INTRODUCTIONTop

The Atlantic goliath grouper Epinephelus itajara (Lichtenstein, 1822) is the largest grouper in the Atlantic Ocean, where it is found in tropical and subtropical waters ranging from the state of Florida in the USA to southern Brazil, as well as on the western coast of Africa from the Congo Republic to Senegal (Craig et al. 2012Craig M.T., Sadovy de Mitcheson Y.J., Heemstra P.C. 2012. Groupers of the World: A Field and Market Guide, CRC Press. 424 pp.). This grouper is listed as critically endangered by the IUCN (International Union for Conservation of Nature), based on criterion A2d (IUCN 2015IUCN. 2015. The IUCN (International Union for Conservation of Nature) Red List of Threatened Species. Version 2015.2. Downloaded on 27 July 2015. www.iucnredlist.org).

Slow growth, longevity and behavioural traits such as the tendency to form breeding groups make E. itajara populations susceptible to fishing pressure (Bullock et al. 1992Bullock L.H., Murphy M.D., Godcharles M.F., et al. 1992. Age, growth, and reproduction of jewfish Epinephelus itajara in the eastern Gulf of Mexico. Fish. Bull. 90: 243-249.). Migration to key locations for the purposes of breeding, observed in most species of the subfamily Epinephelinae (Coleman et al. 2011Coleman F.C., Scanlon K.M., Koenig C.C. 2011. Groupers on the Edge: Shelf Edge Spawning Habitat in and Around Marine Reserves of the Northeastern Gulf of Mexico. Prof. Geogr. 63(4): 1-19.), has not been fully clarified in E. itajara (Mann et al. 2008Mann D.A., Locascio J.V., Coleman F.C., et al. 2008. Goliath grouper Epinephelus itajara sound production and movement patterns on aggregation sites. Endang. Species Res. 7: 229-236. ) and, in fact, both adults and juveniles exhibit location fidelity (Eklund and Schull 2001Eklund A.M., Schull J. 2001. A stepwise approach to investigate the movement patterns and habitat utilization of goliath grouper, Epinephelus itajara, using conventional tagging, acoustic telemetry and satellite tracking. In: Sibert J.R. and Nielsen J.L. (eds) Electronic tagging and tracking in marine fisheries. Springer-Verlag, New York. pp. 189-216., Koenig et al. 2007Koenig C.C., Coleman F.C., Eklund A.M., et al. 2007. Mangroves as Essential Nursery Habitat for Goliath grouper (Epinephelus itajara). Bull. Mar. Sci. 80(3): 567-586.). However, there is evidence based on individual markings that E. itajara breeding groups occur at specific locations and that the adults are capable of travelling long distances to areas far from the coast (Pina-Amargós and Gonzaléz-Sansón 2009Pina-Amargós F., González-Sansón G. 2009. Movement patterns of goliath grouper Epinephelus itajara around southeast Cuba: implications for conservation. Endang. Species Res. 7: 243-247.).

The dispersal of pelagic larvae is correlated with connectivity among different environments and likely depends on local oceanographic patterns, the biological attributes of the larvae, and specific aspects of the reproductive behaviour of the adults, which facilitate local recruitment (Mora and Sale 2002Mora C., Sale P.F. 2002. Are populations of coral reef fishes open or closed? Trends Ecol. Evolut. 17: 422-428.). The long larval phase suggests a considerable dispersal capacity, which may result in a lack of stock structuring on oceanic, regional and subregional scales (Aboim et al. 2005Aboim M.A., Menezes G.M., Schlitt T., et al. 2005. Genetic structure and history of populations of the deep-sea fish Helicolenus dactylopterus (Delaroche, 1809) inferred from mtDNA sequence analysis. Mol. Ecol. 14: 1343-1354. ). The hypothesis of highly vagile E. itajara larvae is supported by the presence of a broad genetic-evolutional unit along the Atlantic coast of South America (Benevides et al. 2014Benevides E.A., Vallinoto M., Filho A.F., et al. 2014. When physical oceanography meets population genetics: the case study of the genetic/evolutionary discontinuity in the endangered Goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species. Biochem. Sys. Ecol. 56: 255-266.). Larval retention by species with aggregated spawning may be selectively advantageous to ensure the access of larvae and juveniles to necessary resources, but a high degree of dispersal and connectivity may not necessarily ensure homogeneous gene flow (Portnoy et al. 2013Portnoy D.S., Hollenbeck C.M., Renshaw M.A., et al. 2013. Does mating behaviour affect connectivity in marine fishes? Comparative population genetics of two protogynous groupers (Family Serranidae). Mol. Ecol. 22: 301-313.).

The first molecular data on E. itajara highlighted the phylogenetic position of the species in the subfamily (Epinephelidae sensu stricto) (Craig and Hastings 2007Craig M.T., Hastings P.A. 2007. A molecular phylogeny of the groupers of the subfamily Epinephelinae with a revised classification of the tribe Epinephelini. Ichthyol. Res. 54: 1-17.) and the genetic features of some populations in northern Brazil, based on the mitochondrial control region (Silva-Oliveira et al. 2008Silva-Oliveira G.C., Rêgo P.S., Schneider H., et al. 2008. Genetic characterisation of populations of the critically endangered Goliath grouper (Epinephelus itajara, Serranidae) from the Northern Brazilian coast through analyses of mtDNA. Genet. Mol. Biol. 31(4): 988-994.). It was subsequently determined that goliath groupers in the Pacific constitute a cryptic species (E. quinquefasciatus) in comparison with the populations of the Atlantic (E. itajara), as demonstrated by their cytochrome B sequences (Craig et al. 2009Craig M.T., Graham R.T., Torres R.A., et al. 2009. How many species of goliath grouper are there Cryptic genetic divergence in a threatened marine fish and the resurrection of a geopolitical species. Endang. Species Res. 7: 167-174. ). Silva-Oliveira et al. (2012)Silva-Oliveira G.C., Silva A.B.C., Oliveira Y., et al. 2012. New nuclear primers for molecular studies of Epinephelidae fishes. Conservation Genet. Resour. 5: 165-168., Seyoum et al. (2013)Seyoum S., Tringali M.D., Barthel B.L., et al. 2013. Isolation and characterization of 29 polymorphic microsatellite markers for the endangered Atlantic goliath grouper (Epinephelus itajara), and the Pacific goliath grouper (E. quinquefasciatus). Conservation Genet. Resour. 5: 729-732. and Silva-Oliveira et al. (2014)Silva-Oliveira G.C., Silva A.B.C., Blanchard F., et al. 2014. Primers for the amplification of the MHC IIβ chain exon 2 in the Atlantic goliath grouper (Epinephelus itajara). Conservation Genet. Resour. 6: 523-525. developed specific markers for E. itajara to establish a more effective database for population studies with evolutionary models aimed specifically at the protection of the species. Torres et al. (2013)Torres R.A., Feitosa R.B., Carvalho D.C., et al. 2013. DNA barcoding approaches for fishing authentication of exploited grouper species including the endangered and legally protected goliath grouper Epinephelus itajara. Sci. Mar. 77(3): 409-418. developed a molecular identification protocol for monitoring the fishing of groupers, including E. itajara, E. morio, Mycteroperca bonaci and M. marginata. Using simple repeat sequence markers, Benevides et al. (2014)Benevides E.A., Vallinoto M., Filho A.F., et al. 2014. When physical oceanography meets population genetics: the case study of the genetic/evolutionary discontinuity in the endangered Goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species. Biochem. Sys. Ecol. 56: 255-266. confirmed the low degree of genetic variation in E. itajara populations from ten locations, including the Atlantic coast of South America, and revealed a strong population division in southern Brazil in comparison with populations found in the eastern and northern regions of the country.

