Abstract

Mixed stocks are described for Chelonia mydas and the frequency of haplotypes in feeding areas can aid understanding of the genetic and ecological diversity, since with this information it is possible to identify the origin of the individuals. The current study aims to characterize and compare genetic diversity along the coast of Paraná with 17 other feeding areas in the Atlantic Ocean. A total of 285 samples from juveniles were DNA sequenced in the control region, resulting in the identification of 12 haplotypes, with a predominance of the CMA8 haplotype (69%) and the first registration of CMA23. For the study subjects, haplotypic and nucleotide diversity were 0.469 ± 0.032 and 0.00189 ± 0.00020, respectively, and comparisons with other feeding areas presented significant values for the majority of FST and ΦST. The results point to the importance of this region and provide evidence that over the years a mixed stock has used the region as a feeding area. This variation could be related to sea currents, climatic changes, and oceanographic characteristics that may alter the availability of food, water temperature, and the presence of turtles. The current results can be considered in conservation plans for Chelonia mydas.

Keywords:
Threatened species; foraging area; genetic diversity; mitochondrial DNA; mixed stocks

Introduction

The green turtle, Chelonia mydas, is affected by multiple threats, and thousands of juveniles are found stranded dead along the Brazilian coast annually (Monteiro et al., 2016Monteiro DS, Estima SC, Gandra TBR, Silva AP, Bugoni L, Swimmer Y, Seminoff JA and Secchi ER (2016) Long-term spatial and temporal patterns of sea turtle strandings in southern Brazil. Mar Biol 163:247.; Tagliolatto et al., 2019Tagliolatto AB, Goldberg DW, Godfrey MH and Monteiro‐Neto C (2019) Spatio‐temporal distribution of sea turtle strandings and factors contributing to their mortality in south‐eastern Brazil. Aquat Conserv 30:331-350.; Cantor et al., 2020Cantor M, Barreto AS, Taufer RM, Giffoni B, Castilho PV, Maranho A, Beatriz C, Kolesnikovas C, Godoy D, Rogério DW et al. (2020) High incidence of sea turtle stranding in the southwestern Atlantic Ocean. ICES J Mar Sci 77:1864-1878.). However, although the species has circumglobal distribution and has been classified as ‘endangered’ by the IUCN (Seminoff, 2004Seminoff JA (2004) Chelonia mydas. The IUCN Red List of Threatened Species, The IUCN Red List of Threatened Species, https://doi.org/10.2305/IUCN.UK.2004.RLTS.T4615A11037468.en . (accessed 10 May 2020).
https://doi.org/10.2305/IUCN.UK.2004.RLT... ), for the Southwestern Atlantic Ocean, including Brazilian waters, the management unit was recently updated to ‘least concern’ (IUCN, 2019IUCN (2019) Chelonia mydas (South Atlantic subpopulation). The IUCN Red List of Threatened Species 2019 The IUCN Red List of Threatened Species 2019 https://doi.org/10.2305/IUCN.UK.2019-2.RLTS.T142121866A142086337.en . (accessed 29 May 2020)
https://doi.org/10.2305/IUCN.UK.2019-2.R... ). The IUCN assessment is based primarily on criteria focusing on genetic population stocks and adult abundance estimates at rookery sites, and juvenile mortality is not considered for classifying the risk assessment. Nevertheless, identifying the geographic origin of juveniles is crucial for understanding connectivity and for planning conservation strategies (Wildermann et al., 2018Wildermann NE, Gredzens C, Avens L, Barrios-Garrido HA, Bell I, Blumenthal J, Bolten AB, McNeill JB, Casale P, Domenico MD et al. (2018) Informing research priorities for immature sea turtles through expert elicitation. Endanger Species Res 37:55-76.).

The green turtle exhibits migration and habitat use patterns that are associated with different ocean zones, depending on the development phase (stage of the animal’s life cycle) and genetic origin. The species uses pelagic and oceanic areas during, approximately, its first 2 or 3 years of life, and then moves to demersal and neritic areas in its juvenile and adult phases (Musick and Limpus, 1997Musick JA and Limpus CJ (1997) Habitat utilization and migration in juvenile sea turtles. In: Lutz PL and Musick JA (eds) The biology of sea turtle. CRC Press, Florida, pp 137-163.). Individuals from different nesting areas congregate in feeding areas, and mixed genetic stocks are recorded in these specific areas. The stocks are identified because they feature specific mitochondrial DNA (mtDNA) haplotypes for each nesting site (Naro-Maciel et al., 2007Naro-Maciel E, Becker JH, Lima EHSM, Marcovaldi MA and DeSalle R (2007) Testing dispersal hypotheses in foraging green sea turtles (Chelonia mydas) of Brazil. J Hered 98:29-39.; 2008Naro-Maciel E, FitzSimmons MLNN, Amato G (2008) Evolutionary relationships of marine turtles: A molecular phylogeny based on nuclear and mitochondrial genes. Mol Phylogenet Evol. 49(2):659-62.; Shamblin et al., 2012Shamblin BM, Bjorndal KA, Bolten AB, Hillis‐Starr ZM, Lundgren IAN, Naro-Maciel E and Nairn CJ (2012) Mitogenomic sequences better resolve stock structure of southern Greater Caribbean green turtle rookeries. Mol Ecol 21:2330-2340.); a pattern which is a consequence of the philopatric behavior described for males and females (Jensen et al., 2013Jensen MP, Limpus CJ, Whiting SD, Guinea ML, Prince RIT, Dethmers KEM, Adnyana W, Kennett R and FitzSimmons NN (2013) Defining olive ridley turtle Lepidochelys olivacea management units in Australia and assessing the potential impact of mortality in ghost nets. Endanger Species Res 21:241-253.).

