the Yucatan Peninsula, called Campeche Bank (CB). On the CB, red hind and yellowfin grouper are caught only by the Mexican semi-industrial fishery [29]. Distributed between 2 and 140 m depth, these two grouper species inhabit rocky or coral reefs [2], and are protogynous hermaphrodites [30,31]. In the latest update of the IUCN Red List for groupers, red hind was classified as Least Concern and yellowfin grouper as Near Threatened [32,33].

Since the 2000s, management tools have been implemented for the Mexican grouper fishery in the Gulf of Mexico and the Caribbean Sea, principally a seasonal closure for all grouper species and a minimum catch size for red grouper (Epinephelus morio, Valenciennes, 1828). However, these actions have been ineffective in promoting the recovery of the CB grouper fishery [28,34]. After a production peak in the early 1970s (~19,000 t), grouper catches have continuously declined [34]. For example, grouper landings in 2023 were estimated at 5092 t, a 73% drop from the 1970s volumes [35]. Concurrently, two marine protected areas (MPAs) have been implemented to preserve some of the most ecologically important reef systems on CB [36]: Alacranes Reef (AR), established in 1994 [37], and Bajos del Norte (BN), established in 2024 [38]. Prior research identified spawning aggregations of red hind and yellowfin grouper in AR and BN [26]. Within this context, the objective of the present study was to generate original data on the main reproductive traits of red hind and yellowfin grouper at both reefs, with the aims of (1) recommending species-specific conventional management options, such as seasonal closure and minimum catch size, and (2) assessing the benefits provided by both MPAs, as non-conventional management options, in promoting grouper fishery sustainability on CB.

2. Materials and Methods

2.1. Study Area

Red hind and yellowfin grouper specimens were collected on two reef banks in the northern and north-eastern CB: AR, approx. 130 km north of the coast of Yucatan (22°21′44″ N–22°35′12″ N; 89°36′30″ W–89°48’00″ W), and BN, approx. 254 km north of Yucatan (23°20′ N, 88°45′ W) (Figure 1). Alacranes Reef, considered a platform reef, is an emerged bank with five sandy islands. Bajos del Norte is a totally submerged bank composed of submarine pinnacles with crests at 5 to 20 m depth [36,39,40,41]. The coral reefs on CB, where various reef-associated groupers live, are currently considered to be in a good state of conservation [42]. Indirect, continental-origin anthropogenic impacts (suspended sediments and contamination) are minimal at these reefs due to their great distance from the Yucatan coast. High-value carnivorous fish fishing is the principal direct impact affecting these ecosystems [40]. Before both reefs were declared MPAs, the Yucatan semi-industrial fleet fished groupers between July and February at AR and between February and May at BN [43,44,45].

Alacranes Reef was declared an MPA in 1994 and a National Park (NP) in 2000 [37,47]. It has had a management plan in effect since 2007 [46]. It covers 333,768 hectares (ha), with a 302,098 ha buffer zone and north and south core zones, both measuring 31,670 ha (9.5% of total NP area) (Figure 1). In early 2024, BN was declared an MPA with the category of NP, although it does not yet have a management plan. Its total area is 1,304,114 ha, which includes a 959,647 ha buffer zone and a 344,467 ha core zone (26% of total NP area) [37] (Figure 1). The core zones of both NPs are managed in a way resembling the IUCN Ia category (strict nature reserve) [48] and the no-take reserve concept [49].

Three grouper spawning aggregation sites have been previously identified at both reefs: (1) a red hind-only site in the AR northern core zone, (2) a tiger grouper (Mycteroperca tigris, Valenciennes, 1833) only site in the BN core zone, and (3) a multi-species (red hind/yellowfin grouper) site in the BN core zone [26].

2.2. Specimen Samples

In AR, specimens were collected in fishery-independent surveys at five sampling sites: one on the windward side for red hind, where it is known to form spawning aggregations [26], and four on the leeward side for yellowfin grouper (Figure 1). Specimens were collected with a spear gun and SCUBA diving, between 15 and 27 m depth, and from an 8 m long fiberglass boat with a 55 hp outboard motor. Collections were carried out during daylight hours (0900–1600 h), from February 2008 to October 2009, except for March and May 2008 due to adverse weather conditions. Monthly collection visits lasted six days on average. A total of 410 red hind and 234 yellowfin grouper specimens were collected at AR (

Table 1. Sex and maturity, number of individuals, median and mean of total length (TL), TL range, and whole-body weight (WW) range of red hind and yellowfin grouper collected between January 2008 and October 2009 at Alacranes Reef and Bajos del Norte, Campeche Bank, southern Gulf of Mexico. SE: standard error.

Sex	Maturity Status	Number	Size (cm TL)			Weight Range (g WW)
			Median	Mean (±SE)	Range
Red hind
Alacranes Reef
Female	Immature	41	36.0	34.9 ± 1.0	20.5–47.0	140–1840
	Mature	176	35.2	35.7 ± 0.3	25.2–48.5	240–1620
	Total	217	35.2	35.5 ± 0.3	20.5–48.5	140–1620
Transitional		2	33.8	33.8 ± 1.8	32.0–35.5	460–700
Male	Mature	191	40.0	39.5 ± 0.3	21.1–49.0	160–2220
Bajos del Norte
Female	Immature	15	35.5	37.1 ± 1.3	28.0–48.0	400–1500
	Mature	478	39.0	39.3 ± 0.2	24.0–51.0	150–1800
	Total	493	39.0	39.2 ± 0.2	24.0–51.0	150–1800
Transitional		6	42.8	43.5 ± 1.9	38.0–50.0	600–2000
Male	Mature	890	43.0	43.2 ± 0.1	28.0–57.5	300–2900
Yellowfin grouper
Alacranes Reef
Female	Immature	189	50.3	50.8 ± 0.9	20.3–80.0	210–7460
	Mature	41	64.0	62.7 ± 1.9	31.2–85.3	380–7800
	Total	230	52.0	53.0 ± 0.8	20.3–85.3	210–7800
Transitional		0	-	-	-	-
Male	Mature	4	70.0	66.9 ± 13.4	35.5–92.0	660–10,040
Bajos del Norte
Female	Immature	143	53.5	54.2 ± 0.6	35.6–81.0	550–7140
	Mature	1080	60.0	61.0 ± 0.3	37.0–94.0	400–9500
	Total	1223	59.5	60.2 ± 0.3	35.6–94.0	400–9500
Transitional		3	55.0	59.3 ± 9.2	46.0–77.0	1100–5500
Male	Mature	184	76.9	76.6 ± 0.7	44.8–100.0	800–12,000

).