Silva-Oliveira et al. (2008)Silva-Oliveira G.C., Rêgo P.S., Schneider H., et al. 2008. Genetic characterisation of populations of the critically endangered Goliath grouper (Epinephelus itajara, Serranidae) from the Northern Brazilian coast through analyses of mtDNA. Genet. Mol. Biol. 31(4): 988-994. used a mitochondrial marker to study E. itajara populations on the northern coast of Brazil, while Benevides et al. (2014)Benevides E.A., Vallinoto M., Filho A.F., et al. 2014. When physical oceanography meets population genetics: the case study of the genetic/evolutionary discontinuity in the endangered Goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species. Biochem. Sys. Ecol. 56: 255-266. used a nuclear marker to study populations on the northern and eastern coasts. Both studies have provided important insights into the population genetics of the species. However, mitochondrial markers have not yet been used to investigate populations along the whole of the vast Brazilian coastline, despite the importance of data on both nuclear and mitochondrial markers for the development of management strategies for endangered species (Toews and Brelsford 2012Toews D.P.L., Brelsford A. 2012. The biogeography of mitochondrial and nuclear discordance in animals. Mol. Ecol. 21: 3907-3930.). A recent study of E. itajara on the Brazilian coast (Giglio et al. 2014Giglio V.J., Adelir-Alves J., Gerhardinger L.C., et al. 2014. Habitat use and abundance of goliath grouper Epinephelus itajara in Brazil: a participative survey. Neotrop. Ichthyol. 12(4): 803-810.) identified local and resident breeding groups, which point to the possibility of the formation of local, temporary populations. The aim of the present study was to evaluate gene flow using a portion of the genome (mitochondrial control region) subject to more accelerated molecular evolution (Tang et al. 2006Tang Q., Liu H., Mayden R., et al. 2006. Comparison of evolutionary rates in the mitochondrial DNA cytochrome b gene and control region and their implications for phylogeny of the Cobitoidea (Teleostei: Cypriniformes). Mol. Phylogenet. Evol. 39: 347-357. ) to determine signs of heretofore unrecognized E. itajara population divisions. The detection of population divisions is important for conservation, as it may reveal evolutionary significant units and management units, which would facilitate genetic rescue programmes and lead to an increase in the effective population size of this species.

MATERIALS AND METHODSTop

Sampling

The 218 samples of E. itajara analysed in the present study were collected at six different localities: Bragança (Pará: PA) (N=10), Tamandaré (Pernambuco: PE) (N=25), Vaza Barris (Sergipe: SE) (N=14), Caravelas (Bahia: BA) (N=50), Conceição da Barra (Espírito Santo: ES) (N=114) and Babitonga (Santa Catarina: SC) (N=5) (Fig. 1). The DNA was extracted from the caudal fin using the method described by Wasko et al. (2003)Wasko A.P., Martins C., Oliveira C., et al. 2003. Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales. Hereditas 138: 161-165.. Each specimen was weighed and measured (total and standard lengths), marked, and then released at the capture site. This procedure was determined by the licenses issued by the Sistema de Autorização e Informação em Biodiversidade (SISBIO/ICMBIO/MMA) of the Brazilian government, under numbers 25088-7 (authentication code 11928433) and 15080-2 (authentication code 84636263). All the samples were conserved in 95% ethanol and stored at –20°C in the Aquatic Vertebrates and Animal Genetics and Conservation laboratories at CEUNES/UFES.

Additional sequences of Atlantic goliath grouper were obtained in GenBank under numbers FJ176303 to FJ176329. These sequences refer to specimens collected in Bragança + Ajuruteua (Pará: PA) (N=92), called in this study by ‘Bragança’, Parnaíba (Piauí: PI) (N=12), Fortaleza (Ceará: CE) (N=2) and Natal (Rio Grande do Norte: RN) (N=9) (Silva-Oliveira et al. 2008Silva-Oliveira G.C., Rêgo P.S., Schneider H., et al. 2008. Genetic characterisation of populations of the critically endangered Goliath grouper (Epinephelus itajara, Serranidae) from the Northern Brazilian coast through analyses of mtDNA. Genet. Mol. Biol. 31(4): 988-994.) (Table 1).