Between 2-4 years of age, juvenile green turtles are recruited from oceanic to coastal zones, and different factors (e.g. ocean currents, genetic origin) influence migratory patterns (Naro-Maciel et al., 2014Naro-Maciel E, Reid BN, Alter SE, Amato G, Bjorndal KA, Bolten AB, Martin M, Nairn CJ, Shamblin B and Pineda-Catalan O (2014). From refugia to rookeries: phylogeography of Atlantic green turtles. J Exp Mar Biol Ecol 461:306-316.; Andrade et al., 2016Andrade MF, Domit C, Broadhurst MK, Tolhurst DJ and Silva-Souza ÂT (2016) Appropriate morphometrics for the first assessment of juvenile green turtle (Chelonia mydas) age and growth in the south-western Atlantic. Mar Biol 163:254.; Coelho et al., 2018Coelho VF, Domit C, Broadhurst MK, Prosdocimi L, Nishizawa H and Almeida FS (2018) Intra-specific variation in skull morphology of juvenile Chelonia mydas in the southwestern Atlantic Ocean. Mar Biol 165:174.). Ontogenetic alterations, including morphological and ecological patterns (Gama et al., 2016Gama LR, Domit C, Broadhurst MK, Fuentes MMPB and Millar RB (2016) Green turtle Chelonia mydas foraging ecology at 25º S in the western Atlantic: Evidence to support a feeding model driven by intrinsic and extrinsic variability. Mar Ecol Prog Ser 542:209-219.; Coelho et al., 2018Coelho VF, Domit C, Broadhurst MK, Prosdocimi L, Nishizawa H and Almeida FS (2018) Intra-specific variation in skull morphology of juvenile Chelonia mydas in the southwestern Atlantic Ocean. Mar Biol 165:174.), are also associated with diversity in the genetic stocks (Naro-Maciel et al., 2016Naro-Maciel E, Hart KM, Cruciata R and Putman NF (2016) DNA and dispersal models highlight constrained connectivity in a migratory marine megavertebrate. Ecography 40:586-597.). Studies on the origin of these haplotypes, their presence and frequency in different feeding areas, and the potential new migration routes followed by individuals provide crucial information, both ecological and related to the biogeography of the species. These studies help in establishing and evaluating management and conservation plans aimed at maintaining the genetic and ecological diversity of species (Bowen and Karl, 2007Bowen BW and Karl SA (2007) Population genetics and phylogeography of sea turtles. Mol Ecol 16:4886-4907.).

Juveniles from different nesting sites are recorded using the Southern and Southeastern coasts of Brazil (Proietti et al., 2012Proietti MC, Reisser JW, Kinas PG, Kerr RD, Monteiro DS, Marins LFF and Secchi ER (2012) Green turtle Chelonia mydas mixed stocks in the western South Atlantic, as revealed by mtDNA haplotypes and drifter trajectories. Mar Ecol Prog Ser 447:195-209.; Jordão et al., 2015Jordão JC, Bondioli ACV, Guebert FM, Thoisy BD and Toledo LFA (2015) Green turtle (Chelonia mydas) genetic diversity at Paranaguá estuarine complex feeding grounds in Brazil. Genet Mol Biol 38:346-352.) for feeding and development (Almeida et al., 2011Almeida AP, Santos AJB, Thomé JCA, Belini C, Baptistotte C, Marcovaldi MÂ, dos Santos ASS and Lopez M (2011) Avaliação do estado de conservação da tartaruga marinha Chelonia mydas (Linnaeus, 1758) no Brasil. BioBrasil 1:12-19.; Wallace et al., 2011Wallace BP, Dimatteo AD, Bolten AB, Chaloupka MY, Hutchinson BJ, Abreu-Grobois FA, Mortimer JA, Seminoff JA, Amorocho D, Bjorndal KA et al. (2011) Global conservation priorities for marine turtles. PLoS One 6:e24510.; Andrade et al., 2016Andrade MF, Domit C, Broadhurst MK, Tolhurst DJ and Silva-Souza ÂT (2016) Appropriate morphometrics for the first assessment of juvenile green turtle (Chelonia mydas) age and growth in the south-western Atlantic. Mar Biol 163:254.; Gama et al., 2016Gama LR, Domit C, Broadhurst MK, Fuentes MMPB and Millar RB (2016) Green turtle Chelonia mydas foraging ecology at 25º S in the western Atlantic: Evidence to support a feeding model driven by intrinsic and extrinsic variability. Mar Ecol Prog Ser 542:209-219.; Lenz et al., 2017Lenz AJ, Avens L and Borges-Martins M (2017) Age and growth of juvenile green turtles Chelonia mydas in the western South Atlantic Ocean. Mar Ecol Prog Ser 568:191-201.). Off the Paraná coast, in Southern Brazil, juvenile green turtles occur annually in waters of the continental shelf, estuaries, and coastal islands (Guebert-Bartholo et al., 2011Guebert-Bartholo FM, Barletta M, Costa MF and Monteiro-Filho ELA (2011) Using gut contents to assess foraging patterns of juvenile green turtles Chelonia mydas in the Paranaguá Estuary, Brazil. Endanger Species Res 13:131-143.; Gama et al., 2016Gama LR, Domit C, Broadhurst MK, Fuentes MMPB and Millar RB (2016) Green turtle Chelonia mydas foraging ecology at 25º S in the western Atlantic: Evidence to support a feeding model driven by intrinsic and extrinsic variability. Mar Ecol Prog Ser 542:209-219.). This region is an important feeding area for juveniles, aggregating individuals aged 2-8 years (Andrade et al., 2016Andrade MF, Domit C, Broadhurst MK, Tolhurst DJ and Silva-Souza ÂT (2016) Appropriate morphometrics for the first assessment of juvenile green turtle (Chelonia mydas) age and growth in the south-western Atlantic. Mar Biol 163:254.), migrating from multiple areas (Carman et al., 2012Carman VG, Falabella V, Maxwell S, Albareda D, Campagna C and Mianzan H (2012) Revisiting the ontogenetic shift paradigm: The case of juvenile green turtles in the SW Atlantic. J Exp Mar Biol Ecol 429:64-72.; Fuentes et al., 2020Fuentes MMPB, Wildermann N, Gandra TBR and Domit C (2020) Cumulative threats to juvenile green turtles in the coastal waters of southern and southeastern Brazil. Biodivers Conserv 29:1783-1803.); however, the area presents high mortality rates and is an anthropogenically threatened hotspot (Cantor et al., 2020Cantor M, Barreto AS, Taufer RM, Giffoni B, Castilho PV, Maranho A, Beatriz C, Kolesnikovas C, Godoy D, Rogério DW et al. (2020) High incidence of sea turtle stranding in the southwestern Atlantic Ocean. ICES J Mar Sci 77:1864-1878.; Fuentes et al., 2020Fuentes MMPB, Wildermann N, Gandra TBR and Domit C (2020) Cumulative threats to juvenile green turtles in the coastal waters of southern and southeastern Brazil. Biodivers Conserv 29:1783-1803.).

