sm80n4-4407

Population genetic structure of an estuarine and a reef fish species exploited by Brazilian artisanal fishing

Regina H.G. Priolli 1,4, Miklos M. Bajay 2, Renato A.M. Silvano 1,3, Alpina Begossi 1,4

1 Fisheries and Food Institute - FIFO, PPG Ecomar, UNISANTA, R. Cesário Mota 08, CEP: 11045-040, Santos, SP, Brazil. E-mail: rhpriolli@gmail.com
2 Department of Genetics, ESALQ, USP, Av. Pádua Dias 11, CEP: 13400-970, Piracicaba, SP, Brazil.
3 Department of Ecology, UFRGS, CP 15007, CEP: 91501-970, Porto Alegre, RS, Brazil.
4 Nepa, Capesca, UNICAMP, Av. Albert Einstein 291, CEP: 13083-852, Campinas, SP, Brazil.

Summary: In this study, we used microsatellite markers to examine the genetic structures of Centropomus undecimalis (Bloch, 1792) and Epinephelus marginatus (Lowe, 1834) populations collected from artisanal fishing sites along a stretch of coastline in southeastern Brazil. Based on F-statistics, there was no significant genetic differentiation evident in any C. undecimalis samples (FST=0.012). However, Bayesian clustering, principal component analysis (PCA) and discriminant analysis of principal components (DAPC) results suggested that there were most likely two clusters, with no relation to geographic areas. The bottleneck results showed no significant values and the effective population sizes (Ne) for the two genetically differentiated groups were large and similar. In contrast, for E. marginatus populations, the microsatellite loci showed no population subdivisions. The FST value was low and non-significant (FST=0.008), a Bayesian analysis indicated one cluster, and a PCA showed that all samples from different geographical sites shared the same genetic structure. The bottleneck results exhibited significant differences, and a low Ne was observed. The results of the genetic study of these two species along the southeastern Brazilian coast suggest that the distinct genetic structure of each species should be taken into account as management units for the conservation of their genetic diversities.

Keywords: small-scale fisheries; microsatellites; genetic diversity; common snook; dusky grouper; bottleneck.

Estructura genética poblacional de dos especies piscícolas, una de estuario y una de arrecife, explotados por la pesca artesanal brasilera

Resumen: En este estudio se han utilizado marcadores microsatélites para examinar la estructura genética de Centropomus undecimalis (Bloch, 1792) y Epinephelus marginatus (Lowe, 1834) en localidades de pesca artesanal localizadas a lo largo de la costa del sudeste de Brasil. Los estadísticos de diferenciación poblacional (F-statisctics) no presentaron diferenciación genética entre las muestras de C. undecimalis (FST=0.012). Sin embargo, los resultados de los análisis basados en clústeres bayesianos, componentes principales (PCA) y análisis discriminante de componentes principales (DAPC) sugieren la posible presencia de dos clústeres diferenciados genéticamente, pero sin ninguna relación con las áreas geográficas. Los resultados del análisis de cuello de botella poblacional no son significativos con valores de tamaño efectivo poblacional (Ne) elevados y similares entre los dos clústeres. Por el contrario, en E. marginatus, los análisis basados en microsatélites no muestran ningún patrón de subdivisión genética. El valor de FST es bajo y no significativo (FST=0.008), el análisis Bayesiano indicia un solo clúster y el PCA determina que todas las muestras de las diferentes localidades geográficas comparten la misma estructura genética. El análisis de cuello de botella muestra diferencias significativas con la observación de una baja Ne. Los resultados de los análisis genéticos en estas dos especies a lo largo de la costa sudeste de Brasil sugieren diferentes estructuras genéticas para cada especie y estos resultados deben tenerse en consideración para la estipulación de las medidas de conservación de la diversidad genética.

Palabras clave: pesquerías a pequeña escala; microsatélites; diversidad genética; róbalo; mero; cuello de botella.

