Bush, P.G.

Jackson, A.

Nemeth, R.S.

Semmens, B.X.

Aquatic Commons

 Proceedings of the 63rd Gulf and Caribbean Fisheries Institute     November 1 - 5, 2010   San Juan, Puerto Rico 
 
Genetic Connectivity of Nassau Grouper Aggregations in the Caribbean Sea 
 
ALEXIS M. JACKSON1*, BRICE X. SEMMENS2, RICHARD S. NEMETH3 , and PHILLIP G. BUSH4  
1Department of Ecology and Evolutionary Biology, University of California Santa Cruz,  
100 Shaffer Road, Santa Cruz, California 95060 USA.  * jackson@biology.ucsc.edu .   
 2Reef Environmental Education Foundation (REEF), PO Box 246, Key Largo, Florida 33037 USA. 
3Center for Marine and Environmental Studies, University of the Virgin Islands,  
#2 John Brewer’s Bay, St. Thomas, Virgin Islands 00802 USA.      
4Cayman Islands Department of the Environment, PO Box 486GT, Grand Cayman, Cayman Islands. 
 
ABSTRACT  
Nassau grouper (Epinephelus striatus) form spatially and temporally predictable spawning aggregations that make them 
particularly susceptible to overfishing.  It is the objective of this study to determine how sustainable aggregation-based fisheries are 
by determining the extent of genetic connectivity between aggregations.  Genomic DNA was extracted from samples collected in 
aggregations in the Cayman Islands and U.S. Virgin Islands.  Three mitochondrial markers (12S, ATP synthase, cytochrome b) were 
sequenced and 9 polymorphic microsatellite loci were isolated.  Statistical analyses were performed on mitochondrial sequences to 
determine population structure and genetic connectivity.  Preliminary results reveal high genetic connectivity and no statistically 
significant genetic structure at either the aggregation level or regional level between aggregations in the Cayman Islands and the 
U.S. Virgin Islands (fst = 0.01283, p-value = 0.74585).  Such results suggest that spawning aggregations do not represent distinct 
populations. 
 
KEY WORDS:  Genetic connectivity, Epinephelus striatus, spawning aggregations, U.S. Virgin Islands, Cayman Islands 
 
 
Conectividad Genética de Nassau Grouper Agregaciones en el Mar Caribe 
 
PALABRAS CLAVE:   Conectividad genética, Epinephelus striatus, agregaciones, Islas Vírgenes de E.U  
 
 
Connectivité Génétique des Agrégations Nassau Grouper dans la Mer des Caraïbes 
 
MOTS CLÉS:  Connectivité génétique, Epinephelus striatus, agrégations, Îles Vierges Américaines  
connectivity must be quantified to determine which 
aggregations are most at risk of extinction and which are 
supplying fish to other aggregations within the region.  
Analyses will determine at what spatial scale genetic 
variation is partitioned for Nassau grouper throughout the 
Caribbean.  Increased knowledge of spawning aggregation 
dynamics will also benefit other species of aggregating 
marine fish, as reproductive aggregation behavior has been 
observed in at least 11 other families of reef fish (Domeier 
and Colin 1997). 
 
MATERIALS AND METHODS   
This study utilized samples collected from aggrega-
tions in the Cayman Islands and the U.S. Virgin Islands.  
The first set was collected from three aggregation sites in 
the Cayman Islands from 2005 to 2009.  A total of 102 fin 
clips were collected, with 8 from the aggregation at Grand 
Cayman, 70 from the aggregation at Little Cayman and 27 
from the aggregation at Cayman Brac, and stored in 
sarcosyl urea.  The second set was collected from one 
aggregation site in the U.S. Virgin Islands in March 2010.  
A total of 78 fin clips were collected and stored in 95% 
ethanol.   
Genomic DNA was extracted from sarcosyl urea 
INTRODUCTION  
Nassau grouper (Epinephelus striatus) form spatially 
and temporally predictable spawning aggregations, 
comprised of hundreds to thousands of fish, which are 
specifically targeted by small-scale fisheries (Smith 1972, 
Aguilar-Perera and Aguilar-Davila 1996, Sadovy and 
Eklund 1999, Ehrhardt and Deleveaux 2007). These 
ecologically important top predators regulate species 
composition and abundances of coral reef species. Studies 
also show that increased presence of groupers on reefs 
increases the likelihood of recruitment on reefs for other 
coral reef fish species (Stallings 2008).  Thus increased 
fishing of Nassau grouper potentially makes marine 
communities more vulnerable to natural and anthropogenic 
disturbances (Friedlander and DeMartini 2002).  Reports 
by fisherman since the 1980s have suggested depleted 
catch (Whaylen et al. 2006) and aggregation extinctions 
(Aguilar-Perera and Aguilar-Davila 1996, Aguilar-Perera 
2006) despite limited understanding of – 1) ultimate loss of 
genetic diversity due to overfishing and 2) the extent of 
genetic connectivity between spawning aggregations.  It is 
the objective of this study to determine how sustainable 
aggregation-based fisheries are for Nassau grouper.  In 
order to address this question, the extent of genetic 
Page 162  63rd Gulf and Caribbean Fisheries Institute  
 
