Genetic diversity in two threatened species of guitarfish (Elasmobranchii: Rhinobatidae) from the Brazilian and Argentinian coasts: an alert for conservation

Vanessa P. Cruz Aisni M.C. L. Adachi Pablo H. Oliveira Giovana S. Ribeiro Fabilene G. Paim Bruno C. Souza Alexandre S.F. Rodrigues Marcelo Vianna Sergio M. Delpiani Juan Martín Díaz de Astarloa Matheus M. Rotundo Fernando F. Mendonça Claudio Oliveira Rosangela P. Lessa Fausto Foresti About the authors

Abstract

The guitarfishes Pseudobatos horkelii and Pseudobatos percellens meet the criteria for threatened status as Critically Endangered (CR) and Endangered (EN), respectively. Both species occur in the Southern Atlantic Ocean. Considering the lack of data on the genetic structure of these species, the present study evaluated the genetic variability and population structure of the P. horkelii and P. percellens in the southern region of Brazil and the northern coast of Argentina, based on sequences of mitochondrial DNA, Control Region (D-loop). Samples of P. horkelii (n = 135) were analyzed in six localities situated in Northern Argentina, along the Brazilian states’ coast. The mean of nucleotide diversity was 0.0053, the ΦST was 0.4277 and demographic analysis of P. horkelii suggests the existence of stability of the populations, with D = 0.9929, FS = 2.0155, SSD = 0.0817, R = 0.2153. In P. percellens (n = 101) were analyzed from six Brazilian localities along the coast of Santa Catarina, Paraná, and São Paulo. The mean nucleotide diversity was 0.0014 and ΦST value of 0.2921, the demographic analysis indicates a high migration rate of P. percellens among the localities evaluated, with D = 0.5222, FS = 0.3528, SSD = 0.01785, R = 0.3890.

Keywords:
D-loop; Endangered species; Populations; Pseudobatos horkelii; Pseudobatos percellens

Resumo

As raias violas Pseudobatos horkelii e Pseudobatos percellens, são listados como “Criticamente em Perigo” (CR) e “Em Perigo” (EN), respectivamente. Ambas as espécies ocorrem no Sul do Oceano Atlântico. Considerando a falta de dados sobre a estrutura genética dessas espécies, o presente estudo avaliou a variabilidade genética e a estrutura populacional de P. horkelii e P. percellens na região sudeste do Brasil e litoral norte da Argentina, com base em sequências de DNA mitocondrial, região de controle (D-loop). Amostras de 135 indivíduos de P. horkelii analisados em seis localidades, situadas no norte da Argentina e ao longo da costa dos estados brasileiros. A média da diversidade nucleotídica foi de 0.0053, o índice ΦST foi de 0.4277 e a análise demográfica de P. horkelii, indicou a existência de estabilidade das populações, com D = 0.9929, Fus = 2.0155, SSD = 0.0817, R = 0.2153. Em 101 exemplares de P. percellens, foram analisados em seis localidades brasileiras ao longo do litoral de Santa Catarina, Paraná e São Paulo. A diversidade nucleotídica média foi de 0.0014 e o valor ΦST de 0.2921, a análise demográfica indicou uma alta taxa de migração de P. percellens entre as localidades analisadas, com D = 0.5222, FS = 0.3528, SSD = 0.01785, R = 0.3890.

Palavras-chave:
D-loop; Espécies ameaçadas; Populações; Pseudobatos horkelii; Pseudobatos percellens

INTRODUCTION

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A quarter of all Elasmobranch species are thought to be threatened by overfishing, either as fishery targets or as bycatch, according to the Red List of International Union for Conservation of Nature (IUCN) Dulvy et al., 2014Dulvy NK, Fowler SL, Musick JA, Cavanagh RD, Kyne PM, Harrison LR et al. Extinction risk and conservation of the world’s sharks and rays. eLIFE. 2014; 3:e00590. ). In general, elasmobranchs have a complex life history characterized by low rates of survival and population growth, which reinforces their sensitivity to mortality (Worm et al., 2013Worm B, Davis B, Kettemer L, Ward-Paige CA, Chapman D, Heithaus MR et al. Global catches, exploitation rates, and rebuilding options for sharks. Mar Policy. 2013; 40:194–204. https://doi.org/10.1016/j.marpol.2012.12.034
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). The crucial life history characteristics include slow growth, late maturity, relatively long life expectancy, and low fecundity and reproductive frequency (Cortés, 2000Cortés E. Demographic analysis as an aid in shark stock assessment and management. Fish Res. 1998; 39(2):199–208. https://doi.org/10.1016/S0165-7836(98)00183-0
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). It is considered that the reduction of elasmobranch populations may have a wide range of negative consequences for both ecological and economic systems (Stevens et al., Stevens JD, Bonfil R, Dulvy NK, Walker PA. The effects of fishing on sharks, rays, and chimaeras (Chondrichthyans), and the implications for marine ecosystems. ICES J Mar Sci. 2000; 57(3):476–94. https://doi.org/10.1006/jmsc.2000.0724
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).