Anthropogenic activities interfere in the life cycle stages of sea turtles by causing loss of spawning and feeding areas, increasing interaction with fisheries and mortality, and also contributing to habitat degradation, through discarded pollutants and non-biodegradable waste. Therefore, the Brazilian national plan for sea turtle conservation highlights the importance of increasing scientific and management efforts in regions such as Southeastern and Southern Brazil, where the coastal zone is intensely used for activities that are potentially hazardous to sea turtles and their habitats (Sanches, 1999Sanches TM (1999) Avaliação e ações prioritárias para a conservação da biodiversidade da zona costeira e marinha: tartarugas marinhas. Termo de Referência nº155/98. http://www.bdt.org.br/workshop/costa/tartaruga/diagnostico. (accessed 22 June 2020)
http://www.bdt.org.br/workshop/costa/tar... ; Fuentes et al., 2020Fuentes MMPB, Wildermann N, Gandra TBR and Domit C (2020) Cumulative threats to juvenile green turtles in the coastal waters of southern and southeastern Brazil. Biodivers Conserv 29:1783-1803.). Improvement in ecological and genetic knowledge will support conservation plans and measures for establishing active initiatives that reduce the threat of exposure and impacts on the species (Mazaris et al., 2009Mazaris AD, Matsinos G and Pantis JD (2009) Evaluating the impacts of coastal squeeze on sea turtle nesting. Ocean Coast Manage 52:139-145.; López-Barrera et al., 2012López-Barrera EA, Longo GO and Monteiro-Filho ELA (2012) Incidental capture of Green turtle (Chelonia mydas) in gillnets of small-scale fisheries in the Paranaguá Bay, Southern Brazil. Ocean Coast Manage 60:11-18.). Particularly for the immature life stages of sea turtles, studies on population dynamics and survivorship can contribute information on threats and add support to conservation plans (Wilderman et al., 2018Wildermann NE, Gredzens C, Avens L, Barrios-Garrido HA, Bell I, Blumenthal J, Bolten AB, McNeill JB, Casale P, Domenico MD et al. (2018) Informing research priorities for immature sea turtles through expert elicitation. Endanger Species Res 37:55-76.).

Evaluating the genetic diversity in feeding areas in Southern Brazil and the variations in different genetic contributions over time is fundamental to understanding and mapping which genetic stocks are most impacted. This knowledge is crucial to assess genetic variability, which includes the potential resilience of a species to continuous degradation of marine ecosystems. Therefore, the present study aimed primarily to characterize green turtles stranded on the Paraná coast from 2007 to 2014 by studying their mtDNA. The secondary objective of this study was to compare the diversity in this area with that recorded in other feeding areas in the Atlantic Ocean.

Material and Methods

Area of the study

The Paraná coast in Southern Brazil (north 25°13’ S, 48°1’ W; south 25°58’ S, 48°35’ W), comprises a remnant of Atlantic Forest and a UNESCO Natural World Heritage site (Lana et al., 2000Lana PC, Marone E, Lopes RM and Machado EC (2000) The subtropical Estuarine Complex of Paranaguá Bay, Brazil. In: Seeliger U and Kjerfve B (eds) Coastal marine ecosystems of Latin America. Springer, Berlin, pp 131-145.). It is the transition zone between the tropical and temperate areas of the Southwestern Atlantic Ocean and is under the influence of the warm-water Brazil Current and cold-water Malvinas Current (Miranda et al., 2015Miranda TP, Genzano GN and Marques AC (2015) Areas of endemism in the Southwestern Atlantic Ocean based on the distribution of benthic hydroids (Cnidaria: Hydrozoa). Zootaxa 4033:484-506.). This area is composed of estuarine areas, bays, and a shallow continental shelf which are influenced by the El Niño and La Niña weather phenomena and strongly influenced by rainfall (Vanhoni and Mendonça, 2008Vanhoni F and Mendonça F (2008) O clima do litoral do estado do Paraná. Rev Bras Climatol 3:49-63.).

Estuarine areas are crucial because of their ecological role and the Paraná Estuarine Complex is an important foraging and migratory area for juvenile sea turtles. However, the region presents intensive human activity (marine traffic, ports, artisanal fisheries, urban areas, and industrial zones), exposing the species to multiple threats (Fuentes et al., 2020Fuentes MMPB, Wildermann N, Gandra TBR and Domit C (2020) Cumulative threats to juvenile green turtles in the coastal waters of southern and southeastern Brazil. Biodivers Conserv 29:1783-1803.).