Citation/Como citar este artículo: Priolli R.H.G., Bajay M.M., Silvano R.A.M., Begossi A. 2016. Population genetic structure of an estuarine and a reef fish species exploited by Brazilian artisanal fishing. Sci. Mar. 80(4): 467-477. doi: http://dx.doi.org/10.3989/scimar.04407.17A

Editor: J. Viñas.

Received: January 27, 2016. Accepted: September 27, 2016. Published: October 20, 2016.

Copyright: © 2016 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License.

Contents

Summary
Resumen
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References

INTRODUCTIONTop

A recent report on snappers and groupers (Amorim and Westmeyer 2016Amorim P., Westmeyer M. 2016. Snapper and Grouper: SFP Fisheries Sustainability Overview 2015. Sustainable Fisheries Partnership Foundation. 18 pp. Available from: http://www.fishsource.com) shows that information on stocks is unclear, especially in developing countries that do not have a reporting system based on catch and effort data. This is particularly the case in Brazil, where there is no systematic data collection on the stocks of snook (Centropomus spp.) and groupers (Epinephelus spp.). To address this lack, we present here new data on the genetic diversities of Centropomus undecimalis (Bloch, 1792) and Epinephelus marginatus (Lowe, 1834) from fishing spots off the southeastern coast of Brazil.

These two biologically different species are among the most exploited fish in two ecologically relevant habitats (estuaries and reefs) in the western Atlantic. The common snook, C. undecimalis, is a protandric hermaphrodite and a diadromous, euryhaline and estuarine fish. The development of this fish includes an inshore larval stage, with juveniles colonizing estuarine environments, including mangroves, drainage areas, sea grass beds and shallow waters (Taylor et al. 2000Taylor R.G., Whittington J.A., Grier H.J., et al. 2000. Age, growth, maturation, and protandric sex reversal in common snook, Centropomus undecimalis, from the east and west coasts of South Florida. Fish. Bull. 98: 612-624.). Interviews with local fishermen indicate that this fish spawns in summer and spring (Begossi et al. 2012aBegossi A., Salivonchyk S.V., Hanazaki N., et al. 2012a. Fishers (Paraty, RJ) and fish manipulation time: a variable associated to the choice for consumption and sale. Braz. J. Biol. 72: 973-975.). Its capture is associated with migratory movements to freshwater ecosystems (Silvano et al. 2006Silvano R.A.M., MacCord P.F.L., Lima R.V., et al. 2006. When does this fish spawn? Fishermen’s local knowledge of migration and reproduction of Brazilian coastal fishes. Environ. Biol. Fish. 76: 371-386), and its capture is also linked to spawning events in the coastal zone (Able 2005Able, K.W. 2005. A re-examination of fish estuarine dependence: Evidence for connectivity between estuarine and ocean habitats. Estuar. Coast. Shelf Sci. 64: 5-17.). These situations can promote decreases in fishery resources, with potential detrimental effects on population size (Perera et al. 2011Perera M.A., Mendoza M., Contreras W.M., et al. 2011. Reproductive biology of common snook Centropomus undecimalis (Perciformes: Centropomidae) in two tropical habitats. Rev. Biol. Trop. 59: 669-681.).

Nuclear markers such as microsatellites or simple sequence repeats (SSRs) have been used to study genetic differences among populations, and these studies have confirmed the hypothesis that restricted gene flow between common snook originated from the Atlantic and Gulf of Mexico off the coast of Florida (Seyoum et al. 2005Seyoum S., Tringali M.D., Sullivan J.G. 2005. Isolation and characterization of 27 polymorphic microsatellite loci for the common snook, Centropomus undecimalis. Mol. Ecol. Notes 5: 924-927., Tringali et al. 2008Tringali M.D., Seyoum S., Wallace E.M., et al. 2008. Limits to the use of contemporary genetic analyses in delineating biological populations for restocking and stock enhancement. Rev. Fish. Sci. 16: 111-116.). Studies of microsatellite loci also found that snook from contrasting environments, marine and freshwater, belong to the same genetic stock (Hernandez-Vidal et al. 2014Hernandez-Vidal U., Lesher-Gordillo J., Contreras-Sanchez W.M., et al. 2014. Genetic variability of the Common Snook Centropomus undecimalis (Perciformes: Centropomidae) in connected marine and riverine environments. Rev. Biol. Trop. 62: 627-636.).