The guitarfish Pseudobatos horkelii (Müller & Henle, 1841) and Pseudobatos percellens (Walbaum, 1792) are demersal species, usually found on sandy or loamy bottoms of the continental shelf, feeding mainly on small fish, crustaceans, and small invertebrates (Bigelow, Schroeder, 1953Bigelow HB, Schroeder WC. Fishes of the Gulf of Maine. Fish Bull. 1953; 53(1):577. http://cybrary.friendsofmerrymeetingbay.org/fgom/Default.htm
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; McEachran, Carvalho, 2002McEachran JD, Carvalho MR. Batoid fishes. In: Carpenter KE, editor. The living marine resources of the Western Central Atlantic. Rome: FAO; 2002. p.1–599. vol 1, Introduction, mollusks, crustaceans, hagfishes, sharks, batoid fishes and chimaeras. (FAO Species Identification Guide for Fishery Purposes and American Society of Ichthyologists and Herpetologist; Special Publication 1).; Bornatowski et al., 2010Bornatowski H, Robert MDC, Costa L. Feeding of guitarfish Rhinobatos percellens (Walbaum, 1972) (Elasmobranchii, Rhinobatidae), the target of artisanal fishery in Southern Brazil. Braz J Oceananogr. 2010; 58(1):45–52. https://doi.org/10.1590/S1679-87592010000100005
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). Pseudobatos horkelii, currently listed by the IUCN as “Critically Endangered” (Pollom et al., 2020aPollom R, Barreto R, Charvet P, Chiaramonte GE, Cuevas JM, Herman K et al. Pseudobatos horkelii. The IUCN Red List of Threatened Species 2020a: e.T41064A2951089.https://dx.doi.org/10.2305/IUCN.UK.2020-3.RLTS.T41064A2951089.en
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), is found between Rio de Janeiro in Brazil and Mar del Plata in Argentina (Miranda, Vooren, 2003) and Pseudobatos percellens, currently listed as “Endangered” (Pollom et al., 2020bPollom R, Charvet P, Avalos C, Blanco-Parra MP, Derrick D, Espinoza E et al.Pseudobatos percellens. The IUCN Red List of Threatened Species 2020b: e.T161373A887217.https://dx.doi.org/10.2305/IUCN.UK.2020-3.RLTS.T161373A887217.en
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), is distributed from the Gulf of Mexico to Northern Argentina (McEachran, Carvalho, 2002McEachran JD, Carvalho MR. Batoid fishes. In: Carpenter KE, editor. The living marine resources of the Western Central Atlantic. Rome: FAO; 2002. p.1–599. vol 1, Introduction, mollusks, crustaceans, hagfishes, sharks, batoid fishes and chimaeras. (FAO Species Identification Guide for Fishery Purposes and American Society of Ichthyologists and Herpetologist; Special Publication 1).).

The guitarfish species of the family Rhinobatidae have been under fishing pressure mainly because they are bycatch of trawling fisheries such as beach seine, single and double trawling, and gillnets (Garstin et al., 2018Garstin A, Oxenford HA. Reducing Elasmobranch bycatch in the Atlantic Sea bob (Xiphopenaeus kroyeri) trawl fishery of Guyana. Gulf Caribb Rese. 2018; 29(1):GCFI10–GCFI20. https://doi.org/10.18785/gcr.2901.04
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), primarily off the coast of southern and southeastern Brazil, Uruguay, and Argentina (Martins, Schwingel, 2003Martins RR, Schwingel PR. Variação espaço-temporal da CPUE para o gêneroRhinobatos(Rajiformes, Rhinobatidae) na costa sudeste e sul do Brasil. Notas Téc. Facimar. 2003; 7:119–29. https://doi.org/10.14210/bjast.v7n1.p119-129
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; Massa et al., 2004Massa AM, Lucifora LO, Hozbor NM. Condrictios de la region costera bonaerense y uruguaya. In: Boschi EE, editor. El Mar Argentino y sus recursos pesqueros. Mar del Plata: Publicaciones especiales INIDEP; 2004. p.85–99. (Los peces marinos de interes pesquero. Caracterizacion biologica y evaluación del estado de explotación; v. 4).; Costa, Chaves, 2006Costa L, Chaves PTC.Elasmobrânquios capturados pela pesca artesanal na costa sul do Paraná e norte de Santa Catarina, Brasil. Biota Neotrop. 2006; 6(3): bn02706032006. https://doi.org/10.1590/S1676-06032006000300007
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; Bornatowski et al., 2010Bornatowski H, Robert MDC, Costa L. Feeding of guitarfish Rhinobatos percellens (Walbaum, 1972) (Elasmobranchii, Rhinobatidae), the target of artisanal fishery in Southern Brazil. Braz J Oceananogr. 2010; 58(1):45–52. https://doi.org/10.1590/S1679-87592010000100005
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). The stocks of P. horkelii were reduced by more than 50% between 1994 and 1999 by fisheries operating in the coastal waters of Argentina and Uruguay (Massa et al., 2004Massa AM, Lucifora LO, Hozbor NM. Condrictios de la region costera bonaerense y uruguaya. In: Boschi EE, editor. El Mar Argentino y sus recursos pesqueros. Mar del Plata: Publicaciones especiales INIDEP; 2004. p.85–99. (Los peces marinos de interes pesquero. Caracterizacion biologica y evaluación del estado de explotación; v. 4).). Overfishing in Southern Brazil also led to the near exhaustion of P. horkelii stocks in the 1980s. Currently, this species is thought to be at 16% of its original stocks, a level considered far below the maximum sustainable yield (Miranda, Vooren, 2003Miranda LV, Vooren CM. Captura e esforço da pesca de elasmobrânquios demersais no Sul do Brasil nos anos de 1975 a 1997. Frente Marítimo. 2003; 19(B):217–31). In 2004, the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) of the Brazilian Ministry of the Environment banned the fishing and sale of this species through normative instruction MMA 5/2004.