Sampling site

The individuals were found either stranded dead, debilitated, or accidentally captured in fishing nets. Samples of liver, epithelial or muscular tissues from juvenile green turtles were collected along the Paraná coast throughout the year from 2007 to 2014 (Figure 1).

Figure 1 ‒
Map of the sample location on the coast of Paraná (PR), Southern Brazil (symbolized by a star). The other feeding areas of the Atlantic Ocean, the North Atlantic group, including North Carolina/USA (NC), Florida/USA (FL), Bahamas (BH), Nicaragua (NI), Barbados (BB), and Cape Verde (CV) indicated by circles; and the South Atlantic group, including Almofala/BR (AL), Fernando de Noronha/BR (FN), Rocas Atoll/BR (RA), Bahia/BR (BA), Espírito Santo/BR (ES), Ubatuba/BR (UB), Arvoredo Island/BR (AI), Cassino Beach/BR (CB), and Buenos Aires Province/AR (PB) represented by lozenges.

The collected tissues were identified according to the animal’s stage of decomposition, adapted from Geraci and Lounsbury (2005Geraci JR and Lounsbury VJ (2005) Marine mammals ashore: A field guide for strandings. National Aquarium in Baltimore, Maryland, 371p.), and stored in 100% ethanol until sample preparation for molecular analysis. The curved carapace length (CCL, cm) was measured in all individuals with complete bodies (Wyneken, 2001Wyneken J (2001) The anatomy of sea turtles. U.S. Department of Commerce NOAA Technical Memorandum NMFS-SEFSC-470, Miami, Florida, 172 p.).

Molecular techniques

Genomic DNA was extracted according to the phenol-chloroform protocol described by Almeida et al. (2001Almeida FS, Fungaro MHP and Sodré LMK (2001) RADP and isoenzyme analysis of genetic variability in three allied species of catfish (Siluriformes: Pimelodidae) from the Tibagi river. J Zool 253:113-120.). The amplification of the control mtDNA region was performed using primers LCM15382 and H950 (Abreu-Grobois et al., 2006Abreu-Grobois A, Horrocks J, Formia A, Dutton P, Leroux R, Vélez-Zuazo X, Soares L and Meylan P (2006) New mtDNA Dloop primers which work for a variety of marine turtle species may increase their solution of mixed stock analyses. In: Proceedings of the 26th Annual Symposium on Sea Turtle Biology, Island of Crete, Greece, p 179.), with a final volume of 15 μL (1× PCR buffer, 0.2 mM of each dNTP, 1.5 mM MgCl₂, 0.5 μM of each primer, 3 U of Taq DNA polymerase [Invitrogen] and 5-50 ng of the DNA sample). The mixture was incubated at 94°C for 5 min; followed by 35 cycles of 40 s at 94°C, 30 s at 51°C, and 1 min at 72°C; and finally, a further 9 min at 72°C. The samples amplified successfully were read in an ABI PRISM 3500xL automatic sequencer (Applied Biosystems).

Data analysis

The quality of the sequences was checked using the Electropherogram Quality Analysis online software (Togawa and Brigido, 2003Togawa RC and Brigido MM (2003) PHPH: Web based tool for simple electropherogram quality analysis. In: 1st International Conference on Bioinformatics and Computational Biology-IcoBiCoBi, Ribeirão Preto, pp 492-497.). The alignment and manual edition of sequences were performed on MEGA6 (Tamura et al., 2013Tamura K, Stecher G, Peterson D, Filipski A and Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725-2729.). The haplotypes in this study were identified according to the Archie Carr Center for Sea Turtle Research database (available online from http://accstr.ufl.edu/genetics.html). Haplotype diversity (h), nucleotide diversity (π), and haplotype counts were calculated using DnaSP 5.1 software (Librado and Rozas, 2009Librado P and Rozas J (2009) DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451-1452.).