The dusky grouper, E. marginatus, is a reef fish that inhabits rocky bottoms, from shallow waters to depths of more than 200 metres, and lives in caves (Andrade et al. 2003Andrade A.B., Machado L.F., Hostim-Silva M., et al. 2003. Reproductive biology of the dusky goruper Epinephelus marginatus (Lowe, 1834). Braz. Arch. Biol. Techn. 46: 373-381.). It is a monandric, protogynous hermaphrodite, is sedentary and shows slow growth. It has been reclassified as a protected species in the Mediterranean (Bouchereau et al. 1999Bouchereau J.L., Body P., Chauvet C. 1999. Growth of the dusky grouper Epinephelus marginatus (Linnnaeus, 1758) (Teleostei, Serranidae), in the natural marine reserve of Lavezzi Islands, Corsica, France. Sci. Mar. 63: 71-77.) and has been listed as endangered by the International Union for the Conservation of Nature (IUCN) since 1996. Large predators, including E. marginatus, have important ecological functions in reef ecosystems (Mumby et al. 2011Mumby P.J., Harborne A.R., Brumbaugh D.R. 2011. Grouper as a Natural Biocontrol of Invasive Lionfish. PLoS One 6: e21510.). These fish species can also be good indicators of the effectiveness of biological conservation efforts, such as those that occur in marine protected areas (Anderson et al. 2014Anderson A.B., Bonaldo R.M., Barneche D.R., et al. 2014. Recovery of grouper assemblages indicates effectiveness of a marine protected area in Southern Brazil. Mar. Ecol. Prog. Ser. 514: 207-215.).

Studies of E. marginatus populations have been carried out using selected microsatellite markers developed for Mycteroperca microlepis (Chapman et al. 1999Chapman R.W., Sedberry G.R., Koenig C.C., et al. 1999. Stock identification of gag, Mycteroperca microlepis, along the southeast coast of the United States. Mar. Biotechnol. 1: 137-146.), Epinephelus quernus (Rivera et al. 2003Rivera M.A.J., Graham G.C., Roderick G.K. 2003. Isolation and characterization of nine microsatellite loci from the Hawaiian grouper Epinephelus quernus (Serranidae) for population genetic analyses. Mar. Biotechnol. 5: 126-129.) and Epinephelus guttatus (Ramirez et al. 2006Ramirez M.A., Patricia-Acevedo J., Planas S., et al. 2006. New microsatellite resources for groupers (Serranidae). Mol. Ecol. Notes 6: 813-817.). Genetic differentiation between the populations from the Atlantic and Mediterranean waters, as well as those within the Mediterranean (De Innocentiis et al. 2001De Innocentiis S., Sola L., Cataudella S., et al. 2001. Allozyme and microsatellite loci provide discordant estimates of population differentiation in the endangered dusky grouper (Epinephelus marginatus) within the Mediterranean Sea. Mol. Ecol. 10: 2163-2175., Schunter et al. 2011Schunter C., Carreras-Carbonell J., Planes S., et al. 2011. Genetic connectivity patterns in an endangered species: The dusky grouper (Epinephelus marginatus). J. Exp. Mar. Biol. Ecol. 401: 126-133.), has been observed. In a previous work on the genetic diversity of E. marginatus populations in Paraty (Rio de Janeiro, Brazil), the same microsatellite markers showed high diversity (Priolli et al. 2014Priolli R.H.G., Stabelini N.S., Bajay M.M. 2014. Diversidade genética de uma espécie em perigo de extinção: a garoupa Epinephelus marginatus. In Begossi A., Lopes P.F.M. (eds), Comunidades pesqueiras de Paraty sugestões para manejo. RiMa Editora, São Carlos, pp. 27-40.).