The available studies on P. horkelli and P. percellens include research mainly on reproductive biology and population dynamics (Lessa et al., 1999Lessa R, Santana FM, Paglerani R. Age, growth and stock structure of the oceanic whitetip shark,Carcharhinus longimanus, from the southwestern equatorial Atlantic. Fish Res. 1999; 42(1–2):21–30. https://doi.org/10.1016/S0165-7836(99)00045-4
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; Rocha, Gadig, 2013Rocha F, Gadig OBF. Reproductive biology of the guitarfish Rhinobatos percellens (Chondrichthyes, Rhinobatidae) from the São Paulo Coast, Brazil, western South Atlantic Ocean. J Fish Biol. 2013; 82(1):306–17. https://doi.org/10.1111/j.1095-8649.2012.03493.x
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; Pasquino et al., 2016Pasquino AF, Martins MF, Gadig OBF. Length–weight relationship of Rhinobatos horkelii Müller and Henle, 1841 and Zapteryx brevirostris (Müller and Henle, 1841) off Brazil, southwestern Atlantic Ocean. J Appl Ichthyol. 2016; 32(6):1282–83. https://doi.org/10.1111/jai.13171
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; Martins et al., 2018Martins MF, Pasquino AF, Gadig OBF. Reproductive biology of the Brazilian guitarfish, Pseudobatoshorkelii (Müller and Henle, 1841) from southeastern Brazil, western South Atlantic. J Appl Ichthyol. 2018; 34(3):646–52. https://doi.org/10.1111/jai.13652
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), feeding habits, and distribution (Menni, Stehmann, 2000Menni RC, Stehmann MFW. Distribution, environment and biology of batoid fishes of Argentina, Uruguay and Brazil. A review. Rev Mus Argent Cienc Nat. 2000; 2(1):69–109. Available from: http://revista.macn.gob.ar/ojs/index.php/RevMus/article/view/126/118
http://revista.macn.gob.ar/ojs/index.php...
; Bornatowski et al., 2010Bornatowski H, Robert MDC, Costa L. Feeding of guitarfish Rhinobatos percellens (Walbaum, 1972) (Elasmobranchii, Rhinobatidae), the target of artisanal fishery in Southern Brazil. Braz J Oceananogr. 2010; 58(1):45–52. https://doi.org/10.1590/S1679-87592010000100005
https://doi.org/10.1590/S1679-8759201000...
; Carmo et al., 2015Carmo WP, Bornatowski H, Oliveira EC, Fávaro LL. Diet of the chola guitarfish, Rhinobatos percellens (Rhinobatidae), in the Paranaguá Estuarine complex. An Acad Bras Ciênc. 2015; 87(2):721–31. http://dx.doi.org/10.1590/0001-3765201520140121
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; Rezende et al., 2020Rezende TM, Barreto R, Felizola KM. Record of Pseudobatos horkelii (Rhinopristiformes: Rhinobatidae) off the state of Sergipe, Brazil, Southwestern Atlantic Ocean. PanAm J Aquat Sci. 2020; 15(1):23–27. Available from: https://panamjas.org/pdf_artigos/PANAMJAS_15(1)_23-27.pdf
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). However, no data are available on the genetic diversity, population genetic structure or gene flow of either species. These data are fundamental to the development of regulatory programs for the conservation of natural stocks and recovery of endangered species (Ovenden et al., 2015Ovenden JR, Berry O, Welch DJ, Buckworth RC, Dichmont CM. Ocean’s eleven: a critical evaluation of the role of population, evolutionary and molecular genetics in the management of wild fisheries. Fish Fish. 2015; 16(1):125–59. https://doi.org/10.1111/faf.12052
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; Carrier et al., 2018Carrier JC, Heithaus MR, Simpfendorfer CA. Shark research: emerging technologies and applications for the field and laboratory. Boca Raton: CRC Press; 2018.; Domingues et al., 2018Domingues RR, Hilsdorf AWS, Gadig OBF. The importance of considering genetic diversity in shark and ray conservation policies. Conserv Genet. 2018; 19:501–25. https://doi.org/10.1007/s10592-017-1038-3
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). When the stock of a species declines, there may be dramatic effects on evolutionary processes such as inbreeding and genetic drift, highlighting the importance of genetic data to develop conservation initiatives. These possible impacts on the adaptive potential of a population confronted by environmental changes, irrespective of other active factors, contribute to a predictable increase in effective extinction risk (Ovenden et al., 2015Ovenden JR, Berry O, Welch DJ, Buckworth RC, Dichmont CM. Ocean’s eleven: a critical evaluation of the role of population, evolutionary and molecular genetics in the management of wild fisheries. Fish Fish. 2015; 16(1):125–59. https://doi.org/10.1111/faf.12052
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).

Since P. horkelli and P. percellens have been overfished in recent years, and given the lack of data on the genetic structure of their populations, the present study aimed to identify the genetic diversity and population structure of these two species along the area from Southeastern Brazil to Northern Argentina using DNA sequences of the mitochondrial Control Region (D-loop), as well as their population dynamics and demographic history and test the hypothesis of panmixia. This study’s outcomes will be of great value to a better understanding of the species and populations’ structure and distribution and contribute to establishing appropriate programs of management and conservation for these species.

MATERIAL AND METHODS

Sample collection. Samples of the P. horkelii (n = 135) and P. percellens (n = 101) were collected by artisanal fishers at different localities along the Atlantic Ocean coast from north of Argentina to Southeastern Brazil (S1). Samples of P. horkelii were collected in Mar del Plata, Argentina (AR, n = 12) and in five localities in Brazil, being 41 in Torrinha (RS); 22 in Florianópolis (SC); 16 in Pontal do Paraná (PR); 20 in Santos (SP); and 24 in Rio de Janeiro (RJ). Samples of P. percellens were collected in three localities in Brazil, being 19 in Florianópolis (SC); 25 in Pontal do Paraná (PR); and in three points in the State of São Paulo, being 16 in Cananéia (SP); 12 in Mongaguá (SP2); and 29 in Santos (SP).