In BN, red hind and yellowfin grouper specimens were collected by fishery-dependent surveys, in the same site where both species gather to spawn [26] (Figure 1). The specimens were captured with spear guns and hookah diving, between 10 and 18 m depth, from a 15 m-long vessel of the semi-industrial fishing fleet. Collections were carried out during daylight hours (0700–1500 h) from January 2008 to October 2009. Monthly collection visits lasted 15 to 17 days. A total of 1389 red hind and 1410 yellowfin grouper specimens were collected at BN (Table 1).

2.3. Specimen and Gonad Analysis

Fish collected at AR were processed upon landing at the main reef island (Perez Island) after being kept on ice post-capture. Those collected at BN were processed immediately after capture, on board the fishing vessel. For each individual, measurements were taken of total and fork length (TL and FL; nearest 0.1 cm), whole-body weight and gutted weight (WW and GW; nearest 1 g), as well as fresh gonad weight (gW; nearest 0.01 g). For histological analysis, a ~1 cm³ of fresh tissue was sampled from the central portion of the left ovary or testis of each organism and fixed in Bouin solution [50]. To estimate fecundity, mature ovaries with hydrated oocytes identifiable by the naked eye were placed in numbered plastic bags and kept on ice during transport to the laboratory.

The Bouin-fixed gonad samples were dehydrated in successive, increasing alcohol concentrations (70%, 90%, and 100%), treated with Clearance solvent, and infiltrated and embedded in Paraplast (melting point 56 °C, Leica). The embedded samples were cut to 6 μm thickness using a semi-automatic microtome. The resulting sections were stretched in a hot water bath (43–45 °C) before being placed on slides and stained using Gabe and Martoja’s one-step trichrome technique [50]. Finally, the stained sections were mounted in a synthetic resin (Ultrakitt, J.T. Baker) and dried in an oven at 60 °C for two to three days.

One fresh ovary sample (FOS) was taken from each mature ovary kept on ice, weighed (_FOSW; range: 15–30 g), and conserved in a 10% buffered formalin solution (preserved ovary sample; POS) [51].

2.4. Reproductive Trait Analysis

2.4.1. Sex and Gonad Development

Sex and sexual maturity were identified by histological examination of the gonads. Based on established criteria [52,53], fish were identified as functional males or females, or as individuals in the transition phase (i.e., sex change). Gametogenesis developmental stages used in this study were based on histological features of oogenesis [54,55,56,57] and spermatogenesis [57]. Based on the most advanced gametogenesis stage observed in the gonads, each specimen was classified into one of the following reproduction phases or sub-phases [58]: immature phase (females only), early developing sub-phase, developing phase, spawning capable phase, actively spawning and past-spawner sub-phases (females only), regressing phase, regenerating phase, and transition phase. Regenerating and immature females were differentiated using established histological signs indicative of previous reproductive activity [16,59]. Except for individuals identified as immature or undergoing sexual transition, all fish classified in any of the other reproductive phases or sub-phases were considered mature specimens. Individuals in the early developing, developing, spawning capable, actively spawning and past-spawner phases or subphases were considered active mature specimens. Active mature individuals classified as spawning capable, actively spawning, and past-spawner were considered representative of the spawning population [60]. Finally, individuals in regeneration or regression were considered inactive mature specimens.

2.4.2. Size Frequency and Sex Ratio

Median sizes of females and males were compared using the Mann–Whitney non-parametric test. Female and male size frequency distributions were compared using the Kolmogorov–Smirnov test. The Sturges rule was applied to identify the number and interval of size classes analysed per species [61]. Transitional individuals were excluded from the calculation of the overall and size class sex ratio (F:M, number of females per male). The Pearson Chi-square goodness-of-fit test (χ²) was applied to determine whether the calculated sex ratio values corresponded to a balanced sex ratio (1:1).

2.4.3. Spawning Seasonality

Each species’ spawning season was analysed by sex and by monthly evolution of the average gonadosomatic index (GSI) value:

$$\mathrm{G}\mathrm{S}\mathrm{I}=100\times ({\displaystyle \frac{\mathrm{g}\mathrm{W}}{\mathrm{G}\mathrm{W}}}),$$

(1)

where gW is the fresh gonad weight and GW the gutted weight of fish, both in g.

The monthly evolution of the frequency of individuals in each reproductive phase or sub-phase was also analysed. Immature and transitional specimens were excluded from these analyses. To track seasonal GSI and reproductive phase/sub-phase frequencies during a single year (i.e., January to December), all sampled individuals were grouped by month of collection. Each species’ annual “peak” spawning period was established as the months in which 50% or more of the collected females were in spawning-capable phase, actively spawning sub-phase or in the past-spawner sub-phase [62].

2.4.4. Size at Maturity and at Transition

The minimum size at which females begin to mature (Lmin) corresponded to the length of the smallest mature female in the collection. The Lmin/Lmax ratio (Lmax = length of the largest female in the collection) was calculated to define the proportion of the maximum size of the species at which females reach maturity [62]. The length at which 50% of females were mature (L50) and the length at which 50% of females had changed sex (PL50) were calculated using a binary logistic regression model [63]:

L50 or PL50 = e^z [1 + e^z]⁻¹,

(2)

where z = a + b × log₁₀TL.

To calculate L50, variation in the percentage of mature females (i.e., active and inactive) was analysed by size class. To calculate PL50, variation in the percentage of females was analysed by size class, excluding individuals in transition. When L50 and PL50 values could not be estimated for individuals from each reef due to inadequate data for the logistic regression, these parameters were calculated by pooling the data from both reefs.

2.4.5. Ovarian Organisation and Spawning Pattern

Three types of ovarian organisation patterns (synchronous, group-synchronous, and asynchronous) and two types of spawning patterns (synchronous/total spawner and asynchronous/batch spawner) can be identified in teleosts [54,64,65]. In the present study, these patterns were characterised by observing the developmental stages of secondary growth oocytes (SG) and the incidence of postovulatory follicles (POFs) in histological sections of ovaries of mature active females in the spawning phase (i.e., spawning capable, actively spawning, and past-spawner).