DNA extraction, amplification and sequencing

The DNA was extracted using the saline protocol described by Aljanabi and Martinez (1997)Aljanabi S.M., Martinez I. 1997. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Res. 25: 4692-4693.. The control region was amplified in a total reaction volume of 12.5 µL: 1X of Taq DNA polymerase buffer (200 mM Tris-HCl, pH 8.4, and 500 mM of KCl), 1.5 mM of MgCl₂, 0.2 mM of dNTP, 1 unit of Taq DNA polymerase enzyme buffer (PHT Phoneutria^®), 10 ng/µL of DNA and 0.2 µM of the primers. The control region primers were L-Pro-1 (5’- ACT CTC ACC CCT AGC TCC CAA AG – 3’) and H-DL-C-1 (5’- CCT GAA GTA GGA ACC AGA TGC CAG – 3’) described by Ostellari et al. (1996)Ostellari L., Bargelloni L., Penzo E., et al. 1996. Optimization of single-strand conformation polymorphism and sequence analysis of the mitochondrial control region in Pagellus bogaraveo (Sparidae, Teleostei): rationalized tools in fish population biology. Anim. Genet. 27: 423-427.. The parameters for the polymerase chain (PCR) reactions was 95°C for 5 min, 34 cycles of denaturation at 95°C for 1 min, hybridization at 50°C for 1 min and extension at 68°C for 1 min, followed by a final extension at 68°C for 5 minutes.

The sequencing reaction (Sanger et al. 1977Sanger F., Air G.M., Barrell B.G., et al. 1977. Nucleotide sequence of bacteriophage φX174 DNA. Nature 265: 687-695.) was prepared for a final volume of 7 µL with 0.35 µL of primer, 1.05 µL of BigDye^® Terminator buffer v3.1 5X for sequencing, 0.7 µL of BigDye^® Terminator v3.1 Cycle and 1.0 µL of DNA. The reaction was placed for sequencing PCR with incubation at 96°C for 2 min, followed by 35 cycles of denaturation at 96°C for 30 s, annealing at 54°C for 15 s and extension at 60°C for 4 min. The amplified fragments were visualized under a blue light transilluminator in 1% agar gel in 1X TAE (Tri-Acetate EDTA) with GelGreen (Biotium^®). The sequenced DNA fragments were read in an ABI Prism 3130 (Applied Biosystems) automatic sequencer.

Alignment

The sequences were analysed and edited using the Geneious program v.4.8.5 (Drummond et al. 2009Drummond A.J., Ashton B., Cheung M., et al. 2009. Geneious v4. 7, Available from http://www.geneious.com) (http://www.geneious.com/). Mitochondrial sequences of the control region of Epinephelus itajara were validated with a search of the GenBank database for similar sequences (http://www.ncbi.nih.gov/BLAST). All sequences were aligned using the Muscle program (www.ebi.ac.uk/Tools/msa/muscle/) (Edgar 2004Edgar R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32(5): 1792-1797.). The sequences of the mtDNA control region of this study were deposited in GenBank under accession numbers KP331716 to KP331745.

Molecular analyses and Analysis of structuring and diversity

Nucleotide composition and intra-species genetic distances were estimated using the two-parameter Kimura model with 10000 bootstrap pseudoreplicates with the aid of the Mega program v.5.2 (Tamura et al. 2011Tamura R., Peterson D., Peterson N., et al. 2011. MEGA 5: Molecular Evolutionary Genetics Analysis using Maximum Likehood, Evolutionary Distance and Maximum Parsimony Methods. Mol. Biol. Evol. 28: 2732-2739.).

Nine a priori sampling units were defined according to geographical criteria: Bragança, Parnaíba, Fortaleza, Natal, Tamandaré, Vaza Barris, Caravelas, Conceição da Barra and Babitonga. For the analyses, the samples were grouped for each unit using the DnaSP v.5 program (Librado and Rozas 2009Librado P., Rosas J. 2009. DnaSP v.5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 1451-1452.). The units of Fortaleza (N=2) and Babitonga (N=5) were not considered for analyses of population structure due to the low number of samples. Intra-population diversity was analysed by estimating the haplotype diversity (h) and nucleotide diversity (π) in Arlequin v.3.5 (Excoffier et al. 2005Excoffier L., Laval L.G., Schneider S. 2005. Arlequin ver. 3.5: an integrated software package for population genetics data analysis. Evol. Bioinform. Online 1: 47-50.). Genetic differentiation between each pair of sampling groups was tested using the F_ST index (Excoffier et al. 2005Excoffier L., Laval L.G., Schneider S. 2005. Arlequin ver. 3.5: an integrated software package for population genetics data analysis. Evol. Bioinform. Online 1: 47-50.). We applied a simulated grouping approach in order to maximize the variance among the population units, using spatial analysis of molecular variance (SAMOVA) software to test for possible statistical deviations consistent with population structuring among the sampling units. Population structuring is indicated by high and significant Φ_CT values (p<0.05). Population analyses were run in the SAMOVA v.2.0 program (Dupanloup et al. 2002Dupanloup I., Schneider S., Excoffier L. 2002. A simulated annealing approach to define the genetic structure of populations. Mol. Ecol. 11(12): 2571-2581.).

The relationship among haplotypes was established through the Haploviewer program (http://www.broad.mit.edu/mpg/haploview/), which provides computation of linkage disequilibrium (LD) statistics and population haplotype patterns (Barrett et al. 2005Barrett J.C., Fry B., Maller J., et al. 2005. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 21: 263-265.).

The “isolation by distance” model was analysed using the Mantel test (BioEstat v.5.3; Ayres et al. 2007Ayres M., Ayres Jr. M., Ayres D.L., et al. 2007. BioEstat. Versão 5.3, Sociedade Civil Mamirauá, MCT – CNPq, Belém, Pará, Brasil.) among matrices of the log-transformed genetic divergence and geographic distances between population units. The hypothesis of the neutrality of mutations over time was tested using Tajima’s D and Fu’s Fs with 1000 permutations, run in Arlequin v.3.5.

RESULTSTop

Genetic variability

The control region was sequenced in 330 individuals (338 bp). A total of 319 bps were conserved sites, 19 were variable and 12 were informative for the parsimony analyses. Nucleotide frequencies were T=0.331, C=0.145, A=0.316 and G=0.208. The overall haplotype diversity of the control region was moderate, with the lowest values being found in the Babitonga, Caravelas and Parnaíba populations and the highest ones in the Tamandaré, Conceição da Barra, Bragança, Vaza Barris and Natal populations (range across all populations: 0.400 to 0.861). Total haplotype diversity was 0.757. However, nucleotide diversity was low in all population units, ranging from 0.001 to 0.005 (Table 2).