A small fragment of muscle tissue (< 1 cm2) was collected from each animal, placed in 2ml sample vials, and preserved in 96% ethanol. All samples were collected in strict accordance with the regulations of the Brazilian Federal Animal Ethics Committee (SISBIO 13843–1), and the analyses followed the International Guidelines for Animal Experiments, as authorized by CEEAA IBB/UNESP, protocol number 556. The tissues were deposited in the collection of the Laboratory of Fish Biology and Genetics – UNESP in Botucatu, São Paulo, Brazil.

The DNA was extracted using the NucleoSpin Tissue XS kit (Macherey and Nagel, Dand, Germany). Partial sequences of the control region of the mitochondrial DNA (D-loop) were obtained by polymerase chain reaction (PCR) using the Dloop2F (CAA AGC CWA GAT TTT TAT TAA AC) and Dloop5R (RCW WAT TAA TAG GAC GGT AMT GGA Y), in hypervariable region II, developed specifically for this research. The samples were amplified in reactions of 12.5 µl containing: 10.35 ul of ultrapure water; 0.90 ul buffer (Tris–HCl 20 mM pH 8.4 and KCl 50 mM), 0.75 μl dNTPs (2mM); of 0,1 μl of Taq DNA polymerase Taq, 0.2 μl of each primer. The cycling conditions used in PCR reactions (Veriti® 96-well Thermal Cycler, Biosystems TM Applied or Mastercycler® EPGradient, Eppendorf) were performed using the following thermal temperatures: initial denaturation at 94°C for 5 min, followed by 35 cycles including denaturation at 94°C of 30s, annealing at 52°C for 30s, extension at 72°C for 1min, and a final extension at 72°C for 10 min. PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) to sequence the samples in an automated ABI 3130xl (Applied Biosystems). The sequences were edited in GENEIOUS 6.0 (Kearse et al., 2012Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28(12):1647–49. https://doi.org/10.1093/bioinformatics/bts199
https://doi.org/10.1093/bioinformatics/b...
) and aligned using the Muscle algorithm (Edgar, 2004Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32(5):1792–797. https://doi.org/10.1093/nar/gkh340
https://doi.org/10.1093/nar/gkh340...
) run in Geneious 6.0. The haplotype sequences were deposited in GenBank under accession numbers MK809354-MK809366.

Population analysis. The relative nucleotide composition, the number of polymorphic sites, the number and relative frequency of haplotypes, haplotype diversity (Hd), nucleotide diversity (π), and the pairwise nucleotide differences between populations were all calculated in ARLEQUIN 3.5.1.3 (Excoffier, Lischer, 2010Excoffier L, Lischer HE. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour. 2010; 10(3):564–67. https://doi.org/10.1111/j.1755-0998.2010.02847.x
https://doi.org/10.1111/j.1755-0998.2010...
). A haplotype network was obtained by the Median-Joining method (Bandelt et al., 1999Bandelt HJ, Forster P, Röhl A. Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol. 1999; 16(1):37–48. https://doi.org/10.1093/oxfordjournals.molbev.a026036
https://doi.org/10.1093/oxfordjournals.m...
) using the software PopART 1.7 (Leigh, Bryant, 2015Leigh JW, Bryant D. Popart: full‐feature software for haplotype network construction. Methods Ecol Evol. 2015; 6(9):1110–16. https://doi.org/10.1111/2041-210X.12410
https://doi.org/10.1111/2041-210X.12410...
).

The pairwiseΦST diversity index was used to estimate the levels of genetic divergence between the localities for P. horkelii and P. percellens, that were tested non-parametrically using 1000 bootstrap replicates (Felsenstein, 1985Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985; 39(4):783–91. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x
https://doi.org/10.1111/j.1558-5646.1985...
), runned in ARLEQUIN 3.5.1.3 and adjusted for simultaneous pairwise comparisons using the sequential Bonferroni procedure (Rice, 1989Rice WR. Analyzing tables of statistical test. Evolution. 1989; 43(1):223–25. https://doi.org/10.2307/2409177
https://doi.org/10.2307/2409177...
). Indeed, genetic divergence was calculated by the Molecular Analysis of Variance, or AMOVA (Excoffier et al., 1992Excoffier L, Smouse PE, Quattro JM. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics. 1992; 131(2):479–91.), with an a priori criterion to test panmixia among samples, and differentiation was assessed among (ΦCT) and within (ΦSC) regions. For P. horkelii we grouped into Northern Argentina and Southern Brazil (AR, RS, SC, PR), and between Southeastern Brazil (SP3, RJ), whereas for P. percellens, we grouped into Southern Brazil (SC, PR) and between Southeastern Brazil (SP1, SP2, SP3).

The demographic parameters Fu’s F (Fu, 1996Fu YX. New statistical tests of neutrality for DNA samples from a population. Genetics. 1996; 143(1):557–70. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1207287/pdf/ge1431557.pdf
https://www.ncbi.nlm.nih.gov/pmc/article...
) and Tajima’s D test (Tajima, 1989Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 1989; 123(3):585–95.), as implemented in Arlequin, was used to test departures from neutrality due to recent population size expansions, or alternatively because of selection, and were obtained from nucleotide mismatch distributions with the Arlequin software to examine the possibility of demographic expansion, with the Sum of Square Deviation (SSD) and Raggedness index.

We used BAPS v 6.0 (Corander et al., 2013Corander J, Cheng L, Marttinen P, Sirén J, Tang J. BAPS: Bayesian analysis of population structure. Manual v 6.0. Bioinformatics. 2013; 28:2537–39.) to identify discrete genetic clusters within the dataset, with the most probable number of genetic groups formed by the sequences being inferred by a Bayesian analysis of the population structure.