For comparisons of genetic diversity with other feeding areas of the Southwestern Atlantic Ocean, previously published results were considered, and the 17 included feeding areas were divided into two groups: i) the North Atlantic group, comprising the areas of North Carolina/USA (NC, Bass et al., 2006Bass AL, Epperly SP and Braun-Mcneill J (2006) Green turtle (Chelonia mydas) foraging and nesting aggregations in the Caribbean and Atlantic: Impact of currents and behavior on dispersal. J Hered 97:346-354.), Florida/USA (FL, Bass and Witzell, 2000Bass AL and Witzell WN (2000) Demographic composition of immature green turtles (Chelonia mydas) from the east central Florida coast: Evidence from mtDNA markers. Herpetologica 56:357-367.), Bahamas (BH, Lahanas et al., 1998Lahanas PN, Bjorndal KA, Bolten AB, Encalada SE, Miyamoto MM, Valverde RA and Bowen BW (1998) Genetic composition of a green turtle (Chelonia mydas) feeding ground population: Evidence for multiple origins. Mar Biol 130:345-352.), Nicaragua (NI, Bass et al., 1998Bass AL, Lagueux CJ and Bowen BW (1998) Origin of green turtles, Chelonia mydas, at “Sleeping Rocks” off the northeast coast of Nicaragua. Copeia 4:1064-1069.), Barbados (BB, Luke et al., 2004Luke K, Horrocks JA, Leroux RA and Dutton PH (2004) Origins of green turtle (Chelonia mydas) feeding aggregations around Barbados, West Indies. Mar Biol 144:799-805.), and Cape Verde (CV, Monzón-Argüello et al., 2010Monzón‐Argüello C, López‐Jurado LF, Rico C, Marco A, López P, Hays GC and Lee PLM (2010) Evidence from genetic and Lagrangian drifter data for transatlantic transport of small juvenile green turtles. J Biogeogr 37:1752-1766.) and ii) the South Atlantic group, comprising the areas of Almofala/BR (AL, Naro-Maciel et al., 2007Naro-Maciel E, Becker JH, Lima EHSM, Marcovaldi MA and DeSalle R (2007) Testing dispersal hypotheses in foraging green sea turtles (Chelonia mydas) of Brazil. J Hered 98:29-39.), Fernando de Noronha/BR (FN, Naro-Maciel et al., 2012Naro-Maciel E, Bondioli ACV, Martin M, De Pádua Almeida A, Baptistotte C, Bellini C, Marcovaldi MA, Santos AJB and Amato G (2012) The interplay of homing and dispersal in green turtles: A focus on the southwestern Atlantic. J Hered 103:792-805.), Rocas Atoll/BR (RA, Naro-Maciel et al., 2012Naro-Maciel E, Bondioli ACV, Martin M, De Pádua Almeida A, Baptistotte C, Bellini C, Marcovaldi MA, Santos AJB and Amato G (2012) The interplay of homing and dispersal in green turtles: A focus on the southwestern Atlantic. J Hered 103:792-805.), Bahia/BR (BA, Naro-Maciel et al., 2012), Espírito Santo/BR (ES, Naro-Maciel et al., 2012), Ubatuba/BR (UB, Naro-Maciel et al., 2007), Paraná/BR (PR, this study), Arvoredo Island/BR (AI, Proietti et al., 2012Proietti MC, Reisser JW, Kinas PG, Kerr RD, Monteiro DS, Marins LFF and Secchi ER (2012) Green turtle Chelonia mydas mixed stocks in the western South Atlantic, as revealed by mtDNA haplotypes and drifter trajectories. Mar Ecol Prog Ser 447:195-209.), Cassino Beach/BR (CB, Proietti et al., 2012Proietti MC, Reisser JW, Kinas PG, Kerr RD, Monteiro DS, Marins LFF and Secchi ER (2012) Green turtle Chelonia mydas mixed stocks in the western South Atlantic, as revealed by mtDNA haplotypes and drifter trajectories. Mar Ecol Prog Ser 447:195-209.), and the Province of Buenos Aires/AR (PB, Prosdoscimi et al., 2012Prosdocimi L, Carman VG, Albareda DA and Remis MI (2012) Genetic composition of green turtle feeding grounds in coastal waters of Argentina based on mitochondrial DNA. J Exp Mar Biol Ecol 412:37-45.).

The level of total genetic divergence among sub-populations, according to haplotype frequency (F_ST) and the Kimura 2P genetic distance parameters (Kimura, 1980Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111-120.) (Φ_ST), was calculated using Arlequin 3.5.2.2 software (Excoffier and Lischer, 2010Excoffier L and Lischer HEL (2010) Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10:564-567.). The same software was used to execute the AMOVA to estimate genetic variance partitioning between the stipulated populations and groups, with significance estimates of 5,000 permutations and p ≤ 0.05. The AMOVA was executed considering only individuals from the Paraná coast, separated by year of sample collection, using the same criteria for the estimates.

Results

In the sampling period, approximately 1500 sea turtles were recorded along 40 km of the Paraná coast and estuarine areas according to researchers at Laboratório de Ecologia e Conservação (CEM-UFPR). Tissue samples for genetic analysis were collected from 436 green turtles and DNA samples were sequenced in 285 individuals (~65%). From the mtDNA samples analyzed, 12 haplotypes were defined according to a 485pb region, and 11 of these haplotypes presented 14 polymorphic sites between them, whereas one haplotype (CMA42) had a 4pb insertion/deletion (indel).

Haplotype CMA8 was the most frequently observed haplotype, identified in 195 individuals (69%), followed by CMA5 in 52 individuals (18%). The other 10 haplotypes occurred with a frequency of less than 5%, with only haplotype CMA8 being identified in all years (Table 1).

The CCLs of the analyzed individuals ranged from 27.5 to 47.6 cm (mean±SD = 39.95 ± 7.32 cm), indicating that all individuals are classified as juveniles (<65 cm CCL) (Bresette et al., 2010Bresette MJ, Witherington BE, Herren RM, Bagley DA, Gorham JC, Traxler SL, Crady CK and Hardy R (2010) Size-class partitioning and herding in a foraging group of green turtles Chelonia mydas. Endanger Species Res 9:105-116.). No variation was observed among the mean CCLs over the years (p = 0.809), and no differences were detected among the mean CCLs of individuals identified as having different haplotypes (p = 0.114).

However, it was observed that the diversity of haplotypes is affected by the number of samples collected in the same year; 2012 (n = 74) had the greatest number of samples, with seven (7) identified haplotypes, whereas 2007 (n = 4) had the smallest number of samples, with only two (2) identified haplotypes (Table 1). On the other hand, even though the year 2008 had few samples, haplotypes CMA23 and CMA32 were sampled only in that year. The AMOVA revealed that the difference between sampling years was 3.44% and within years was 96.56%. Haplotype (h) and nucleotide (π) diversities were 0.469 ± 0.032 and 0.00189 ± 0.00020, respectively (Table 2).

Concerning haplotype occurrence in the 17 Southwestern Atlantic Ocean feeding areas and their classification into two major groups (North and South Atlantic), the AMOVA results indicated that the greatest difference is actually between the groups, whereas the smallest difference is between feeding areas within the groups (Table 3). Comparing all the feeding areas, 81 of 120 F_ST comparisons and 73 of 120 Φ_ST comparisons were statistically significant, considering p ≤ 0.05 (Table 4). Particularly for the Paraná coast feeding area, F_ST comparisons presented non-significant values for BA, ES, UB, AI, CB, and PB, whereas Φ_ST presented non-significant values for FN, RA, BA, ES, UB, AI, CB, and PB (Table 4).