2.4.6. Spawning Interval

Female red hinds and yellowfin groupers may spawn more than once during their spawning season [13,14,19]. In response, the spawning interval (SI), the period of time elapsed between two successive spawning events [55,56], was estimated for both species using the hydrated oocyte method [66,67]. Females with the presence in their ovaries of oocytes in late maturation [OLMs] stage, oocytes in late germinal vesicle migration and/or in germinal vesicle breakdown and/or in hydration (=females in actively spawning sub-phase), were considered representative of the spawning fraction. At the population level, SI was estimated monthly by calculating the reciprocal of the spawning fraction [55,56,66]. The spawning fraction (proportion of females in the actively spawning sub-phase) was calculated considering the spawning population (i.e., spawning-capable, actively spawning, and past-spawner).

2.4.7. Fecundity

Because female red hinds and yellowfin groupers exhibit multiple spawning, batch fecundity (BF) was estimated using the gravimetric technique with the hydrated oocyte method [68], counting the OLMs present in the ovaries [58,68,69]. Females with OLMs were selected by histological ovary examination. To avoid underestimating fecundity, those with POFs were eliminated from the analysis [66]. The POS were weighed before beginning the analysis (_POSW; nearest 0.01 g). Three subsamples (SS) of known weight were taken from the POS (_POSSW; range: 0.030–0.050 g). Each POSS was placed on a glycerine-soaked slide to facilitate OLM separation and count (N_OLM) using a stereomicroscope [66]. Because total ovary weight was measured when fresh, and due to the possible effect caused by formalin fixation on _POSW, a correction factor (CF) was calculated for each female using the formula CF = _POSW/_FOSW. The CF value was used to estimate the fresh ovary weight (FO) of each POSS: _FOSSW = _POSSW × CF [68]. Finally, BF was calculated as follows:

$$\mathrm{B}\mathrm{F}={\displaystyle \frac{{\sum }_{i=1}^n\frac{{\mathrm{N}}_{\mathrm{O}\mathrm{L}\mathrm{M}i}}{{}_{\mathrm{F}\mathrm{O}\mathrm{S}\mathrm{S}i}\mathrm{W}}}{n}}\times \mathrm{g}\mathrm{W},$$

(3)

where N_OLMi is the number of oocytes in late maturation contained in the ith preserved ovary subsample, _FOSSiW is the estimated fresh weight of the ith ovary subsample, gW is the fresh gonad weight, both in g, and n = 3 (number of preserved ovary subsamples analysed).

The BF estimates were analysed considering female TL and WW, applying a linear model (y = a + bx) adjusted with the least squares method. Relative fecundity (RF) was calculated by dividing BF by WW of the corresponding female.

All statistical calculations were run with the SYSTAT 8.0 (www.grafiti.com) and INFOSTAT (www.infostat.com.ar) programs, using an α = 0.05 significance level.

3. Results

3.1. Size and Sex-Ratio

Individuals of both species from AR were smaller than those caught at BN (Table 1 and Figure 2). Red hind length-frequency distributions and median sizes differed between the reefs for both females (Kolmogorov–Smirnov, KS = 0.37, p < 0.01; and Kruskal–Wallis, H = 84.9, p < 0.0001) and males (Kolmogorov–Smirnov, KS = 0.39, p < 0.01; and Kruskal–Wallis, H = 117.4, p < 0.0001). For yellowfin grouper, size differed between reefs for females (Kolmogorov–Smirnov, KS = 0.31, p < 0.01; and Kruskal–Wallis, H = 64.5, p = 0.0001). Due to the small number of male yellowfin groupers caught at AR (n = 4; Table 1), no comparisons could be performed for male size between reefs.

At each reef, red hind females were smaller than males (Table 1; Figure 2). The length-frequency distributions and median sizes of female red hind differed from those of male at both AR (Kolmogorov–Smirnov, KS = 0.38, p < 0.01; and Kruskal–Wallis, H = 70.8, p < 0.0001) and BN (Kolmogorov–Smirnov, KS = 0.47, p < 0.01; and Kruskal–Wallis, H = 282.9, p < 0.0001). For yellowfin grouper, size differed between females and males at BN (Kolmogorov–Smirnov, KS = 0.62, p < 0.01 and Kruskal–Wallis, H = 222.4, p < 0.0001). Due to the small number of male yellowfin groupers caught at AR, no comparisons could be performed for yellowfin grouper female to male sizes at this reef.

For red hind, the overall sex ratio was balanced in AR (1.14:1.00; Chi-square test, χ² = 1.66, df = 1, p > 0.05) but skewed towards males at BN (0.55:1.00; Chi-square test, χ² = 113.96, df = 1, p < 0.05). The overall sex ratio for yellowfin grouper at BN was skewed towards females (6.65:1.00; Chi-square test, χ² = 767.25, df = 1, p < 0.05). Red hind at AR and BN, and yellowfin grouper at BN, exhibited a sex ratio skewed towards females in smaller size classes and towards males in larger size classes (

Table 2. Sex ratio by length class for red hind and yellowfin grouper collected at Alacranes Reef and Bajos del Norte, on the Campeche Bank, Yucatan, Mexico, between January 2008 and October 2009. TL: total length. F:M: number of females for each male. χ²: Pearson chi-square test. *: value did not differ significantly from 1:1 sex ratio (χ²_{0.05 (1)} = 3.841). When the expected frequency was < 5 for a size class, values of two or three consecutive size classes were pooled for sex ratio and χ² calculation.