To test for isolation by distance (IBD) patterns were examined in guitarfish species using Mantel tests in GENEPOP 4 (Raymond, Rousset, 1995Raymond M, Rousset F. Genepop (Version-1.2): population genetics software for exact tests and ecumenicism. J Hered. 1995; 86(3):248–49. https://doi.org/10.1093/oxfordjournals.jhered.a111573
https://doi.org/10.1093/oxfordjournals.j...
; Rousset, 2008Rousset F. Genepop’007: a complete re-implementation of the genepop software for Windows and Linux. Mol Ecol Resour. 2008; 8(1):103–06.) to determine the correlation between geographical distances and genetic distances (Mantel, 1967Mantel N. The detection of disease clustering and a generalized regression approach. Cancer Res. 1967; 27:209–20. Available from: https://cancerres.aacrjournals.org/content/canres/27/2_Part_1/209.full.pdf
https://cancerres.aacrjournals.org/conte...
; Slatkin, 1993Slatkin M. Isolation by distance in equilibrium and nonequilibrium populations. Evolution. 1993; 47(1):264–79. https://doi.org/10.2307/2410134
https://doi.org/10.2307/2410134...
). Geographical distance was calculated as from sampling points using GPS and were estimated in km from Google Earth Pro (http://earth.google.co.uk).

RESULTS

Pseudobatos horkelii. A total of 135 consensus D-loop sequences of 702 base pairs in length were obtained for P. horkelii from localities situated along the northern coast of Argentina (AR), and Southern (RS, SC, PR) and Southeastern Brazil (SP3, RJ). The nucleotide frequencies of these sequences were A = 32.1%; C = 11.2%; G = 23.5% and T = 33.1%. Ten polymorphic sites (S) were detected (Tab. 1), with nucleotide diversity (π) ranging from 0.0013±0.0006 in AR to 0.0043±0.0018 in RS, with a mean of 0.0053. In total, 16 haplotypes (H) were identified as Ph1 to Ph16 (Fig. 1A).

TABLE 1 |
Polymorphisms and frequency found in haplotypes of Pseudobatos horkelii and P. percellens, based on D-loop. Regions of the North of Argentina coast and South-eastern / Southern regions of Brazil. AR – Argentina, RS – Rio Grande do Sul, SC – Santa Catarina, PR – Paraná, SP1 – Cananéia/SP, SP2 – Mongaguá/SP, SP3 – Santos /SP, RJ – Rio de Janeiro.

FIGURE 1 |
Median-joining network of mtCR haplotypes for A. Pseudobatos horkelii and B. Pseudobatos percellens. Haplotypes are represented by circles with size proportional to frequency in the total sample. All hatch marks correspond to one mutation. Samples from northern Argentina (AR), Torrinha/RS (RS), Florianópolis/SC (SC), Pontal do Paraná/PR (PR), Cananéia/ SP (SP1), Mongaguá/SP (SP2), Santos/SP (SP3), Rio de Janeiro/RJ(RJ).

The haplotypes that were present in a great number of specimens were Ph8 (15.5%) and Ph12 (17%) (Tab. 1). The less frequent haplotypes Ph 11 and Ph 12 were found in RJ and RS, respectively with a unique haplotype observed in only one geographic sample. Ten among the 16 haplotypes analyzed with an overall haplotype diversity (Hd) of 0.8992, are composed of specimens from RS which presented the greatest Hd = 0.8902±0.0214 (Tab. 2). The geographic region with the lowest number of haplotypes was PR, with Hd = 0.5333±0.0456.

The pairwise ΦST were estimated based on the control region, the values ranged from 0.0969 between AR and RS, to 0.6427 between AR and SP3 (S2), and a higher level of genetic differentiation with statistically significant ΦST was found between all localities (P < 0.05). The molecular variance analysis (AMOVA) resulted in a partition of the genetic variation into Northern of Argentina/Southern of Brazil (AR, RS, SC, PR) to Southeastern (SP3, RJ) of Brazil (ΦCT = 0.2576; P = 0.0000), and significant differences between locations within regions of ΦSC = 0.2664; P = 0.0000 (Tab. 3). The overall ΦST value was 0.4277 (P = 0.0000), thus rejecting a hypothesis of panmixia.

TABLE 2 |
Population statistics of Pseudobatos horkelii and P. percellens, based on D-loop. Number of individuals (n), polymorphic sites (S), number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), mean number of pairwise differences (K).
TABLE 3 |
Hierarchical AMOVA for the control region of Pseudobatos horkelii and P. percellens. Samples of P. horkelii were grouped into northern Argentina (AR), southern Brazil (BRA: RS, SC, PR) and southeastern Brazil (BRA: SP3, RJ). Whereas for P. percellens, were grouped into southern Brazil (BRA: SC, PR) and southeastern Brazil (BRA: SP1, SP2, SP3).

The demographic analysis considering the locality or all samples as a local population, showed non-significant values of neutrality by Tajima’s D and Fu’s FS tests (S3). Similar results represented by non-significant values were found by the AMOVA neutrality tests when two groups as AR, RS, SC, PR and SP3, RJ were considered. Although the Raggedness and SSD indices are significant (p> 0.05) for almost all analysis in different tests by localities or groups, the graph of mismatch showed differences in observed and expected curves, representing a bimodal pattern between all samples (S4). The Bayesian analysis generated three groups which did not correspond to the geographic localities (Fig. 2A). Mantel test results a positive value, withal not significant for P. horkelii (r = 0.059; P = 0.4210).