The results indicate moderate levels of haplotype diversity in Bragança (0.806), Natal (0.861), Vaza Barris (0.846), Conceição da Barra (0.798) and Tamandaré (0.710), and the lowest levels in Parnaíba (0.530), Caravelas (0.516) and Babitonga (0.400). While nucleotide diversity was extremely low (0.001 to 0.005) for all populations, the highest values were recorded for TAM, VBS and CBR, and much lower ones for Bragança, Parnaíba and Babitonga (Table 2).

Patterns of genetic differentiation

The pairwise F_ST values for comparisons between populations varied considerably, between 0.005 and 0.833. However, Caravelas (Bahia) was significantly different from six populations: Bragança, Parnaíba, Fortaleza, Natal, Vaza Barris, and Conceição da Barra (Table 3). No significant correlation was found between the genetic divergence and genetic distance of the population units based on the control region marker (r=0.0501; p=0.7719), indicating that geographic distance does not explain the genetic divergence among the Atlantic goliath grouper population units sampled along the coast of Brazil (Fig. 1).

The SAMOVA indicated incipient genetic structuring among the Brazilian populations (Φ_ST=0.083 to 0.121; p<0.001), with three to five distinct populations (k) being observed (Table 4), and significant population structuring (Φ_CT=0.090 to 0.083; p<0.05). In three different groupings (k3, k4, and k5), the Parnaíba and Caravelas remained structured in comparison with all other Brazilian localities (Table 4). Considering the values of the pairwise F_ST and the Φ_CT of the SAMOVA, three populations were defined: 1) Parnaíba; 2) Caravelas; 3) Bragança + Tamandaré + Vaza Barris + Conceição da Barra.

A total of 30 haplotypes were identified, none of which was present in all locations. However, haplotypes 19 and 23 were widely distributed and common in all population units, except those for which the samples were small, i.e. Fortaleza and Babitonga (Fig. 2).

The Bragança, Conceição da Barra, and Tamandaré units showed the greatest number of haplotypes (76.7%, 40.0% and 26.7% of the total, respectively). Overall, 19 (63%) of the haplotypes were identified in a single unit, of which 12 were rare. The Bragança (12 haplotypes) and Tamandaré (5 haplotypes) units had the greatest number of exclusive haplotypes (56%) in comparison with other populations (Fig. 2). However, the results demonstrate the sharing of haplotypes among all units, even the most geographically distant ones (Table 5).

Demographic history and neutrality

Tajima’s D values were negative for the majority of E. itajara population units, but not statistically significant. Likewise, Tajima’s D for the grouped units was non-significant (D=–1.269; p>0.10), so the hypothesis of neutrality in the E. itajara populations of the Brazilian coast was rejected. The results of Fu’s Fs test based on the distribution of haplotypes also demonstrated non-significant negative values for all population units, except Bragança (Table 2).

DISCUSSIONTop

Genetic variation

The analysis of the mitochondrial control region revealed moderate to low genetic diversity (Grant and Bowen 1998Grant W.S., Bowen B.W. 1998. Shallow population histories in deep evolutionary lineages of marine fishes: Insights from sardines and anchovies and lessons for conservation. J. Hered. 89: 415-426., Silva-Oliveira et al. 2008Silva-Oliveira G.C., Rêgo P.S., Schneider H., et al. 2008. Genetic characterisation of populations of the critically endangered Goliath grouper (Epinephelus itajara, Serranidae) from the Northern Brazilian coast through analyses of mtDNA. Genet. Mol. Biol. 31(4): 988-994.) in the Atlantic goliath grouper populations on the coast of Brazil, as demonstrated by the haplotype diversity (h) and very low nucleotide diversity (π) values. These genetic diversity values differ from the high values reported by Rivera et al. (2004)Rivera M.A., Kelley C.D., Roderic G.K. 2004. Subtle population genetic structure in the Hawaiian grouper, Epinephelus quernus (Serranidae) as revealed by mitochondrial DNA analyses. Biol. J. Linnean Soc. 81: 449-468. for E. quernus in Hawaii (USA) and by Souza et al. (2015)Souza A.S., Dias Júnior E.A., Galetti P.M., et al. 2015. Wide-range genetic connectivity of Coney, Cephalopholis fulva (Epinephelidae), through oceanic islands and continental Brazilian coast. An. Acad. Bras. Cienc. 87(1): 121-136. for Cephalopholis fulva (Epinephelidae) on the Brazilian coast, although they are similar to the values reported for other species of reef fish (Zhang et al. 2006Zhang J., Cai Z., Huang L. 2006. Population genetic structure of crimson snapper Lutjanus erythropterus in East Asia, revealed by analysis of the mitochondrial control region. ICES J. Mar. Sci. 63: 693-704., Hobbs et al. 2013Hobbs J-P.A., Herwerden L.V., Jerry D.R., et al. 2013. High Genetic Diversity in Geographically Remote Populations of Endemic and Widespread Coral Reef Angelfishes (genus: Centropyge). Diversity 5: 39-50.). The reduced genetic diversity of the Atlantic goliath grouper has been related to overfishing and the loss of habitats (Silva-Oliveira et al. 2008Silva-Oliveira G.C., Rêgo P.S., Schneider H., et al. 2008. Genetic characterisation of populations of the critically endangered Goliath grouper (Epinephelus itajara, Serranidae) from the Northern Brazilian coast through analyses of mtDNA. Genet. Mol. Biol. 31(4): 988-994.), and the accentuated decline of the populations of this species throughout its area of distribution in recent years (McClenachan 2009McClenachan L. 2009. Historical declines of goliath grouper populations in South Florida, USA. Inter-Research. ESR. 7: 175-181. , Coleman and Koenig 2010Coleman F.C., Koenig C.C. 2010. The Effects of Fishing, Climate Change, and Other Anthropogenic Disturbances on Red Grouper and Other Reef Fishes in the Gulf of Mexico. Integr. Comp. Biol. 50 (2): 201-212.) may exert a negative influence on its genetic diversity in the near future, further underscoring its status as a critically endangered species. Low genetic diversity in E. itajara has been suggested in microsatellite loci (Seyoum et al. 2013Seyoum S., Tringali M.D., Barthel B.L., et al. 2013. Isolation and characterization of 29 polymorphic microsatellite markers for the endangered Atlantic goliath grouper (Epinephelus itajara), and the Pacific goliath grouper (E. quinquefasciatus). Conservation Genet. Resour. 5: 729-732.), segments of immunological genes (Silva-Oliveira et al. 2014Silva-Oliveira G.C., Silva A.B.C., Blanchard F., et al. 2014. Primers for the amplification of the MHC IIβ chain exon 2 in the Atlantic goliath grouper (Epinephelus itajara). Conservation Genet. Resour. 6: 523-525.), and nuclear markers anchored in microsatellite regions (Benevides et al. 2014Benevides E.A., Vallinoto M., Filho A.F., et al. 2014. When physical oceanography meets population genetics: the case study of the genetic/evolutionary discontinuity in the endangered Goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species. Biochem. Sys. Ecol. 56: 255-266.), further reinforcing the threatened status of the species on the Brazilian coast.