Size Class (cm TL)	Alacranes Reef								Bajos del Norte
	Number of Fish						Sex Ratio (F:M)	χ2	Number of Fish					Sex Ratio (F:M)	χ2
	Female	Male	Total						Female	Male	Total
Red hind
19.5–22.4	2	1	3	}					0	0	0			-	-
22.5–25.4	2	0	2			14	6.00:1.00	7.14	2	0	2			-	-
19.5–28.4	8	1	9						3	1	4	}	12	11.00:1.00	8.33
28.5–31.4	30	4	34				7.50:1.00	19.88	8	0	8
31.5–34.4	45	16	61				2.80:1.00	13.79	35	4	39			8.75:1.00	24.64
34.5–37.4	51	30	81				1.70:1.00	5.44	128	30	158			4.27:1.00	60.78
37.5–40.4	48	52	100				0.92:1.00	0.16 *	141	158	299			0.89:1.00	0.97 *
40.5–43.4	21	59	80				0.36:1.00	18.05	102	295	397			0.35:1.00	93.83
43.5–46.4	8	21	29 9	}		38	0.36:1.00	8.53	50	228	278			0.22:1.00	113.97
46.5–49.4	2	7							18	132	150			0.14:1.00	86.64
49.5–52.4	0	0	0				-	-	6	37	43			0.16:1.00	22.35
52.5–55.4	0	0	0				-	-	0	3	3			-	-
55.5–58.4	0	0	0				-	-	0	2	2			-	-
Total	217	191	408				1.14:1.00	1.66 *	493	890	1383			0.55:1.00	113.96
Yellowfin grouper
19.5–26.4	3	0	3				-	-	0	0	0			-	-
26.5–33.4	11	0	11				-	-	0	0	0			-	-
33.5–40.4	20	1	21				-	-	6	0	6			-	-
40.5–47.4	45	0	45				-	-	64	2	66			32.00:1.00	58.24
47.5–54.4	53	1	54				-	-	267	4	271			66.75:1.00	255.24
54.5–61.4	37	0	37				-	-	375	6	381			62.50:1.00	357.38
61.5–68.4	31	0	31				-	-	284	21	305			13.52:1.00	226.78
68.5–75.4	21	0	21				-	-	157	50	207			3.14:1.00	55.31
75.5–82.4	7	0	7				-	-	56	54	110			1.04:1.00	0.04 *
82.5–89.4	2	1	3				-	-	12	32	44			0.38:1.00	9.09
89.5–96.4	0	1	1				-	-	2	11	13			0.18:1.00	6.23
96.5–103.4	0	0	0				-	-	0	4	4			-	-
Total	230	4	234				-	-	1223	184	1407			6.65:1.00	767.25

The brackets are intended to indicate that the number of fish from the first three size classes (19.5–28.4), were joined for further analysis. Such is the case for classes (43.5–49.4) in AR, and for BN (19.5–31.4).

).

The sex ratio was balanced in the intermediate size classes for red hind (37.5–40.4 cm TL) at AR and BN, and for yellowfin grouper (75.5–82.4 cm TL) at BN (Table 2). Due to the small number of male yellowfin groupers caught at AR, the overall and size class sex ratios could not be calculated for individuals captured at AR (Table 2).

3.2. Spawning Seasonality

The observed increases in mean monthly GSI values for female red hinds indicate that their reproductive season starts in December at both reefs but ends in April at AR and in June at BN (Figure 3).

Maximum mean GSI values (±standard error, SE) for females were observed in February (10.82 ± 0.66%, n = 46) and March (9.42 ± 0.97%, n = 41) at AR, and in March (11.62 ± 2.09%, n = 16) and April (9.51 ± 1.37%, n = 33) at BN.

Male red hinds at both reefs exhibited similar monthly mean GSI values variation, with maximum mean values recorded in February and March at both AR (1.57 ± 0.15%, n = 13 and 1.10 ± 0.08%, n = 20, respectively) and BN (0.92 ± 0.04%, n = 127 and 0.79 ± 0.05%, n = 39, respectively) (Figure 3). Minimum mean GSI values are indicative of reproductive inactivity and were observed from June to November for both sexes at both reefs (Figure 3).

In yellowfin grouper females, monthly mean GSI results showed their reproductive season to begin in February–March and end in June at both reefs (Figure 3). Maximum mean GSI values for females were observed in April at both AR (4.74 ± 1.71%, n = 4) and BN (8.71 ± 0.55%, n = 182). The breeding season for male yellowfin groupers was longer than for females at BN, starting in January and ending in July–August, with maximum mean GSI values observed in March (5.44 ± 0.67%, n = 11) and April (4.98 ± 0.39%, n = 18) (Figure 3). Minimum mean GSI values were observed from June/July to December/January for both sexes and at both reefs (Figure 3).

Maximum mean GSI values for females were similar in both species (Figure 3). Maximum individual GSI values for females were also similar in both species: 26.2% (AR) and 33.7% (BN) for red hind and 34.2% (BN) for yellowfin grouper. In contrast, maximum mean GSI values for male red hinds were much lower than those observed for male yellowfin groupers (Figure 3). Maximum individual GSI values for males were 2.2% (AR) and 2.8% (BN) for red hind, and 9.4% (BN) for yellowfin grouper.

Monthly frequencies of the number of females in each reproductive phase or sub-phase showed that both species spawned from winter to early spring. The female red hind spawning peak was longer at BN (January–April) than at AR (February–March) (Figure 4). For female yellowfin grouper, the spawning peak occurred in March and April at both reefs (Figure 4). Compared to females of the same species, both red hind and yellowfin grouper males exhibited a longer reproductive activity period and a shorter reproductive inactivity period (Figure 4).

Capture of mature red hind specimens was generally highest during this species’ seasonal spawning peak at each reef: March 2009 at AR (n = 41) and January 2009 at BN (n = 60) for females; January 2009 at AR (n = 102) and BN (n = 154) for males (Figure 5). The same held true for capture of mature yellowfin grouper specimens at BN, with the highest catches occurring during the seasonal spawning peak: March (n = 67) and April (n = 75) 2008 and March (n = 139), April (n = 112), and May (n = 179) 2009 for females; and April 2008 (n = 27) and May 2009 (n = 32) for males (Figure 5). Outside the spawning season, fewer mature females and males of both species remained at the spawning sites, probably due to migration of most of the spawners to their residence areas (Figure 5). The few immature female red hinds observed at both reefs were captured only outside the species’ spawning season, whereas immature females of yellowfin grouper were present year-round at both reefs (Figure 5).

3.3. Size at Maturity and at Transition

For female red hind, Lmin values were very similar at AR and BN: 25.2 cm TL (female in regression phase) and 24.0 cm TL (female in regeneration phase), respectively. The Lmin/Lmax ratio was 0.52 at AR and 0.47 at BN. The L50 estimated from all female red hind collected at both reefs was 22.0 cm TL (95% CI: 16.0–25.4 cm TL) (Figure 6). The smallest recorded male red hind was 21.1 cm TL (specimen in development phase) at AR and 28.0 cm TL (specimen in spawning-capable phase) at BN. Estimated PL50 values for red hind were very similar between reefs: 38.2 cm TL (95% CI: 37.0–39.4 cm TL) at AR and 39.0 cm TL (95% CI: 38.6–39.5 cm TL) at BN (Figure 6).

In female yellowfin grouper, Lmin was 31.2 cm TL (specimen in early development sub-phase) at AR and 37.0 cm TL (specimen in regeneration phase) at BN. The Lmin/Lmax ratio was 0.37 at AR and 0.39 at BN. Estimated from all females collected at both reefs, L50 was 45.4 cm TL (95% CI: 43.6–46.9 cm TL) (Figure 6). The smallest recorded male yellowfin grouper was 35.5 cm TL (specimen in regression phase) at AR and 44.8 cm TL (specimen in spawning-capable phase) at BN. The PL50 value estimated using data from both reefs was 80.9 cm TL (95% CI: 79.3–83.0 cm TL), very similar to the 79.8 cm TL (95% CI: 78.2–81.7 cm TL) for BN only (Figure 6).