FIGURE 2 |
Graph of the Bayesian analysis of population structure of mtCR for A. Pseudobatos horkelii and B. Pseudobatos percellens. Samples from northern Argentina (AR), Torrinha/RS (RS), Florianópolis/SC (SC), Pontal do Paraná/PR (PR), Cananéia/ SP (SP1), Mongaguá/SP (SP2), Santos/SP (SP3), Rio de Janeiro/RJ(RJ).

Pseudobatos percellens. D-loop sequences of 646 bps were obtained from 101 specimens of P. percellens, from southern (SC, PR) and southeastern (SP1, SP2, SP3) regions in Brazil, with an overall nucleotide composition of A = 34.9%, C = 24.1%, G = 9.6% and T = 31.4%. Only six polymorphic sites were detected, representing eight haplotypes identified as Pp1 to Pp8 (Tab. 1, Fig. 1B). The mean nucleotide diversity (π) found was 0.0014, ranging from 0.0002±0.0000 in PR to 0.0020±0.0009 in SC. The Pp1 haplotype was found in 60.3% of the samples analyzed comprising the largest number among carrier specimens. Four among the eight haplotypes detected are present in the genome of only two specimens, being the haplotype Pp3 from PR, the haplotypes Pp5 and Pp6 from SP3, and the haplotype Pp7 from SP1.

The mean diversity haplotype found was Hd = 0.6049, calculated among samples of the five regions analyzed, and SP3 presented the largest number of haplotypes (five), with Hd = 0.6578+/-0.0663; other two regions presented two haplotypes, being PR with Hd = 0.1533+/- 0.0915 and SP2 with Hd = 0.4848+/- 0.1059 (Tab. 2).

Analysis of pairwise ranged from values of ΦST = 0.0320 between SP2 and SP3, to 0.3760 between PR and SP1 (S5). A high level of genetic differentiation with statistically significant ΦST was found between almost all localities (P < 0.05), excepting the result between SP2 and SP3 that was not significant (ΦST = 0.0320, p > 0.05). Likewise, the results of AMOVA reveal significant differences in the population structuring simulations (Tab. 3), with an overall ΦST value of 0.29218 (P = 0.0000), what rejects a hypothesis of panmixia. The AMOVA analysis also resulted in a partition of the genetic variation into southern (SC, PR) to southeastern (SP1, SP2, SP3) regions of Brazil (ΦCT = 0.2011; P = 0.0000), and revealed significant differences between locations within regions (ΦSC = 0.01972; P = 0.0000) (Tab. 3).

The indices of demographic analysis using the neutrality index showed non-significant values of neutrality by Tajima’s D and Fu’s FS tests (S3). Despite of the Raggedness and SSD indices are significant (p> 0.05) for almost all analysis in different tests, the graph of mismatch showed unimodal pattern between all samples (S4). The Bayesian analysis generated two groups which did not correspond to the geographic localities (Fig. 2A). Mantel test results a negative correlation for P. percellens (r = -0.3160, P = 0.3780).

DISCUSSION

The results of the present study provide interesting insights concerning the genetic diversity of two endangered guitarfish species Pseudobatos horkelii and P. percellens occurring in the Southeastern region of the Atlantic Ocean. The D-loop mitochondrial marker was used to obtain information regarding connectivity, genetic diversity and population structure of the species along the coast of Northern Argentina, Southern and Southeastern Brazil. These indexes indicate that both species present population structure in the different regions analyzed. Our data revealed that populations of P. horkelii showed significant differences among all locations sampled in Mar del Plata in Argentina and in five localities along the coast of the states of Rio Grande do Sul, Santa Catarina, Paraná, São Paulo and Rio de Janeiro in Brazil. Similar results were obtained in the analysis of P. percellens, which showed significant differences among samples obtained along the coast of the states of Santa Catarina, Paraná, and São Paulo, from which individuals from Cananéia, Mongaguá, and Santos were assessed.

The available genetic studies conducted on Pseudobatos species have so far been related to molecular identification of species and fishery products using the DNA barcode methodology as a molecular marker (Franco et al., 2012Franco B, Mendonça FF, Oliveira C, Foresti F. Illegal trade of the guitarfish Rhinobatos horkelii on the coasts of central and southern Brazil: genetic identification to aid conservation. Aquat Conserv. 2012; 22(2):272–76. https://doi.org/10.1002/aqc.2229
https://doi.org/10.1002/aqc.2229...
; Souza et al., 2018Souza FA, Sperb C, Castilho CL, Figueiredo PI, Gonçalves LT, Machado R et al. Molecular identification of shark meat from local markets in Southern Brazil based on DNA barcoding: Evidence for mislabeling and trade of endangered species. Front Genet. 2018; 9:138. https://doi.org/10.3389/fgene.2018.00138
https://doi.org/10.3389/fgene.2018.00138...
), and refer mainly to illegal trade of threatened species in different regions of the Brazilian coast, including locations in the states of Rio de Janeiro, São Paulo, Paraná, Santa Catarina and Rio Grande do Sul. Interestingly, even though guitarfish are listed as endangered species, our study’s data indicate considerable genetic diversity levels. Pseudobatos horkelii, which is currently a “Critically Endangered” (CR) species, showed indices of haplotypic diversity of Hd = 0.6518 and nucleotide diversity of π = 0.0029 as a result of ten mutations of the 702 bps of D-loop region sequenced, that resulted in 16 haplotypes identified in 135 individuals sampled. Alternatively, in P. percellens currently listed as “Endangered” (EN), the average genetic diversity indices are slightly smaller, with Hd = 0.5185 and π = 0.0010, and only six mutations were found, distributed in 646 bps sequenced, with eight haplotypes.