The neutrality tests (Tajima’s D and Fu’s Fs) in the present study lend support to the hypothesis of a low degree of genetic variation. Based on Tajima’s D and Fu’s Fs, there is no evidence that any of the Atlantic goliath grouper populations along the coast of Brazil are in a process of demographic or spatial expansion. Tajima’s D for the pooled data of all units also does not suggest population expansion (D=–1.269; p>0.10). However, Fu’s Fs was significant (Fs=–12.505; p<0.02) for the Bragança (Pará) population, indicating population expansion. Mangroves are important nursery areas for E. itajara (Frias-Torres 2006Frias-Torres S. 2006. Habitat Use of Juvenile Goliath Grouper Epinephelus itajara in the Florida Keys, USA. Endang. Species Res. 1: 1-6., Koenig et al. 2007Koenig C.C., Coleman F.C., Eklund A.M., et al. 2007. Mangroves as Essential Nursery Habitat for Goliath grouper (Epinephelus itajara). Bull. Mar. Sci. 80(3): 567-586.). Recent events, such as the Holocene fluctuations in sea level with the associated changes in the sedimentation patterns of the Amazon basin, modified the distribution of mangrove ecosystems on the northern coast of Brazil (Cohen et al. 2005Cohen M.C.L., Souza-Filho P.W., Lara, R.L., et al. 2005. A model of Holocene mangrove development and relative sea-level changes on the Bragança Peninsula (northern Brazil). Wetl. Ecol. Manag. 13: 433-443.). Furthermore, human impacts on these environments over the past few centuries may have contributed to the loss of habitats in this region (Behling et al. 2001Behling H., Cohen M.C.L., Lara R.J. 2001. Studies on Holocene mangrove ecosystem dynamics of the Bragança Peninsula in north-eastern Pará, Brazil. Palaeogeogr. Palaeocl. 167: 225-242.), which is reflected in the low genetic diversity observed in the present study.

Population structuring

The amplitude of the pairwise genetic differentiation (F_ST) and the comparison of three population groups (1. Parnaíba 2. Caravelas 3. Bragança, Natal, Tamandaré, Vaza Barris and Conceição da Barra) indicate the existence of significant structuring (Φ_CT=0.09, p<0.05) in the E. itajara populations on the Brazilian coast. These results indicate that the gene flow on the Brazilian coast is discontinuous and not totally homogeneous, and no evidence was found of any systematic relationship with geographic patterns. According to Allendorf and Phelps (1981)Allendorf F.W., Phelps S.R. 1981. Use of allelic frequencies to describe population structure. Can. J. Fish Aquat. Sci. 38: 1507-1514., reduced levels of gene flow can homogenize the genetic makeup of populations, and even a small amount of gene flow would be sufficient to annul differences in the frequency of alleles among populations (Palumbi 2003Palumbi S.R. 2003. Population Genetics, Demographic Connectivity, and the Design of Marine Reserves. Ecol. Appl. 13(1, Suppl.): 146-158.). Thus, genetic structuring among the geographically close population units analysed in the present study indicates the occurrence of certain limitations on larval dispersal. A lack of any systematic relationship between genetic patterns and geographic distribution has also been found in genetic studies of reef fish on the Brazilian coast, including Ocyurus chrysurus (Lutjanidae) (Vasconcellos et al. 2008Vasconcellos A.V., Vianna P., Paiva P.C., et al. 2008. Genetic and morphometric differences between yellowtail snapper (Ocyurus chrysurus, Lutjanidae) populations of the tropical West Atlantic. Genet. Mol. Biol. 31(1): 308-316.) and Cephalopholis fulva (Epinephelidae) (Souza et al. 2015Souza A.S., Dias Júnior E.A., Galetti P.M., et al. 2015. Wide-range genetic connectivity of Coney, Cephalopholis fulva (Epinephelidae), through oceanic islands and continental Brazilian coast. An. Acad. Bras. Cienc. 87(1): 121-136.). Significant genetic structuring, with relatively reduced variation between populations (Φ_CT), has been detected in a number of marine fishes (Zhang et al. 2006Zhang J., Cai Z., Huang L. 2006. Population genetic structure of crimson snapper Lutjanus erythropterus in East Asia, revealed by analysis of the mitochondrial control region. ICES J. Mar. Sci. 63: 693-704., Cunha et al. 2014Cunha I.M.C., Souza, A.S., Dias Jr. E.A., et al. 2014. Genetic Multipartitions Based on D-Loop Sequences and Chromosomal Patterns in Brown Chromis, Chromis multilineata (Pomacentridae), in the Western Atlantic. Hindawi BioMed Research International. Article ID 254698, 11 pp.) and may indicate the occurrence of intermittent gene flow (Hébert et al. 2000Hébert C., Danzman R.G., Jones M.W., et al. 2000. Hydrography and population genetic structure in brook charr (Salvelinus fontinalis, Mitchill) from eastern Canada. Mol. Ecol. 9: 971-982.) between the coastal areas analysed in this study.