3.4. Ovarian Organisation and Spawning Pattern

Active mature females of both species displayed several oocyte batches in their ovaries at various development stages (Figure 7 and Figure 8). At the beginning of their spawning-capable phase, females exhibited both a batch of primary growth oocytes (PG) and one of secondary growth oocytes (SG); the latter consisted of cortical alveolar oocytes (CA) as well as primary (Vtg1), secondary (Vtg2), and tertiary (Vtg3) vitellogenic oocytes (Figure 7a and Figure 8a). When the spawning-capable phase was most advanced, Vtg3 oocytes had proliferated significantly, although other SG oocyte stages (CA, Vtg1, and Vtg2 oocytes) were still present (Figure 7b,c and Figure 8b,c). This asynchrony in SG oocyte development was particularly evident in the ovaries of actively spawning females (Figure 7d and Figure 8d) and past-spawner females (Figure 7e and Figure 8e). The presence of POFs in the ovaries of females in these two reproductive subphases indicated that both red hind and yellowfin grouper females produce and release several batches of hydrated oocytes over time. The presence of numerous vitellogenic oocytes in atresia in the ovaries of regressing females (Figure 7f and Figure 8f) indicates that, at the end of the spawning season and in both species, a degradation/reabsorption process of many incompletely developed SG oocytes that were not ovulated occurs.

3.5. Spawning Interval

During their respective spawning seasons, the estimated SI was 1–3 days for female red hind and 1–2 days for female yellowfin grouper (

Table 3. Spawning interval (SI) for female red hind and yellowfin grouper collected during their 2008 and 2009 spawning seasons at Alacranes Reef and Bajos del Norte, Campeche Bank, Yucatan, Mexico. ¹: spawning-capable, actively spawning, and past-spawner females. ²: actively spawning females. ³: reciprocal of the spawning fraction proportion. ⁴: d: day of moon phase; dafm: day after full moon; dbnm: day before new moon; danm: day after new moon; dlq: day of last quarter; dblq: day before last quarter; dalq: day after last quarter. TL: total length. Yellowfin grouper females in actively spawning sub-phase were not captured at Alacranes Reef.

Month/Year		Spawning Population 1					SI 3 (Day)
		Number	Size Range (cm TL)	Spawning Fraction 2
				Number	Size Range (cm TL)	Proportion		Moon Phase 4
Red hind
Alacranes Reef
February	2008	10	30.3–43.0	8	30.3–43.0	0.80	1.3	2 dafm
	2009	29	31.5–41.5	1	36.0	0.04	25.0	4 dafm
March	2009	34	25.5–44.8	31	25.5–44.8	0.91	1.1	3–5 dafm
Bajos del Norte
January	2008	11	30.3–34.6	4	30.3–34.5	0.36	2.8	1 dblq
	2009	55	29.3–45.2	28	30.1–43.6	0.51	2.0	4 db−1 danm
March	2009	17	29.0–47.0	7	37.0–45.5	0.41	2.4	4 dbnm
April	2009	27	34.0–44.5	21	34.0–44.5	0.78	1.3	1 db−1 dalq
Yellowfin grouper
Bajos del Norte
March	2008	63	48.0–72.0	55	48.0–72.0	0.87	1.2	dlq−2 dalq
	2009	105	49.5–75.0	60	50.5–75.0	0.57	1.8	1–4 dalq
April	2008	62	44.3–80.0	33	44.3–74.5	0.53	1.9	1 db−2 dalq/2–3 danm
	2009	95	49.0–81.0	93	49.0–81.0	0.98	1.0	3–1 dblq
May	2008	8	50.1–70.5	1	50.1	0.13	7.7	2 dalq

). Due to an unrepresentative sample size of actively spawning females (n = 1 month⁻¹), the SI values calculated for red hind in February 2009 (SI = 25.0) and for yellowfin grouper in May 2008 (SI = 7.7) were considered inaccurate. In actively spawning females of both species, no close relationship was observed between capture date and a specific phase of the moon. At AR, actively spawning female red hinds were caught on days near the full moon, while at BN, actively spawning females of both species were caught on days near the last quarter or the new moon (Table 3).

3.6. Fecundity

For female red hind at both study sites, BF varied from 8710 to 471,726 oocytes while RF ranged from 15 to 575 oocytes g⁻¹. Average (±SE) BF and RF were 137,289 ± 17,144 oocytes and 224 ± 25 oocytes g⁻¹, respectively (Table 4). For female yellowfin grouper at BN, BF ranged from 25,986 to 1,806,624 oocytes, and RF from 13 to 452 oocytes g⁻¹. Average (±SE) BF and RF were 406,800 ± 48,030 oocytes and 138 ± 15 oocytes g⁻¹, respectively (Table 4). The ratios calculated between BF and TL or WW were, for red hind:

BF = −58,007 + 5455 × TL (R² = 0.04; n = 36)

BF = 46,574 + 138 × WW (R² = 0.11; n = 36)

and for yellowfin grouper:

BF = −1,556,158 + 31,977 × TL (R² = 0.24; n = 61)

BF = −168,866 + 196 × WW (R² = 0.24; n = 61)

The calculated linear regression slopes differed from zero (p < 0.05), except for the BF/TL ratio in red hind.

Table 4. Batch fecundity, relative fecundity, size, and weight of red hind and yellowfin grouper females collected during their 2008 and 2009 spawning seasons at Alacranes Reef and Bajos del Norte, Campeche Bank, Yucatan, Mexico. TL: total length. WW: whole-body weight. SE: standard error. Yellowfin grouper females in actively spawning sub-phase were not captured at Alacranes Reef.