Discussion

A total of 12 haplotypes were identified among the 285 juvenile green turtles sampled along the Paraná coast, Southern Brazil. A predominance of the haplotype CMA8, concerning both number of individuals and continuous presence over the years, reinforces the genetic characteristics recorded in different regions over the Southwestern Atlantic Ocean. The number of haplotypes and the genetic diversity values found for the individuals sampled between 2007 and 2014 highlight the relevance of this region as a feeding and development ground for multiple genetic stocks of juvenile green turtles from the South Atlantic Ocean.

Considering the individuals from the Paraná coast by sampling year, the second most frequent haplotype (CM-A5) was observed for all years except 2007, and the other 10 haplotypes occurred with a relatively low frequency (≤ 5%). Three haplotypes occurred more frequently on the Paraná coast than in the other previously described Atlantic feeding areas; haplotype CMA42 was identified in six individuals, whereas haplotypes CMA39 and CMA46 were identified only in Brazil, once in Arvoredo Island (Santa Catarina state) and once in Ubatuba (São Paulo state), respectively (Naro-Maciel et al., 2007Naro-Maciel E, Becker JH, Lima EHSM, Marcovaldi MA and DeSalle R (2007) Testing dispersal hypotheses in foraging green sea turtles (Chelonia mydas) of Brazil. J Hered 98:29-39.; Proietti et al., 2012Proietti MC, Reisser JW, Kinas PG, Kerr RD, Monteiro DS, Marins LFF and Secchi ER (2012) Green turtle Chelonia mydas mixed stocks in the western South Atlantic, as revealed by mtDNA haplotypes and drifter trajectories. Mar Ecol Prog Ser 447:195-209.). These differences in haplotype occurrence may be associated with biogeographic and migratory behavior, affected by climate and oceanographic conditions. The Paraná coast is humid mesothermal (subtropical) and is influenced by polar air masses in winter and by tropical Atlantic masses in summer (Vanhoni and Mendonça, 2008Vanhoni F and Mendonça F (2008) O clima do litoral do estado do Paraná. Rev Bras Climatol 3:49-63.). These variations, including the food availability and coastal water mass and currents, might influence the presence of animals from different reproductive stocks in feeding areas (Proietti et al., 2009Proietti MC, Lara-Ruiz P, Reisser JW, Pinto LDS, Dellagostin AO and Marins LF (2009) Green turtles (Chelonia mydas) foraging at Arvoredo Island in Southern Brazil: Genetic characterization and mixed stock analysis through mtDNA control region haplotypes. Genet Mol Biol 32:613-618.; Prosdocimi et al., 2012Prosdocimi L, Carman VG, Albareda DA and Remis MI (2012) Genetic composition of green turtle feeding grounds in coastal waters of Argentina based on mitochondrial DNA. J Exp Mar Biol Ecol 412:37-45.).

The haplotypes analyzed in this study are identified mainly from nesting areas in the South Atlantic (coast of Brazil and Africa) (Formia et al., 2006Formia A, Godley BJ, Dontaine J-F and Bruford MW (2006) Mitochondrial DNA diversity and phylogeography of endangered green turtle (Chelonia mydas) populations in Africa. Conser Genet 7:353-369.; Naro-Maciel et al., 2007Naro-Maciel E, Becker JH, Lima EHSM, Marcovaldi MA and DeSalle R (2007) Testing dispersal hypotheses in foraging green sea turtles (Chelonia mydas) of Brazil. J Hered 98:29-39.; Proietti et al., 2009Proietti MC, Lara-Ruiz P, Reisser JW, Pinto LDS, Dellagostin AO and Marins LF (2009) Green turtles (Chelonia mydas) foraging at Arvoredo Island in Southern Brazil: Genetic characterization and mixed stock analysis through mtDNA control region haplotypes. Genet Mol Biol 32:613-618.; Proietti et al., 2012Proietti MC, Reisser JW, Kinas PG, Kerr RD, Monteiro DS, Marins LFF and Secchi ER (2012) Green turtle Chelonia mydas mixed stocks in the western South Atlantic, as revealed by mtDNA haplotypes and drifter trajectories. Mar Ecol Prog Ser 447:195-209.) and only three of them (CM-A3, CM-A5 and CM-A6) are described for nesting areas in the North Atlantic and the Caribbean (Encalada et al., 1996Encalada SE, Lahanas PN, Bjorndal KA, Bolten AB, Miyamoto MM and Bowen BW (1996) Phylogeography and population structure of the Atlantic and Mediterranean green turtle Chelonia mydas: A mitochondrial DNA control region sequence assessment. Mol Ecol 5:473-483.; Naro-Maciel et al., 2007Naro-Maciel E, Becker JH, Lima EHSM, Marcovaldi MA and DeSalle R (2007) Testing dispersal hypotheses in foraging green sea turtles (Chelonia mydas) of Brazil. J Hered 98:29-39.; Proietti et al., 2009Proietti MC, Lara-Ruiz P, Reisser JW, Pinto LDS, Dellagostin AO and Marins LF (2009) Green turtles (Chelonia mydas) foraging at Arvoredo Island in Southern Brazil: Genetic characterization and mixed stock analysis through mtDNA control region haplotypes. Genet Mol Biol 32:613-618.). Future studies addressing biogeographic issues should correlate genetic diversity and oceanographic and climatic factors. This approach would make an important contribution to understanding the potential effects of climate change on mixed stocks using specific foraging grounds.