The greater genetic differentiation between the Caravelas sample and both distant (Bragança and Parnaíba) and adjacent (Vaza Barris and Conceição da Barra) populations may be accounted for by the transgression and regression events that occurred on the Brazilian coast during the Quaternary (Andrade et al. 2003Andrade A.C.S., Dominguez J.M.L., Martin L., et al. 2003. Quaternary evolution of the Caravelas strandplain — Southern Bahia State—Brazil. An. Acad. Bras. Ciênc. 75: 357-382.), which provoked fluctuations in sea levels, in particular over the past 120000 years (Suguio et al. 1985Suguio K., Bittencourt A.C.S.P., Dominguez J.M.L., et al. 1985. Flutuações do nível relativo do mar durante o quaternário superior ao longo do litoral brasileiro e suas implicações na sedimentação costeira. Rev. Bras. Geociênc. 15: 273-286., Bittencourt et al. 2000Bittencourt A.C.S.P., Dominguez J.M.L., Martin L., et al. 2000. Patterns of sediment dispersion coastwise the State of Bahia-Brazil. An. Acad. Bras. Cienc. 72: 21-33.). These oscillations probably hampered the colonization process and may have resulted in the formation of barriers to the dispersal of pelagic larvae in the region of the Abrolhos Ridge in Bahia, in particular on the inner continental shelf through the formation of effective hydrodynamic and geological barriers (Leipe 1999Leipe T., Knoppers B., Marone E., et al. 1999. Suspended matter transport in coral reef waters of the Abrolhos Bank, Brazil. Geo-Marine Letters 19(3): 186-195.) and the reversion of currents (Teixeira et al. 2013Teixeira C.E.P., Lessa G.C., Cirano M., et al. 2013. The inner shelf circulation on the Abrolhos Bank, 18oS, Brazil. Cont. Shelf Res. 70: 13-26.), which would have retained the larvae at the mouth of the Caravelas River, in southern Bahia. Interactions between small (1-10 individuals) resident breeding groups (Giglio et al. 2014Giglio V.J., Adelir-Alves J., Gerhardinger L.C., et al. 2014. Habitat use and abundance of goliath grouper Epinephelus itajara in Brazil: a participative survey. Neotrop. Ichthyol. 12(4): 803-810.) in areas favourable to larval retention may result in reduced gene flow and greater differentiation among populations.

In addition to the fluctuations in sea level, the Parnaíba region is affected by the Intertropical Convergence zone (ITCZ). Between 40-8 thousand years ago (kya), the ITCZ shifted towards the northern coast of Brazil, affecting the surface temperature of the sea and wind intensity, creating semi-arid conditions in northeastern Brazil. These conditions were interrupted by short cycles of higher precipitation, and one long cycle between 15-11 kya, when the ITCZ migrated between the equator and northeastern Brazil (Moura and Shukla 1981Moura A., Shukla J. 1981. On the dynamics of droughts in northeast Brazil: Observations, theory and numerical experiments with a general circulation model. J. Atmos. Sci. 38: 2653-2675., Arz et al. 1998Arz H.W., Pätzold J., Wefer G. 1998. Correlated millennial-scale changes in surface hydrography and terrigenous sediment yield inferred from last-glacial marine deposits off northeastern Brazil. Quat. Res. 50: 157-166., Behling et al. 2000Behling H., Arz H.W., Pätzold J., et al. 2000. Late Quaternary vegetational and climate dynamics in northeastern Brazil, inferences from marine core GeoB 3104-1. Quat Sci Rev. 19: 981-994.). The semi-arid conditions are reflected in the region’s coastal vegetation, especially between Piauí (2°S) and Rio Grande do Norte (5°S). Here, alluvial deposits are limited to a few small areas, due to the exposure of the local rivers to wave energy, and stratigraphic data indicate that mangroves, especially Rhizophora, were established only at the beginning of the Holocene and were probably rare or absent during most of the Last Glacial Maximum (Behling et al. 2000Behling H., Arz H.W., Pätzold J., et al. 2000. Late Quaternary vegetational and climate dynamics in northeastern Brazil, inferences from marine core GeoB 3104-1. Quat Sci Rev. 19: 981-994.). Even then, mangrove forests, which are considered to be essential nursery areas for Epinephelus itajara (Koenig et al. 2007Koenig C.C., Coleman F.C., Eklund A.M., et al. 2007. Mangroves as Essential Nursery Habitat for Goliath grouper (Epinephelus itajara). Bull. Mar. Sci. 80(3): 567-586.), have a limited distribution in this region due to the scarcity of bodies of freshwater, prolonged droughts and high salinity, which restricts the development of these plants (Schaeffer-Novelli et al. 1990Schaeffer-Novelli Y., Cintron-Molero G., Adaime R.R. 1990. Variability of mangrove ecosystems along the brazilian coast. Estuaries 13(2): 201-218.). These unique local conditions may have a fundamental influence on the population dynamics of E. itajara, given that the density of juvenile individuals may be affected by the structural complexity of the mangrove (Frias-Torres 2006Frias-Torres S. 2006. Habitat Use of Juvenile Goliath Grouper Epinephelus itajara in the Florida Keys, USA. Endang. Species Res. 1: 1-6.) and the positive correlation between the abundance of adult groupers and the extension of adjacent areas of mangrove (Sadovy and Eklund 1999Sadovy Y., Eklund A.M. 1999. Synopsis of biological information on the Nassau grouper, Epinephelus striatus, (Bloch 1792), and the jewfish, E. itajara (Lichtenstein 1822). NOAA Technical Report, NMFS 146, and FAO Fisheries Synopsis 157. 65 pp.).