Number	Size (cm TL) Mean ± SE (Range)	Weight (g WW) Mean ± SE (Range)	Batch Fecundity (Oocytes Number) Mean ± SE (Range)	Relative Fecundity (Oocytes Number g−1 of WW) Mean ± SE (Range)
Red hind
Alacranes Reef
20	34.0 ± 0.8 (25.5–41.8)	623 ± 46 (240–1120)	160,061 ± 26,694 (29,793–471,726)	254 ± 34 (58–575)
Bajos del Norte
16	38.0 ± 0.6 (34.0–43.6)	700 ± 75 (300–1300)	108,824 ± 17,819 (8710–278,184)	186 ± 36 (15–454)
Total
36	35.8 ± 0.6 (25.5–43.6)	657 ± 42 (240–1300)	137,289 ± 17,144 (8710–471,726)	224 ± 25 (15–575)
Yellowfin grouper
Bajos del Norte
61	61.4 ± 0.7 (44.3–72.1)	2938 ± 121 (1200–5700)	406,800 ± 48,030 (25,986–1,806,624)	138 ± 15 (13–452)

Table 4. Batch fecundity, relative fecundity, size, and weight of red hind and yellowfin grouper females collected during their 2008 and 2009 spawning seasons at Alacranes Reef and Bajos del Norte, Campeche Bank, Yucatan, Mexico. TL: total length. WW: whole-body weight. SE: standard error. Yellowfin grouper females in actively spawning sub-phase were not captured at Alacranes Reef.

Number	Size (cm TL) Mean ± SE (Range)	Weight (g WW) Mean ± SE (Range)	Batch Fecundity (Oocytes Number) Mean ± SE (Range)	Relative Fecundity (Oocytes Number g−1 of WW) Mean ± SE (Range)
Red hind
Alacranes Reef
20	34.0 ± 0.8 (25.5–41.8)	623 ± 46 (240–1120)	160,061 ± 26,694 (29,793–471,726)	254 ± 34 (58–575)
Bajos del Norte
16	38.0 ± 0.6 (34.0–43.6)	700 ± 75 (300–1300)	108,824 ± 17,819 (8710–278,184)	186 ± 36 (15–454)
Total
36	35.8 ± 0.6 (25.5–43.6)	657 ± 42 (240–1300)	137,289 ± 17,144 (8710–471,726)	224 ± 25 (15–575)
Yellowfin grouper
Bajos del Norte
61	61.4 ± 0.7 (44.3–72.1)	2938 ± 121 (1200–5700)	406,800 ± 48,030 (25,986–1,806,624)	138 ± 15 (13–452)

4. Discussion

The reproductive life history of red hind and yellowfin grouper in the Gulf of Mexico has received minimal attention. Some data on the reproductive season and fecundity of both species are available for the populations of the northeastern Gulf of Mexico (Florida Middle Ground) [27]. Spawning aggregations formed by these grouper species were identified at AR and BN, in the SGM [26]. The present study contains valuable new data on the diverse reproductive traits of the two most abundant reef-associated grouper species from the CB [43,44].

In both species, females and males were smaller at AR than at BN, a difference possibly linked to the distinct morphological characteristics (i.e., emerged vs. submerged bank) at the two reef systems. However, at both reefs, these groupers’ spawning aggregation sites occur within the same depth range (10 to 27 m), and both sites benefit from increases in nutrient supply provided by the seasonal (April–June) upwelling of cold subsurface water from the Caribbean Sea through the Yucatan Channel [26,70].

Therefore, further study of the prevailing environmental factors at each site will be needed before any link to differences in growth can be claimed. Fishing is recognised as one of the main causes of changes in the age/size demographic structure of exploited fish populations because it often selectively eliminates the oldest/largest individuals [71,72]. Prior to the implementation of the AR management plan in 2007, it is quite possible that fishing pressure on exploited species was greater at AR than at BN, due to easier access (i.e., distance from the coast) and shelter (i.e., habitable islands) for semi-industrial and recreational fishers at AR. However, no catch data are available for either of the studied reefs in this earlier period. Sampling at AR was fishery-independent, involving a single diver, whereas at BN it was fishery-dependent, involving several divers. These discrepancies could have led to greater opportunities for capturing larger specimens at BN than at AR. Indeed, the total number of individuals collected at BN was much higher than at AR (see Table 1). Similar female and male size and sample size differences between both reefs have also been observed for the tiger grouper in a previous study [73]. Continued systematic sampling following a strategy like that implemented in the present study could help to eventually explain the observed size differences between reefs.

Overall sex ratio for yellowfin grouper at BN was skewed towards females, which is generally the case for protogynous hermaphroditic fish populations [52,62]. In contrast, for red hind, the ratio was balanced at AR and biased towards males at BN. These are atypical sex ratios for protogynous species and may be related to the different movement patterns of females and males during aggregation [74]. Similar results have been reported for the red hind aggregations at the Bahamas [13] and the Netherlands Antilles [23], and also for the tiger grouper aggregation at BN [73]. It has been observed that, due to their reproductive behaviour, male red hinds arrive earlier and stay longer at spawning aggregation sites than females [19,23,75]. Consequently, for a few days before aggregation formation, the sex ratio is skewed in favour of males, but later becomes balanced and then skews in favour of females when they arrive at spawning sites [76]. Similar temporal changes in sex ratio have also been reported for yellowfin grouper in the USVI [21,22]. The remaining population characteristics analysed here for both species (female sizes vs. male sizes; sex ratio by size classes) were consistent with sexuality patterns previously reported for these two species [14,16,30,31].

At both AR and BN, the spawning season of red hind began earlier (January) than that of yellowfin grouper (March). In addition, the red hind peak spawning period was longer (four months) than that of yellowfin grouper (two months), with the two species spawning simultaneously in March and April at the same aggregation site in BN. These results, based on observations made from 2008 to 2009, confirm previous observations, made from 2001 to 2004 on the spatiotemporal formation of these two-grouper species’ spawning aggregations at these reef systems [26]. More than four years later, red hind and yellowfin grouper continued to spawn at the same sites and during the same season. Although the monthly density of individuals at the studied aggregation sites was not estimated in the present study, the higher number of mature individuals of both species captured during their respective spawning seasons strongly suggests that these groupers continued to form spawning aggregations at the same sites. The reduced number of inactive or active mature individuals captured at the sites after the respective spawning seasons implies that most red hind and yellowfin grouper spawners had abandoned the sites at the end of their spawning seasons. Similar behaviour has been observed in Bermuda and the USVI for red hind spawners [12,77] and in the USVI for yellowfin grouper spawners [21,22]. The red hind spawning seasons observed here at both studied reefs were similar to those reported for red hind populations from the Bahamas [13] and various regions of the Caribbean Sea [15,16,17,18,19,20,23,24,25,77]. However, in Bermuda and in the northeast Gulf of Mexico, red hind has been observed to reproduce later: between May and July, with a spawning peak in June, and between April and August, respectively [12,27,78]. The later spawning season in Bermuda may be explained by prevailing local environmental conditions (temporal variation in temperature and peak of primary production) [12]. The limited data provided for red hind in the northeast Gulf of Mexico (such as the number of individuals analysed monthly) prevents any interpretation or comparison of results [27]. The yellowfin grouper spawning season observed here at both studied reefs was similar to those observed for populations in the north-eastern Gulf of Mexico [27], Bahamas [13], Cuba [14], and the USVI [21,22]. Regardless of region, red hind and yellowfin grouper spawning peaks are generally associated with the full moon [18,20,21,23,25,74,75,77,79,80]. This coincides with the present data for red hind at AR, where actively spawning females were collected from 2 to 5 days after the full moon in February and March. However, red hind and yellowfin grouper at BN did not follow this trend since they spawned on days near the new moon or last quarter moon in January, March, and April. Apparently, spawning synchronisation with a particular lunar phase can vary temporally in groupers. Indeed, in a study performed in Puerto Rico, red hind was found to spawn in January on days close to the new moon, whereas in February it occurred near the full moon [19].