Small-scale fisheries frequently exploit environments that have a high degree of aquatic and fish biodiversity, and these fisheries involve fishing for consumption and sale (McClanahan et al. 2006McClanahan T.R., Marnane M.J., Cinner J.E., et al. 2006. A comparison of marine protected areas and alternative approaches to coral-reef management. Curr. Biol. 16: 1408-1413., Begossi et al. 2012aBegossi A., Salivonchyk S.V., Hanazaki N., et al. 2012a. Fishers (Paraty, RJ) and fish manipulation time: a variable associated to the choice for consumption and sale. Braz. J. Biol. 72: 973-975.). However, there is evidence that even artisanal fishing can impact target fish populations (Pinnegar and Engelhard 2008Pinnegar J.K., Engelhard G.H. 2008. The ‘shifting baseline’ phenomenon: a global perspective. Rev. Fish. Biol. Fish. 18: 1-16.). Artisanal fisheries account for about 50% of the overall Brazilian fish catch, but there is a scarcity of basic data on these catches, which is an obstacle for making temporal comparisons (Begossi et al 2012aBegossi A., Salivonchyk S.V., Hanazaki N., et al. 2012a. Fishers (Paraty, RJ) and fish manipulation time: a variable associated to the choice for consumption and sale. Braz. J. Biol. 72: 973-975.). Also, there is often no information available about the status of vulnerable species that are commercially important (Begossi et al. 2012bBegossi A., Salyvonchyk S., Nora V., et al. 2012b. The paraty artisanal fishery (southeastern Brazilian coast): ethnoecology and management of a social-ecological system (SES). J. Ethnobiol. Ethnomed. 8: 22-40.).

In this study, we used microsatellite markers to examine the genetic structure of the C. undecimalis and E. marginatus populations along a stretch of the southeastern Brazilian coastline. Information about the genetic diversities and population structures of these species can eventually be translated into management and conservation strategies. Our goals were to (1) test the alternative hypotheses of panmixia versus genetic population subdivisions for each species, (2) assess the genetic diversity, bottleneck and effective population size of each species, and (3) provide insights for the conservation of these species that takes their genetic diversities into account.

MATERIALS AND METHODSTop

Sample collection and DNA extraction

Samples from the two species (one sample per individual fish) were collected from the artisanal fishery landing points in Paraty (Rio de Janeiro State, Brazil). At this specific site only, during two weeks per month, fishermen were approached as soon as they arrived at one of two main landing points (Fig. 1). They were asked for their permission to weigh and measure their catch, which was then identified using local names. A questionnaire was used to determine the main fishing spots and target species and to confirm fish identifications (Begossi et al. 2012bBegossi A., Salyvonchyk S., Nora V., et al. 2012b. The paraty artisanal fishery (southeastern Brazilian coast): ethnoecology and management of a social-ecological system (SES). J. Ethnobiol. Ethnomed. 8: 22-40., Lopes et al. 2013Lopes P.F.M, Rosa E.M., Salyvonchyk S., et al. 2013. Suggestions for fixing top-down coastal fisheries management through participatory approaches. Mar. Policy 40: 100-110.). We collected 76 and 122 individual C. undecimalis and E. marginatus, respectively, at two landing points for the Paraty fishery (Table 1).

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Fig. 1. – Map of Paraty artisanal fishing sites on the coast of the states of Rio de Janeiro and São Paulo, Brazil. Red stars indicate landing ports. The fishing spots that were used by the communities of Paraty to catch C. undecimalis (blue or black numerals: 1, Tarituba; 2, Ilha do Pico; 3, Ilha do Araújo; 4, Baía de Paraty; 5, Canto do Morro; 6, Ilha do Ventura; 7, Ilha Rasa; 8, Aracaíba; 9, Laje Sete Cabeças) and E. marginatus (red or black numerals: 1, Tarituba; 2, Ilha do Pico; 3, Ilha do Araújo; 4, Baía de Paraty; 5, Ilha do Algodão; 6, Cajaíba; 7, Joatinga; 8, Ponta Negra; 9, Sono).

Table 1. – Details of the Centropomus undecimalis and Epinephelus marginatus sampling sites, with the number of individuals (N) and their average length and weight. (—) indicates no data collected.