The same level of diversity have also been found in other species of skates of the family Rajidae, in Raja straeleni Poll, 1951 classified as “Deficient Data” (DD) (Smale, 2009Smale MJ.Raja straeleni. The IUCN Red List of Threatened Species 2009: e.T161586A5458059.https://dx.doi.org/10.2305/IUCN.UK.2009-2.RLTS.T161586A5458059.en
https://dx.doi.org/10.2305/IUCN.UK.2009-...
), the values found for haplotypic diversity were Hd = 0.67, and for nucleotide diversity π = 0.0025; and in Raja clavata Linnaeus, 1758 classified as “Near Threatened” (NT) (Ellis, 2016Ellis J. 2016.Raja clavata. The IUCN Red List of Threatened Species 2016: e.T39399A103110667.https://dx.doi.org/10.2305/IUCN.UK.2016-3.RLTS.T39399A103110667.en
https://dx.doi.org/10.2305/IUCN.UK.2016-...
), the value of Hd was 0.55 and π was 0.0023 (Pasolini et al., 2011Pasolini P, Ragazzini C, Zaccaro Z, Cariani A, Ferrara G, Gonzalez EG et al. Quaternary geographical sibling speciation and population structuring in the Eastern Atlantic skates (suborder Rajoidea) Raja clavata and R. straeleni. Mar Biol. 2011; 158(10):2173–86. https://doi.org/10.1007/s00227-011-1722-7
https://doi.org/10.1007/s00227-011-1722-...
). In the family Pristidae, the sawfish Pristis pristis (Linnaeus, 1758) classified as “Critically Endangered” (CR) (Kyne et al., 2013Kyne PM, Carlson J, Smith K.Pristispristis(errata version published in 2019). The IUCN Red List of Threatened Species 2013: e.T18584848A141788242.https://dx.doi.org/10.2305/IUCN.UK.2013-1.RLTS.T18584848A141788242.en
https://dx.doi.org/10.2305/IUCN.UK.2013-...
), Hd was 0.39 and π was 0.0011 (Feutry et al., 2015Feutry P, Kyne PM, Pillans RD, Chen X, Marthick JR, Morgan DL, Grewe PM. Whole mitogenome sequencing refines population structure of the Critically Endangered sawfish Pristispristis. Mar Ecol Prog Ser. 2015; 533:237–44. https://doi.org/10.3354/meps11354
https://doi.org/10.3354/meps11354...
). In this context, the real situation presented by the levels of genetic diversity in rays requires a most focused attention concerning conservation of wild stocks of the species in dangered situation of disappearance in this group of organisms. The values of Hd and π found for Raja straeleni, a species with deficient data of dangerousness, points to a situation similar as P. horkelii currently listed as “Critically Endangered” (CR). According to Domingues et al., (2018)Domingues RR, Hilsdorf AWS, Gadig OBF. The importance of considering genetic diversity in shark and ray conservation policies. Conserv Genet. 2018; 19:501–25. https://doi.org/10.1007/s10592-017-1038-3
https://doi.org/10.1007/s10592-017-1038-...
, despite an increase in the number of genetics studies in the last decade, only~ 10% of shark and ray species have been investigated in terms of their population genetic structure, genetic diversity and demographic history, theses information’s genetics, could help the determine applications for conservation (Hoban et al., 2013aHoban SM, Hauffe HC, Pérez-Espona S, Arntzen JW, Bertorelle G, Bryja J et al. Bringing genetic diversity to the forefront of conservation policy and management. Conserv Genet Resour. 2013a; 5:593–98. https://doi.org/10.1007/s12686-013-9859-y
https://doi.org/10.1007/s12686-013-9859-...
, bHoban S, Gaggiotti O, Bertorelle G. Sample planning optimization tool for conservation and population genetics (SPOTG): a software for choosing the appropriate number of markers and samples. Methods Ecol Evol. 2013b; 4:299–303. https://doi.org/10.1111/2041-210x.12025
https://doi.org/10.1111/2041-210x.12025...
).

Population structure and demography. Levels of genetic differentiation among guitarfishes revealed patterns of population structure along the Southeastern Atlantic Ocean area for P. horkelii and P. percellens. A hypothesis to explain the genetic structure may be the resident behavior of the species, which performs seasonal migrations only among depth zones between 50 and 150 m during the reproductive period that occurs in the winter and remains together up to 20 m depth (Vooren, 1997Vooren CM. Demersal elasmobranchs. In: Seeliger U, Odebrecht C, Castello JP, editors. Subtropical convergence environment: the coast and the sea in the Southwestern Atlantic. Berlim: Springer; 1997. p.41–146.). Similar findings of population structure in other resident batoids species have been reported with Hypanus americanus (Hildebrand & Schroeder, 1928) (Richards et al., 2019Richards VP, DeBiasse MB, Shivji M. Deep mitochondrial lineage divergence among populations of the southern stingray (Hypanus americanus (Hildebrand & Schroeder, 1928)) throughout the Southeastern United States and Caribbean. Mar Biodiv. 2019; 49(4):1627–34. https://doi.org/10.1007/s12526-018-0930-5
https://doi.org/10.1007/s12526-018-0930-...
). The analysis of 267 individuals of this species sampled from Eastern USA and Caribbean using the D-loop marker revealed a high level of genetic partitioning among localities (ΦST = 0.49; P < 0.0000) that permitted differentiate the samples into three populations. Furthermore, Le Port et al., (2011) also observed a high level of genetic structuring in samples of Bathytoshia brevicaudata (Hutton, 1875) among South Africa, Australia, and New Zealand populations, with an overall ФST = 0.67, P < 0.001, this structuring may be result of that the deep oceanic basins (and tropical waters) act as a major barrier to stingray dispersal.