Overall, 10 haplotypes were identified with low frequency in the present study; this finding is consistent with other studies conducted along the Brazilian coast (Naro-Maciel et al., 2007Naro-Maciel E, Becker JH, Lima EHSM, Marcovaldi MA and DeSalle R (2007) Testing dispersal hypotheses in foraging green sea turtles (Chelonia mydas) of Brazil. J Hered 98:29-39.; Naro-Maciel et al., 2012Naro-Maciel E, Bondioli ACV, Martin M, De Pádua Almeida A, Baptistotte C, Bellini C, Marcovaldi MA, Santos AJB and Amato G (2012) The interplay of homing and dispersal in green turtles: A focus on the southwestern Atlantic. J Hered 103:792-805.; Proietti et al., 2012Proietti MC, Reisser JW, Kinas PG, Kerr RD, Monteiro DS, Marins LFF and Secchi ER (2012) Green turtle Chelonia mydas mixed stocks in the western South Atlantic, as revealed by mtDNA haplotypes and drifter trajectories. Mar Ecol Prog Ser 447:195-209.; Jordão et al., 2015Jordão JC, Bondioli ACV, Guebert FM, Thoisy BD and Toledo LFA (2015) Green turtle (Chelonia mydas) genetic diversity at Paranaguá estuarine complex feeding grounds in Brazil. Genet Mol Biol 38:346-352.). Considering h and π values, this study presents results similar to those presented for other feeding areas across the whole Atlantic Ocean (Bass et al., 1998Bass AL, Lagueux CJ and Bowen BW (1998) Origin of green turtles, Chelonia mydas, at “Sleeping Rocks” off the northeast coast of Nicaragua. Copeia 4:1064-1069.; Lahanas et al., 1998Lahanas PN, Bjorndal KA, Bolten AB, Encalada SE, Miyamoto MM, Valverde RA and Bowen BW (1998) Genetic composition of a green turtle (Chelonia mydas) feeding ground population: Evidence for multiple origins. Mar Biol 130:345-352.; Bass and Witzell, 2000; Luke et al., 2004Luke K, Horrocks JA, Leroux RA and Dutton PH (2004) Origins of green turtle (Chelonia mydas) feeding aggregations around Barbados, West Indies. Mar Biol 144:799-805.; Bass et al., 2006Bass AL, Epperly SP and Braun-Mcneill J (2006) Green turtle (Chelonia mydas) foraging and nesting aggregations in the Caribbean and Atlantic: Impact of currents and behavior on dispersal. J Hered 97:346-354.; Naro-Maciel et al., 2007Naro-Maciel E, Becker JH, Lima EHSM, Marcovaldi MA and DeSalle R (2007) Testing dispersal hypotheses in foraging green sea turtles (Chelonia mydas) of Brazil. J Hered 98:29-39., 2012Naro-Maciel E, Bondioli ACV, Martin M, De Pádua Almeida A, Baptistotte C, Bellini C, Marcovaldi MA, Santos AJB and Amato G (2012) The interplay of homing and dispersal in green turtles: A focus on the southwestern Atlantic. J Hered 103:792-805.; Monzón-Argüello et al., 2010Monzón‐Argüello C, López‐Jurado LF, Rico C, Marco A, López P, Hays GC and Lee PLM (2010) Evidence from genetic and Lagrangian drifter data for transatlantic transport of small juvenile green turtles. J Biogeogr 37:1752-1766.; Proietti et al., 2012Proietti MC, Reisser JW, Kinas PG, Kerr RD, Monteiro DS, Marins LFF and Secchi ER (2012) Green turtle Chelonia mydas mixed stocks in the western South Atlantic, as revealed by mtDNA haplotypes and drifter trajectories. Mar Ecol Prog Ser 447:195-209.; Prosdoscimi et al., 2012Prosdocimi L, Carman VG, Albareda DA and Remis MI (2012) Genetic composition of green turtle feeding grounds in coastal waters of Argentina based on mitochondrial DNA. J Exp Mar Biol Ecol 412:37-45.). Comparing the results of the present study with those of a previous study conducted on the Paraná coast between 2005 and 2008 (Jordão et al., 2015Jordão JC, Bondioli ACV, Guebert FM, Thoisy BD and Toledo LFA (2015) Green turtle (Chelonia mydas) genetic diversity at Paranaguá estuarine complex feeding grounds in Brazil. Genet Mol Biol 38:346-352.), the diversity of haplotypes increased: six haplotypes were identified in both studies, six are new for this region, and only one was not sampled again (CM-A1). These results reinforce the importance of this feeding area for multiple stocks of green turtles, and also that maintaining management and conservation actions is crucial to guarantee the genetic variability along the Southwestern Atlantic Ocean.

The difference between the North and South Atlantic groups (AMOVA between groups of 64.56%) may be associated with the geographical distances between the feeding areas and the influence of sea currents; the relatively warm currents that flow off the Brazilian coast (Equatorial Atlantic and Central South Atlantic) form a direct front against the cold-water Malvinas Current (Tomczak and Godfrey, 2003Tomczak M and Godfrey JS (2003) Regional oceanography: An introduction. Daya Publishing House, Delhi.). These different currents interfere with the presence of individuals in the feeding areas because, reportedly, individuals remain on the coast of Argentina and Uruguay during summer and autumn and migrate to Southern Brazil where waters are warmer in winter (Carman et al., 2012Carman VG, Falabella V, Maxwell S, Albareda D, Campagna C and Mianzan H (2012) Revisiting the ontogenetic shift paradigm: The case of juvenile green turtles in the SW Atlantic. J Exp Mar Biol Ecol 429:64-72.). The values found for genetic differences could also result from colonization events that influence the distribution of animals among nesting areas and the location of the feeding areas (Bass et al., 2006Bass AL, Epperly SP and Braun-Mcneill J (2006) Green turtle (Chelonia mydas) foraging and nesting aggregations in the Caribbean and Atlantic: Impact of currents and behavior on dispersal. J Hered 97:346-354.).