Species Sites Coordinates N Date Length (cm) Weight (g)
C. undecimalis Paraty 23°13′04″S, 44°42′47″W 76 December 2009–April 2011 70.96±12.889 3.48±1.585
Ubatuba 23°26′02″S, 45°04′16″W 15 December 2010–April 2011 83.93±9.888 6.26±2.190
E. marginatus Paraty 23°13′04″S, 44°42′47″W 122 December 2009–April 2011 35.93±6.411 0.90±0.551
Rio de Janeiro 22°54′10″S, 43°12′27″W 30 December 2010–April 2011

To increase the sampling area, we collected 15 additional samples of C. undecimalis from artisanal fisheries from one landing point in Ubatuba (São Paulo State) and 30 additional samples of E. marginatus from one landing point in Rio de Janeiro (Rio de Janeiro State). In these cases, the fish collected from fishermen´s catches were mostly bought from the fishers at the landing points, which are also local fish markets. Only Ubatuba catches were weighed and measured.

Samples of muscle or the caudal fin (approximately 2 cm2) of each individual were stored in 70% ethanol at 4°C, shipped on ice to the laboratory and lyophilized for two days under 0.040 mbar and at –50°C (Alpha 1.2 LD Plus, Martin Christ, Osterode, Germany). DNA was isolated from a few milligrams of lyophilized tissue using a protocol developed by Shiozawa et al. (1992)Shiozawa D.K., Kudo J., Evans R.P., et al. 1992. DNA extraction from preserved trout tissues. Gt. Basin Nat. 52: 29-34., with some modifications (Almeida et al. 2001Almeida F.S., Fungaro M.H.P., Sodré L.M.K. 2001. RAPD and isoenzyme analysis of genetic variability in three allied species of catfishes (Siluriformes Pimelodidae) from the Tibagi river, Brazil. J. Zool. 253: 113-120.). DNA quality and concentration were evaluated via electrophoresis in agarose gels following staining with SYBR Safe (Invitrogen, CA, USA).

Microsatellite analyses

Centropomus undecimalis: Fifteen microsatellite loci were amplified using primer pairs specifically described for C. undecimalis (Cun01, Cun03, Cun05B, Cun08, Cun09, Cun10A, Cun11, Cun12, Cun14, Cun16, Cun17, Cun18, Cun19, Cun22 and Cun23) (Seyoum et al. 2005Seyoum S., Tringali M.D., Sullivan J.G. 2005. Isolation and characterization of 27 polymorphic microsatellite loci for the common snook, Centropomus undecimalis. Mol. Ecol. Notes 5: 924-927.). Polymerase chain reaction (PCR) assays were performed in a final volume of 20 μL containing 15 ng DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.9), 250 μM of each dNTP, 0.2 µM BSA, 2.0 mM MgCl2, 0.16 μM forward primer, 0.20 μM reverse primer, 0.13 μM M13 primer (IRDye 700 or IRDye 800, LI-COR Corporate, NE, USA) and 1.0 unit Taq DNA polymerase (Life Technologies, CA, USA). The reactions were amplified using a PTC-200 thermocycler (MJ Research, MA, USA) with the following ‘touchdown’ cycling programme: 95°C for 10 min, followed by 7 cycles of 94°C for 1 min, 67°C decreasing to 53°C at 2°C per cycle for 1 min, and 72°C for 1 min, followed by 24 cycles of 94°C for 30 s, 53°C for 40 s, and 72°C for 1 min, and a final extension step at 72°C for 10 min.

The products of the amplification were separated on a 6.5% (w/v) polyacrylamide gel with a 4300 DNA Analyser (LI-COR Corporate). The data were collected automatically based on the differential fluorescence (700 or 800 nm) of the products, and allele scoring was performed using the program SAGA MX Generation (LI-COR Corporate, NE, USA).