The results of this study showed that haplotypes were widely shared, although the SAMOVA identified significant Φ_ST values, which indicate a certain amount of population differentiation (Wright 1951Wright S. 1951. The genetical structure of populations. Ann. Eugen. 15: 323-354.) based on restrictions to gene flow. However, this genetic structuring—indicated by the Φ_ST value—is much lower than that found in other groupers, such as Epinephelus labriformis (F_ST≤0.661, p=0.001) (Craig et al. 2006Craig M.T., Hastings P.A., Pondella D.J., et al. 2006. Phylogeography of the flag cabrilla Epinephelus labriformis (Serranidae): implications for the biogeography of the Tropical Eastern Pacific and the early stages of speciation in a marine shore fish. J. Biogeogr. 33: 969-979.) and E. akaara (F_ST≤0.379, p<0.001) (Chen et al. 2008Chen S., Liu T., Li Z., et al. 2008. Genetic population structuring and demographic history of red spotted grouper (Epinephelus akaara) in South and East China Sea. Afr. J. Biotechnol. 7: 3554-3562.), although the population structure is similar to that found in other species of reef fish (Zhang et al. 2006Zhang J., Cai Z., Huang L. 2006. Population genetic structure of crimson snapper Lutjanus erythropterus in East Asia, revealed by analysis of the mitochondrial control region. ICES J. Mar. Sci. 63: 693-704., Drew and Barber 2012Drew J.A, Barber P.H. 2012. Comparative Phylogeography in Fijian Coral Reef Fishes: A Multi-Taxa Approach towards Marine Reserve Design. PLoS ONE 7(10): e47710., Cunha et al. 2014Cunha I.M.C., Souza, A.S., Dias Jr. E.A., et al. 2014. Genetic Multipartitions Based on D-Loop Sequences and Chromosomal Patterns in Brown Chromis, Chromis multilineata (Pomacentridae), in the Western Atlantic. Hindawi BioMed Research International. Article ID 254698, 11 pp.). Silva-Oliveira et al. (2008)Silva-Oliveira G.C., Rêgo P.S., Schneider H., et al. 2008. Genetic characterisation of populations of the critically endangered Goliath grouper (Epinephelus itajara, Serranidae) from the Northern Brazilian coast through analyses of mtDNA. Genet. Mol. Biol. 31(4): 988-994. found significant genetic differentiation in pairwise F_ST values for E. itajara between Parnaíba and both Bragança and Natal, but did not detect structuring between Bragança and Natal.

The significant differences in F_ST and the complex haplotype network found for the Bragança, Parnaíba, Caravelas and Conceição da Barra population units indicate that these locations are not isolated. However, the dispersal of larvae or adults may be occasional, with a lower degree of genetic exchange than that required to completely homogenize the population, possibly due to the existence of a barrier to gene flow, which may influence the degree of genetic differentiation among localities (Aboim et al. 2005Aboim M.A., Menezes G.M., Schlitt T., et al. 2005. Genetic structure and history of populations of the deep-sea fish Helicolenus dactylopterus (Delaroche, 1809) inferred from mtDNA sequence analysis. Mol. Ecol. 14: 1343-1354. ). This barrier does not appear to avoid gene flow completely, given the presence of shared haplotypes among these population units. Castro et al. (2007)Castro A.L.F., Stewart B.S., Wilson S.G., et al. 2007. Population genetic structure of Earth’s largest fish, the whale shark (Rhincodon typus). Mol. Ecol. 16: 5183-5192. concluded that the sharing of haplotypes between the farthest geographic extremes of a species’ range is consistent with potential migration among oceanic basins that does not independently alter the size of the different groups. Despite the small sample size for the southernmost population, at Babitonga (Santa Catarina) the common haplotypes are shared with other population units; this population is not isolated from the others and is characterized by reduced genetic diversity. This finding differs from that of Benevides et al. (2014)Benevides E.A., Vallinoto M., Filho A.F., et al. 2014. When physical oceanography meets population genetics: the case study of the genetic/evolutionary discontinuity in the endangered Goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species. Biochem. Sys. Ecol. 56: 255-266., who used a nuclear marker. This incongruent evidence might suggest possible phenomena of sex-biased demography such as male/female phylopatry in the region.

The circulation patterns of the Brazil Current and North Brazil Current in the South Atlantic (Soutelino et al. 2013Soutelino R.G., Gangopadhyay A., Silveira I.C.A. 2013. The roles of vertical shear and topography on the eddy formation near the site of origin of the Brazil Current. Cont. Shelf Res. 70: 46-60.), sedimentation events on the coastal plain that caused fluctuations in the sea level during the Last Glacial Maximum at the beginning of the Holocene (Andrade et al. 2003Andrade A.C.S., Dominguez J.M.L., Martin L., et al. 2003. Quaternary evolution of the Caravelas strandplain — Southern Bahia State—Brazil. An. Acad. Bras. Ciênc. 75: 357-382.), and the effects of the Intertropical Convergence Zone at low latitudes (Zabel et al. 2003Zabel M., Wagner T., deMenocal P. 2003. Terrigenous signals in sediments from Terrigenous Signals in Sediments of the Low-Latitude Atlantic – Indications to Environmental Variations during the Late Quaternary, Part II: Lithogenic Matter. In: Wefer G., Mulitza S., Ratmeyer V. (eds), The South Atlantic in the Late Quaternary: Reconstruction of Mass Budget and Current Systems, Springer-Verlag, Berlin, Heidelberg, New York. pp. 323-345.) may all have influenced the distribution of the species in coastal environments, resulting in asynchrony in the process of population expansion in different geographic areas (Souza et al. 2015Souza A.S., Dias Júnior E.A., Galetti P.M., et al. 2015. Wide-range genetic connectivity of Coney, Cephalopholis fulva (Epinephelidae), through oceanic islands and continental Brazilian coast. An. Acad. Bras. Cienc. 87(1): 121-136.). Regional and local oceanic circulation patterns (Teixeira et al. 2013Teixeira C.E.P., Lessa G.C., Cirano M., et al. 2013. The inner shelf circulation on the Abrolhos Bank, 18oS, Brazil. Cont. Shelf Res. 70: 13-26.) may also play an important role in the dispersal of eggs, as well as the recruitment of larvae and juveniles (Nonaka et al. 2000Nonaka R.H., Matsuura Y., Suzuki K. 2000. Seasonal variation in larval fi sh assemblages in relation to oceanographic conditions in the Abrolhos Bank region off eastern Brazil. Fish. Bull. 98: 767-784.). These factors may also have determined the different levels of habitat complexity observed along the coast (Arz et al. 1998Arz H.W., Pätzold J., Wefer G. 1998. Correlated millennial-scale changes in surface hydrography and terrigenous sediment yield inferred from last-glacial marine deposits off northeastern Brazil. Quat. Res. 50: 157-166.), which play an important role in the selectivity of coastal marine organisms. The modifications in the availability of habitats caused by these events induce demographic reductions and expansions that alter the connectivity and distribution of populations among the different geographic regions (Cunha et al. 2014Cunha I.M.C., Souza, A.S., Dias Jr. E.A., et al. 2014. Genetic Multipartitions Based on D-Loop Sequences and Chromosomal Patterns in Brown Chromis, Chromis multilineata (Pomacentridae), in the Western Atlantic. Hindawi BioMed Research International. Article ID 254698, 11 pp.). However, these factors have not been evaluated adequately for the understanding of the role of these events as a biogeographical filter for the Atlantic goliath grouper on the coast of Brazil.