In addition, other fact that may have influenced the structure of these populations may involve fishing activities. Few abundance estimates are available for the Brazilian guitarfish throughout its range, P. horkelii was highly explored in the Rio Grande do Sul region in the years 1980s and 1990s, with the mean biomass of annual catches ranging from 600 to 1,800 tons (Miranda, Vooren, 2003Miranda LV, Vooren CM. Captura e esforço da pesca de elasmobrânquios demersais no Sul do Brasil nos anos de 1975 a 1997. Frente Marítimo. 2003; 19(B):217–31), and the according the authors, believe that the effects of overfishing, may cause a population decline. However, for P. percellens there are no regular records of fishing exploitation in the region the available data indicate occurrence of capture by bycatch, that is, as accompanying fauna in fishing of other commercially interesting species (Lessa, Vooren, 2007Lessa R, Vooren CM. Rhinobatoshorkelii. The IUCN Red List of Threatened Species. Version 2016.3 [Internet]; 2007. Available from: http://www.iucnredlist.org
http://www.iucnredlist.org...
). Such activity also should determine strong interferences in the structure of populations.

In this case, the tests of neutrality (the Tajima D and Fu FS statistics) were used to check the excess of rare mutations. Non-significant results showed evidence of recent population expansion for P. horkelii. The demographic analysis for the P. horkelii based on D-loop pairwise nucleotide differences showed a bimodal mismatch distribution model and a high Harpending’s raggedness index in values ranging from 0.0183 to 0.7866, with significant P values. The bimodal curve indicates the existence of a population demographically stable (Slatkin, Hudson, 1991Slatkin M, Hudson RR. Pairwise comparisons of mitochondrial DNA sequences in stable and exponentially growing populations. Genetics. 1991; 129(2):555–62. ; Rogers, Harpending, 1992Rogers AR, Harpending H. Population growth makes waves in the distribution of pairwise genetic differences. Mol Biol Evol. 1992; 9(3):552–69. https://doi.org/10.1093/oxfordjournals.molbev.a040727
https://doi.org/10.1093/oxfordjournals.m...
) that has suffered a sudden size reduction, a population bottleneck and should be in a recent population expansion. In this case, the tests of neutrality (the Tajima D and Fu FS statistics) were used to check the excess of rare mutations. Non-significant results showed evidence of recent population expansion for P. horkelii. This could suggest that the bimodal curve characterizes the existence of stable populations, despite the great fishing exploitation that the species has suffered in the last decades.

The mismatch distribution analysis showed unimodal graphic for P. percellens, which could indicate the occurrence of population expansion events or events of expansion of the geographic distribution area, with a high migration rates among close populations (Rogers, Harpending, 1992Rogers AR, Harpending H. Population growth makes waves in the distribution of pairwise genetic differences. Mol Biol Evol. 1992; 9(3):552–69. https://doi.org/10.1093/oxfordjournals.molbev.a040727
https://doi.org/10.1093/oxfordjournals.m...
). If this information is analyzed together with the non-significant data from the neutrality tests, the results could be interpreted as a strong indication that in P. percellens it is possible that a high migration rate is occurring between the analyzed sites.

In the present study, the results of the Bayesian analysis for P. horkelli and P. percellens indicated K = 3 clusters. For P. horkelii, Rio Grande do Sul was the only locality showed the three clusters, Santa Catarina and Paraná showed the same clusters (blue and vermelho), while Argentina showed a single cluster (green). In P. percellens, presented for Paraná and Santos a single cluster (green), while individuals from Santa Catarina and Cananéia showed different clusters, blue and green (SC) and red and green (SP1), respectively.

Even though P. horkelii has suffered a severe population decline of more than 80% due to overfishing (Lessa, Vooren, 2007Lessa R, Vooren CM. Rhinobatoshorkelii. The IUCN Red List of Threatened Species. Version 2016.3 [Internet]; 2007. Available from: http://www.iucnredlist.org
http://www.iucnredlist.org...
), its populations revealed high indices of genetic diversity, suggesting the existence of stable populations of this species, with highest haplotypic diversity occurring mostly in the region of Rio Grande do Sul, where the prohibition on the exploitation of this species should be most effective. Even considering the lack of population or capture data available for P. percellens, this species is captured by intense fishing practices similar to those observed for P. horkelii, in which different types of trawls, gillnets, and seines are used, which has also led to a significant decline in populations of this species in recent years. In this context, it must be considered that the proper management of coastal habitats inhabited by these species, as well as an efficient management and control of fisheries at the regional and national levels should be a priority in planning conservation programs.

ACKNOWLEDGEMENTS

This study was funded by the Brazilian agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES). The authors thank João B. L. Sales for the revision of this manuscript and Bianca S. Rangel for discussion of the results. To the fishers of the ‘‘Projeto Pró-Pesca: pescando o conhecimento” for providing samples from southeastern Brazil.

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ADDITIONAL NOTES

  • HOW TO CITE THIS ARTICLE

    Cruz VP, Adachi AMCL, Oliveira PH, Ribeiro GS, Paim FG, Souza BC, Rodrigues ASF, Vianna M, Delpiani SM, Díaz de Astarloa JM, Rotundo MM, Mendonça FF, Oliveira C, Lessa RP, Foresti F. Genetic diversity in two threatened species of guitarfish (Elasmobranchii: Rhinobatidae) from the Brazilian and Argentinian coasts: an alert for conservation. Neotrop Ichthyol. 2021; 19(2):e210012. https://doi.org/10.1590/1982-0224-2021-0012

Publication Dates

  • Publication in this collection
    05 July 2021
  • Date of issue
    2021

History

  • Received
    11 Jan 2021
  • Accepted
    4 May 2021
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