Epinephelus marginatus: Eight microsatellite loci were amplified from 5 ng of extracted E. marginatus DNA using primer pairs originally developed for Mycteroperca microlepis (GAG007, GAG008, GAG010, GAG023, GAG045 and GA049) (Chapman et al. 1999Chapman R.W., Sedberry G.R., Koenig C.C., et al. 1999. Stock identification of gag, Mycteroperca microlepis, along the southeast coast of the United States. Mar. Biotechnol. 1: 137-146.) and for Epinephelus quernus (CA-3 and CA-6) (Rivera et al. 2003Rivera M.A.J., Graham G.C., Roderick G.K. 2003. Isolation and characterization of nine microsatellite loci from the Hawaiian grouper Epinephelus quernus (Serranidae) for population genetic analyses. Mar. Biotechnol. 5: 126-129.). The DNA extraction and PCR conditions were the same as those for C. undecimalis, with the exceptions of the MgCl2 concentration, which was modified to 1.5 mM, and annealing temperature, which started at 54°C and then decreased to 40°C.

Data analysis

Genotype and allele frequencies of the microsatellite loci from each species were analysed to obtain standard genetic diversity estimates.

The total number of alleles (NA), allelic richness (AR), observed heterozygosity (HO) and expected heterozygosity (HE) were calculated with FSTAT v. 2.9.3 (Goudet 1995Goudet J. 1995. FSTAT (Version 1.2): A computer program to calculate F-statistics. J. Hered. 86: 485-486.). The departures from Hardy-Weinberg equilibrium (HWE) and the linkage disequilibrium between pairs of loci, with significance set at P<0.05, were assessed with GENEPOP v. 4.1 (Raymond and Rousset 1995Raymond M., Rousset F. 1995. GENEPOP (version-1.2) - population-genetics software for exact tests and ecumenicism. J. Heredity 86: 248-249.) and were later adjusted using a sequential Bonferroni correction (Rice 1989Rice W.R. 1989. Analyzing tables of statistical tests. Evolution 43: 223-225.). Variations in allelic frequencies were quantified using F-statistics (f, F, θ) per locus over samples, which correspond to Wright’s FIS, FIT and FST, respectively. The statistical significance of departures from zero was tested using bootstrapping over loci (FSTAT). Null allelic frequencies were calculated based on the method of Brookfield (1996)Brookfield J.F.Y. 1996. A simple new method for estimating null allele frequency from heterozygote deficiency. Mol. Ecol. 5: 453-455., using the program MICRO-CHECKER v. 2.2.3 (Van Oosterhout et al. 2004Van Oosterhout C., Hutchinson W.F., Wills D.P.M., et al. 2004. MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Notes 4: 535-538.) and the expectation maximization algorithm (Dempster et al. 1977Dempster A.P., Laird N.M., Rubin D.B. 1977. Maximum likelihood from incomplete data via em algorithm. J. R. Stat. Soc. Ser. B. Methodol. 39: 1-38.). The influence of null alleles was determined in FREENA (Chapuis and Estoup 2007Chapuis M.P., Estoup A. 2007. Microsatellite null alleles and estimation of population differentiation. Mol. Biol. Evol. 24: 621-631.) by computing the genetic divergence parameter FST values using an ENA (excluding null alleles) correction. After accounting for null allele frequencies, loci with frequencies of ≥0.2 were considered potentially problematic for the calculations.

The genetic structure based on the microsatellite data was further investigated using a Bayesian model-based Markov Chain Monte Carlo clustering method and was implemented using the program STRUCTURE v. 2.3.3 (Pritchard et al. 2000Pritchard J.K., Stephens M., Donnelly P. 2000. Inference of population structure using multilocus genotype data. Genetics 155: 945-959., Hubisz et al. 2009Hubisz M.J., Falush D., Stephens M., et al. 2009. Inferring weak population structure with the assistance of sample group information. Mol. Ecol. Resour. 9: 1322-1332.). This method estimates the number of genetic clusters (K) without any a priori assumptions about the population structure. The following parameters for each species were applied to the analysis: diploid, an admixture model and correlated allele frequencies. Following a burn-in period of 300000, ten independent runs were performed for each K value (from 1 to 10), with 500000 iterations. The true value of K (ΔK) was chosen according to the method of Evanno et al. (2005)Evanno G., Regnaut S., Goudet J. 2005. Detecting the number of clus