Previous research suggested that red hind largely exhibited synchronous ovarian organisation and asynchronous ovulation (batch spawning) [19,25]. The present results confirm asynchronous ovulation in red hind but also suggest asynchronous ovarian organisation. The constant presence of CA, Vtg1, and Vtg2 oocytes in the ovaries of mature spawning-capable, actively spawning, and past-spawner females implies continuous recruitment of new oocytes in SG from the oocyte batch in PG during the spawning season. The spawning frequency in red hind females could be more than once during annual spawning aggregation formation [19]. In the present results, it is estimated that females can spawn repeatedly within a 1-to-3-day interval during their spawning season. At both AR and BN, yellowfin grouper exhibited an asynchronous ovarian organisation and ovulation, a pattern previously observed in Cuba [14] and the USVI [22]. In the latter study, the authors calculated that yellowfin grouper females generally spawn every 2 to 3 nights (SI = 2.3–2.4 days), and some could even spawn daily. This is similar to the frequency observed here for yellowfin grouper on the CB (SI = 1.0–1.9 days).

Gonadosomatic index values in male groupers can function as a proxy index for possible sperm competition between individuals and can provide information on spawner mating patterns at the time of spawning [19]. When in aggregations, groupers can engage in pair spawning or group spawning [81]. In pair spawning, males do not compete to fertilise oocytes released by females and thus have no need to produce large amounts of sperm. When they mature, these males develop testes of reduced size/weight (low GSI values). In group spawning, males compete among themselves to mate with a female. To increase the probability of fertilising the released oocytes, they produce a large amount of sperm and consequently, when mature, have larger/heavier testes (high GSI values) [66]. On the studied reefs, the highest individual GSI value observed for male yellowfin groupers (GSI = 9.4%) was higher than for male red hinds (GSI = 2.2–2.8%). Similar results have been reported for male yellowfin groupers in Cuba (GSI = 6.8%) [14], Jamaica (GSI = 6.3%) [15], and the Bahamas (GSI = 8.3%) [13], as well as for male red hinds in Puerto Rico (GSI < 1%) [19] and Antigua and Barbuda (GSI = 1.6%) [24]. These results suggest that, at spawning, sperm competition would be higher among male yellowfin groupers than among male red hinds. This is supported by reports showing that, when spawning, yellowfin grouper spawners form groups consisting of 7 to 12 males and a single female [22], while red hind spawners form smaller social units consisting of one male with several females or in a one male to one female ratio [18,79,82].

A significant correlation has been observed between female Lmin and Lmax in several grouper populations, and their Lmin/Lmax ratios vary from 0.33 to 0.74 (average = 0.51) [62]. The present Lmin/Lmax results for both species differed minimally between reefs: Lmin/Lmax = 0.52 (AR) and 0.47 (BN) for red hind and Lmin/Lmax = 0.37 (AR) and 0.39 (BN) for yellowfin grouper, and they were consistent with the data range previously reported [62]. Interpreting growth in length using the Lmin/Lmax values indicated that yellowfin grouper females tended to mature earlier (range: 37–39% of maximum size) than red hind females (range: 47–52% of maximum size). For red hind, overall L50 (22.0 cm TL) was unusually lower than the Lmin values for females at AR (25.2 cm TL) and BN (24.0 cm TL). In addition, the smallest male (21.1 cm TL) collected at AR was smaller than the Lmin for females at AR, a normal discrepancy in protogynous species exhibiting diandry (presence of primary males). However, the red hind L50 at the studied reefs did not notably differ from those reported in the Bahamas (L50 = 23.5 cm TL) [13] and in Puerto Rico (L50 = 21.5 cm TL) [19]. It is therefore possible that the scarcity of red hind females smaller than 30 cm TL (n = 19 at AR, n = 8 at BN), in conjunction with the absence of females smaller than 20 cm TL in the overall collections, could have resulted in overestimation of red hind Lmin values. A higher L50 value of 38.0 cm TL has been reported for red hind in Venezuela [25], although this is probably an overestimation because the analysis did not include females < 26.5 cm TL. Sizes at maturity in yellowfin grouper at the reefs studied here (Lmin = 31.2 and 37.0 cm TL and L50 = 43.3 cm TL) were lower than those reported in the Bahamas (Lmin = 40.7 cm TL, L50 = 56.1 cm TL) [13] and Cuba (Lmin = 52.8 cm TL) [14]. Through an adaptive process, selective fishing of the largest females in a fish population can induce maturity in smaller-sized/younger females [83,84]. Therefore, these differences in yellowfin grouper size at maturity could be explained by higher selective fishing mortality at the studied reefs than that experienced in the Bahamas and Cuba. However, the presence of large-sized females and males at both AR and BN (see Table 1) suggests this is not the case. Furthermore, the L50 results observed in the Bahamas [13] and Cuba [14] could be overestimates created by the small number of individuals included in both studies (n = 298 females and n = 135 females or males, respectively) and/or errors in discriminating between immature females and inactive mature females in regeneration phase.

No PL50 values have been published for other red hind or yellowfin grouper populations. However, the PL50 values calculated here for red hind (38.2 and 39.0 cm TL) are generally similar to the estimates of mean size at sex change for red hind populations in Bermuda (35.0 cm TL) [12], Jamaica (38.0 cm TL) [15], Antigua and Barbuda (37.1 cm TL) [24], and the Bahamas (range: 25.7–40.1 cm T) [13]. Yellowfin grouper PL50 values at the studied reefs (79.8 and 80.4 cm TL) were also similar to estimated size at sex change in the Bahamas (range: 71.6–87.1 cm TL) [13].