In studies on South Atlantic feeding areas, non-significant values were observed when comparing the data with other areas geographically close to the Paraná coast, which was expected because these regions have similar conditions in terms of the influence of sea currents and other environmental factors. Similar results were observed for feeding areas of the Province of Buenos Aires on the coast of Argentina, which presented significant differences when compared with the five feeding areas in the Northeastern Atlantic, but also with one northern area in the Southwestern Atlantic Ocean (Prosdoscimi et al., 2012Prosdocimi L, Carman VG, Albareda DA and Remis MI (2012) Genetic composition of green turtle feeding grounds in coastal waters of Argentina based on mitochondrial DNA. J Exp Mar Biol Ecol 412:37-45.). Moreover, on the Brazilian coast, two feeding areas in Southern Brazil (Rio Grande do Sul state) exhibited genetic differences when compared with the North Atlantic areas, but no differences from Ubatuba and Rocas Atoll in Southeastern and Northeastern Brazil (Proietti et al., 2012Proietti MC, Reisser JW, Kinas PG, Kerr RD, Monteiro DS, Marins LFF and Secchi ER (2012) Green turtle Chelonia mydas mixed stocks in the western South Atlantic, as revealed by mtDNA haplotypes and drifter trajectories. Mar Ecol Prog Ser 447:195-209.). The composition of feeding areas might depend on the movement of individuals between feeding areas, the abundance of nesting sites, the distances between the nesting and feeding areas, philopatry, and sea currents (Prosdoscimi et al., 2012Prosdocimi L, Carman VG, Albareda DA and Remis MI (2012) Genetic composition of green turtle feeding grounds in coastal waters of Argentina based on mitochondrial DNA. J Exp Mar Biol Ecol 412:37-45.).

According to Wright’s scale (1978Wright S (1978) Evolution and the genetics of populations volume 4: Variability within and among natural populations. University of Chicago Press, Chicago, Illinois, 590 p.), F_ST and Φ_ST values are significant when considering the comparisons between the Paraná coast and North Atlantic areas, and the comparisons with genetic difference values are either high or very high. Comparing the Paraná coast with other South Atlantic areas, F_ST had significant values for AL, FN, and RA, and Φ_ST had significant values for AL, but genetic difference values were either moderate or small. The genetic difference values obtained between the Paraná coast and other regions could be the result of oceanographic parameters, considering that the sea currents may favor individuals following the direction of the south coast of Brazil or move them away from Brazil with other predominant currents (Naro-Maciel et al., 2007Naro-Maciel E, Becker JH, Lima EHSM, Marcovaldi MA and DeSalle R (2007) Testing dispersal hypotheses in foraging green sea turtles (Chelonia mydas) of Brazil. J Hered 98:29-39.). Phylogeographic analysis in nesting grounds of green turtles distinguished two haplotype groups (Northwestern and Southwestern), and the pattern of distribution is non-random among feeding areas and could be influenced by ocean currents (Prosdocimi et al., 2012Prosdocimi L, Carman VG, Albareda DA and Remis MI (2012) Genetic composition of green turtle feeding grounds in coastal waters of Argentina based on mitochondrial DNA. J Exp Mar Biol Ecol 412:37-45.).

Concerning the samples in the present study according to the sampling year, on average, five haplotypes were identified per year, but the occurrence of haplotype CMA23 was novel. This haplotype is exclusively described for South Atlantic nesting areas (Ascension Island and Rocas Atoll/Fernando de Noronha) (Proietti et al., 2012Proietti MC, Reisser JW, Kinas PG, Kerr RD, Monteiro DS, Marins LFF and Secchi ER (2012) Green turtle Chelonia mydas mixed stocks in the western South Atlantic, as revealed by mtDNA haplotypes and drifter trajectories. Mar Ecol Prog Ser 447:195-209.). The occurrence of this haplotype brings new information on this feeding area and for the entire Regional Management Unit (RMU). By updating information about the species in the RMU, it is possible to improve the protection of the nesting populations, since this considers the overlapping of areas of nesting and feeding, regarding reproduction and threats (Wallace et al., 2011Wallace BP, Dimatteo AD, Bolten AB, Chaloupka MY, Hutchinson BJ, Abreu-Grobois FA, Mortimer JA, Seminoff JA, Amorocho D, Bjorndal KA et al. (2011) Global conservation priorities for marine turtles. PLoS One 6:e24510.). The identification of the occurrence of Chelonia mydas haplotypes in the foraging areas enables identification of the presence of mixed stocks, which makes the foraging areas important in terms of conservation, since their preservation ensures that a suitable place is maintained for the occurrence of individuals from different nesting areas. This survey can be considered in the elaboration of plans for monitoring water quality, food availability, vessel traffic, installations of new projects, and other modifications that may change the conditions of the Paraná coast in the future.

Collectively, the results of the included samples from 2007 to 2014 along the Paraná coast, support the use of this specific foraging ground by juvenile Chelonia mydas from multiple origins, and evidence its considerable genetic variability. The study also highlights the presence of different haplotypes in different years and the importance of developing systematic and continuous monitoring of feeding areas to understand connectivity and migratory patterns. This variation over the years may be related to the sea currents, climatic issues, or other oceanographic characteristics; these and other factors should also be investigated more carefully in the future. Furthermore, future studies should include analysis using different molecular markers to improve knowledge on turtle origins and biogeography. In general, the results contribute crucial information to improve management and conservation plans for Chelonia mydas and its habitats.

Acknowledgements

The authors are grateful to the Laboratório de Ecologia e Conservação research team who provided the samples and information and the Universidade Estadual de Londrina and Universidade Federal do Paraná for technical and logistical support. We would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) for granting a scholarship to Camila Satie Savada (Finance Code 001).

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