The population structure found in the present study indicates limited gene flow, and although the small sample size may also have been important here, the size of the samples analysed in the present study was similar to that used in the studies of Silva-Oliveira et al. (2008)Silva-Oliveira G.C., Rêgo P.S., Schneider H., et al. 2008. Genetic characterisation of populations of the critically endangered Goliath grouper (Epinephelus itajara, Serranidae) from the Northern Brazilian coast through analyses of mtDNA. Genet. Mol. Biol. 31(4): 988-994. and Benevides et al. (2014)Benevides E.A., Vallinoto M., Filho A.F., et al. 2014. When physical oceanography meets population genetics: the case study of the genetic/evolutionary discontinuity in the endangered Goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species. Biochem. Sys. Ecol. 56: 255-266.. These incongruities may be clarified by the investigation of additional molecular markers and further research into the biology and ecology of E. itajara.

This reduced mitochondrial diversity may be related to the faster genetic drift in these haploid genes, inherited maternally, in comparison with the diploid, biparental nuclear genes (Palumbi and Bader 1994Palumbi S.R., Baker C.S. 1994. Contrasting Population Structure from Nuclear Intron Sequences and mtDNA of Humpback Whales. Mol. Biol. Evol. 11(3): 246-435.). In this case, the apparent incongruities between the mitochondrial data recorded in the present study and the results for the nuclear markers presented by Benevides et al. (2014)Benevides E.A., Vallinoto M., Filho A.F., et al. 2014. When physical oceanography meets population genetics: the case study of the genetic/evolutionary discontinuity in the endangered Goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species. Biochem. Sys. Ecol. 56: 255-266. may be the result of demographic events, as mentioned above. As mitochondrial genes generally have higher mutation rates, due to genetic drift (Ingvarsson 2004Ingvarsson P.K. 2004. Population subdivision and the Hudson-Kreitman-Aguade test: testing for deviations from the neutral model in organelle genomes. Genet. Res. 83: 31-39.), the mitochondrial control region will evolve more rapidly than the SNPs of the nuclear genome (Pesole et al. 1999Pesole G., Gissi C., De Chirico A., et al. 1999. Nucleotide substitution rate of mammalian mitochondrial genomes. J. Mol. Evol. 48: 427-434., Kumar and Subramanian 2002Kumar S., Subramanian S. 2002. Mutation rates in mammalian genomes. Proc. Natl. Acad. Sci. USA 99: 803-808.) and the AMOVA demonstrated a greater degree of subdivision in comparison with the nuclear DNA (Ji et al. 2011Ji Y-Q., Wu D-D., Wu G-S., et al. 2011. Multi-Locus Analysis Reveals A Different Pattern of Genetic Diversity for Mitochondrial and Nuclear DNA between Wild and Domestic Pigs in East Asia. PLoS ONE 6(10): e26416.). Further studies of additional markers may help clarify these uncertainties.

Genetic insights for conservation

The data on the haplotypes shared among the population units of the Atlantic goliath grouper along the coast of Brazil emphasize the importance of regional haplotype richness for the genetic diversity of the species. Local impacts can have negative effects on the genetic conservation due to this sharing of haplotypes, which may extend throughout the geographic distribution of E. itajara.

The moderate haplotype diversity and extremely low degree of nucleotide diversity suggest the random fixation of variation and possible genetic drift in the study region, as a consequence of population depression resulting from founder effects or bottleneck events. However, the ample genetic diversity found among population units (0.400-0.861) in the present study indicate that the effects of these events have varied considerably among the different regions, in turn reinforcing the need for the application of conservation measures in priority areas with the main focus on financial limitations.

The results of this study indicate low levels of genetic diversity, primarily in the populations from Parnaíba (Piauí) and Caravelas (southern Bahia). Ceará (Fortaleza) and Babitonga (Santa Catarina) also showed low values, although the sample size was prohibitively low in these cases. The Φ_ST and F_ST values also indicate the genetic differentiation of these populations in relation to the others located along the Brazilian coast. It is important to note, however, that few genetic data are available for the populations from the coast of southern Brazil between Conceição da Barra (Espírito Santo) and Babitonga (Santa Catarina). These populations may be problematic, given the unique geological and oceanographic characteristics of this region, which may cause different patterns of genetic diversity and gene flow in this region. Nevertheless, it does not appear reasonable to assume that the Atlantic goliath grouper of the Brazilian coast is in a state of panmixia, considering the levels of pairwise genetic differentiation found in the present study (F_ST) and the significant differences found among population groups (Φ_CT).

Based on the findings of the present study, and the critically endangered status of the species, the current ban on the harvesting of the Atlantic goliath grouper should be maintained, considering 1) the variation found in the genetic diversity of the subpopulations located along the Brazilian coast, indicating that they have suffered differing degrees of impact and threat; 2) the very low level of nucleotide diversity, which suggests a greatly reduced effective population size and indicates that overfishing and habitat destruction in recent decades may have had negative genetic effects for future generations; 3) the distinct findings for the mitochondrial and nuclear markers, which reinforce the need for analysis of additional molecular markers (i.e. Msats and MHC genes); and 4) the lack of data from some regions of the Brazilian coast, which may be important for understanding the dynamics of the population as a whole. The conservation of the Atlantic goliath grouper will depend on political efforts for the creation of coastal protection areas, especially in mangroves and breeding grounds, and the participation of local communities in the decision-making process.

ACKNOWLEDGEMENTSTop

The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), the Fundação de Amparo à Pesquisa do Espírito Santo (FAPES), the Projeto Meros do Brasil, which is supported by Petrobrás S.A. (through the Programa Petrobrás Ambiental), and Transpetro Transportes S.A. for financial support. R.A. Torres is especially grateful to CNPq for the research fellowship provided (Grant Numbers 306099/2011-0 and 301208/2012-3).

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