Because they attained larger maximum sizes, female yellowfin grouper had higher maximum BF values than female red hind in the studied reefs. The estimated BF for tiger grouper at BN (range: 84,119–1,032,578 oocytes; n = 31) was similar to that observed here for yellowfin grouper since female tiger grouper also attains a large maximum size (Lmax = 71 cm FL) [73]. The present fecundity data were generally difficult to compare to those for grouper populations from other geographic regions. The exception is BF results for USVI yellowfin grouper estimated based on OLM counts [85]. All other studies have attempted to estimate an annual potential fecundity by counting all the developing oocytes in the ovaries [12,15,25,27,86], and none of them have used methodologies that consider that both red hind and yellowfin grouper exhibit asynchronous ovarian organisation. The resulting estimated annual potential fecundity values are therefore probably underestimated.

The Lmax values for red hind and yellowfin grouper at the studied reefs suggest that, in the late 2000s, their populations were still in a good state of conservation. The Lmax values for female and male red hind from AR (48.5 and 49.5 cm TL, respectively) and BN (51.0 and 57.5 cm TL, respectively) exceeded the Lmax values reported for this species at the Red Hind Bank Marine Conservation District (MCD) at St. Thomas, USVI. This MPA was closed seasonally beginning in 1990 and permanently since 1999. In 2000, the female red hind Lmax value was 43.9 cm TL, and that for males was 48.0 cm TL [74], while in 2018–2020, overall Lmax was 48.2 cm TL [76]. This also held true for yellowfin grouper in the sense that Lmax values for females and males at AR (85.3 and 92.0 cm TL, respectively) and BN (94.0 and 100.0 cm TL, respectively) were similar to or higher than the Lmax values reported for this species at the MPA of Mona Island, Puerto Rico (85 cm FL), designated a nature reserve in 1986 [83], and at Grammanik Bank (GB), USVI (88.9 cm TL for females and 91.5 cm TL for males), seasonally closed since 2005 [22].

Many researchers believe MPAs, and particularly no-take fishery reserves, to be an effective management strategy for grouper transient spawning aggregation conservation and even recovery [75,76,87,88,89,90,91,92,93]. At AR, the red hind spawning aggregation site has been fully protected since 2007 because it is located in the NP’s north core zone, where commercial and recreational fishing is prohibited [46]. Collection of spawning-capable and past-spawner yellowfin grouper females at other sites in the AR suggests that this grouper may also aggregate to spawn at this reef. If these aggregation sites are located in the NP’s core zones, they would also be protected. The core zone at BN encompasses a red hind/yellowfin grouper multispecies spawning site, but its recent creation (2024) means this NP does not yet have official management guidelines. Since 2003, red hind and yellowfin grouper reproduction at AR and BN was partially protected by a one-month seasonal grouper fishery closure (15 February–15 March), which, in 2017, was extended to two months (February–March) [94,95]. To date, spawning aggregations of both red hind and yellowfin grouper in BN still remain vulnerable to fishing for several months of their respective spawning seasons: January and April for red hind and April for yellowfin grouper. In the future (once its management program is official), the BN NP’s core zone will offer comprehensive protection to red hind and yellowfin grouper spawning aggregations, as well as to the tiger grouper spawning aggregation that forms between February and May at another site inside its core zone [26,73]. Both NPs may also help protect black grouper during its spawning season. Indeed, the collection of active mature female black groupers at AR and BN [44,45,96] suggests that this species also forms spawning aggregations at both reefs.

Notwithstanding, several studies imply that MPAs are not always the most appropriate tool for protecting fish species that aggregate to spawn, nor for providing direct benefits to the fisheries targeting them [97]. This is especially true for small-surface-area MPAs, which offer less protection for mobile species than for sedentary ones [98,99]. Red hind and yellowfin grouper form transient spawning aggregations, implying that spawners migrate annually for several days to weeks toward aggregation sites [81]. In species that migrate long distances to reach and leave spawning sites, spawners can move out of an MPA’s boundaries into areas where they are vulnerable to fishing. No data are yet available on the movements of red hind and yellowfin grouper on the CB, and it is therefore unknown if the core zones of the AR and BN NPs provide any protection to the migration routes of the spawners as they move between aggregation and residence sites. Most of each NP’s surface area consists of buffer zones (70% at BN, 80% at AR; see Figure 1) where semi-industrial and recreational fishing are or will be authorised, and therefore, it is unlikely that NPs will protect spawners outside of the core zones during their spawning seasons. Red hind has been observed to transit up to 33 km between reefs when migrating towards its spawning aggregation sites in Bermuda [78,100], Puerto Rico [18,19], and the USVI [74,75]. Red hind spawners are also reported to spread over areas of 90 to 500 km² around their aggregation sites in the USVI [74,75]. Yellowfin grouper spawners in the USVI have been observed to move continuously between two MPAs (GB and MCD) 2 km apart, separated from each other by an unprotected zone [101,102]. To increase MPAs’ efficiency, some researchers suggest adding an adaptive approach to the implementation of traditional management measures such as seasonal fishery closures [97]. For example, in Mexico, the two-month CB-wide seasonal closure, applying to all grouper species, may provide some protection to red hind and yellowfin grouper as they migrate towards their aggregation sites in AR and BN.

5. Conclusions

The reproductive conservation status of red hind and yellowfin grouper from Campeche Bank and the efficiency level of the AR and BN NPs for preservation of their spawning aggregations are currently undetermined. Ten years after its implementation, the AR NP management plan is reported to be inefficient for fisheries management [102]. After the conclusion of the collection period in the present study, grouper catches on CB have dropped 42% between 2010 (8728 t) and 2023 (5092 t) [35,103,104]. Nevertheless, the present results suggest that the core zones at both NPs will protect the “source” sites for egg and larvae production for red hind, yellowfin grouper, tiger grouper and eventually black grouper. Furthermore, both AR and BN also probably function as grouper settlement and nursery areas, since several immature female red hind, yellowfin grouper, and tiger grouper have been documented in the aggregation sites at the core zones of both NPs [73]. However, there are no data available on the degree of grouper population connectivity between these two reefs, preventing any conclusions that the current NP designs will allow populations of these three groupers to be self-sustaining at each reef. It is unknown if this protection will guarantee life cycle completion within NP boundaries. Gaps in knowledge on larval drift and the movement patterns of juveniles and adults make it unwise to predict if both NPs contribute to sustaining or improving CB grouper fisheries.