preserved tissues with the use of the Qiagen DNeasy mini 
kit using the manufacturer’s protocols.  Genomic DNA was 
extracted from ethanol preserved tissues using the standard 
chloroform extraction and isopropanol precipitation 
methods (Sambrook et al. 1989). Three mitochondrial 
markers were sequenced for each sample. A 357 bp 
fragment of 12S rRNA was amplified with primers 12Sal 
(5’AAACTGGGATTAGATACCCCACTAT-3’) and 
12Sbr (5’AGAGTGACGGGCGGTGTGT-3’).  A 634 bp 
fragment of the ATP synthase units 6 and 8 were amplified 
with primers L8331 (5’-AAAGCRTYRGCCTTTTAAGC-
3’) and H9236 (5’ GTTAGTGGTCAKGGGCTTGGRTC 
3’).  A 785 bp fragment of cytochrome b was amplified 
with primers gludgL (5’-
TGAYTTGAARAACCAYCGTTG-3’) and CB3H 
(5’GGCAAATAGGAARTATCATTC-3’) (Palumbi et al. 
1991). All markers were sequenced in the forward 
direction using an ABI 3700 sequencer.  Nine polymorphic 
microsatellite loci were cross-amplified using primer pairs 
designed for Epinephelus acanthistius.  Loci isolated 
represent di and tetra repeats of the following kind - (CA)
x  , (CATC)x , (TACA)x and (TAGA)x .  Sequencher 4.5 
was used to align mitochondrial sequences.  MacClade was 
used to identify informative sites.  Arlequin (Excoffier et 
al. 2005) was used to perform an analysis of molecular 
variance (AMOVA) associated with population structure 
and genetic connectivity in order to generate fst values, and 
identify unique haplotypes.  Additionally, Migrate-n was 
used to determine directional gene flow. 
 
RESULTS AND DISCUSSION  
Analysis of mitochondrial sequences identified 14 
informative sites, providing enough power with which to 
recover statistically significant genetic differentiation if 
present.  Informative sites defined 19 haplotypes, which 
exhibit little genetic divergence from one another and 
whose distribution is not associated with any geographic 
patterns (Figure 1).  Preliminary results suggest a lack of 
genetic structure at either the aggregation level or at a 
regional level between the aggregations in the Cayman 
Islands and the U.S. Virgin Islands.  The AMOVA 
revealed high levels of historical gene flow between 
aggregations based on non-significant fst values (fst = 
0.01283, p = 0.74585), demonstrating genetic homogeneity 
and extensive interbreeding among aggregations.  Output 
from Migrate-n revealed that observed panmixia has been 
facilitated by extensive directional gene flow from 
aggregations in the U.S. Virgin Islands to aggregations in 
the Cayman Islands (15,637 ± 3603 migrants).  Lack of 
genetic structure between spawning aggregations has been 
observed in other population genetic studies of aggregation
-forming species (Shulzitski et al. 2009, Shaw et al. 2010), 
and suggests that aggregations do not represent distinct 
populations.  While genotyping of all samples for the nine 
isolated microsatellites could potentially resolve finer scale 
genetic structure among aggregations, studies have shown 
that microsatellites may not reveal any more structure than 
mitochondrial sequences (Haavie et al. 2000), in particular 
in a case where mitochondrial sequences are variable 
enough to determine genetic structure. These results are 
important for management, as aggregations of Nassau 
grouper continue to disappear throughout the Caribbean.  If 
the species is panmictic throughout its range then aggrega-
tion-based fisheries could have less of an impact on loss of 
genetic diversity for the species than if aggregations 
represented genetically distinct populations. 
Figure 1.  Haplotype Network for Nassau grouper 
(Epinephelus striatus) based upon 3 mitochondrial loci. 
Individual sequences from aggregations are color coded as 
follows – blue (Little Cayman), purple (U.S. Virgin Islands), 
green (Grand Cayman) and red (Cayman Brac).  Each 
circle represents a unique haplotype and is proportional in 
size to the number of individuals from the dataset that 
possess it.  Each notch mark represents a single mutational 
change. 
 
CONCLUSIONS  
If spawning aggregations are in fact the most appropri-
ate biological unit by which to define subpopulations for 
Nassau grouper then preliminary results from this study 
suggest genetic homogeneity between aggregations in the 
Cayman Islands and the U.S. Virgin Islands.  Expanding 
the current study to include spawning aggregations from 
other localities in the Caribbean will determine if there is 
any larger scale regional population structure revealed by 
aggregations; it will be important to determine if the 
species is actually panmictic throughout its range.  All 
significant results of the final study should be incorporated 
to regional management plans for Nassau grouper. 
 
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Genetic connectivity of Nassau grouper aggregations in the Caribbean Sea.

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Genetic connectivity of Nassau grouper aggregations in the Caribbean Sea.

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