Journal of Heredity, 2015, 355–365 doi:10.1093/jhered/esv030 Original Article
Original Article
Population Genetic Structure of the Bonnethead Shark, Sphyrna tiburo, from the Western North Atlantic Ocean Based on mtDNA Sequences Downloaded from http://jhered.oxfordjournals.org/ at New York University on June 15, 2015
Elena Escatel-Luna, Douglas H. Adams, Manuel Uribe-Alcocer, Valentina Islas-Villanueva, and Píndaro Díaz-Jaimes From the Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Apdo. Postal 70–305 Ciudad Universitaria, México D.F. 04510, Mexico (Escatel-Luna); Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 1220 Prospect Avenue, Suite 285, Melbourne, FL 32901 (Adams); and Laboratorio de Genética de Organismos Acuáticos, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Apdo. Postal 70–305, México D.F. 04510, México (Uribe-Alcocer, Islas-Villanueva, and Díaz-Jaimes). Address correspondence to Píndaro Díaz-Jaimes at the address above, or e-mail: [email protected]. Received September 5, 2014; First decision October 30, 2015; Accepted April 23, 2015.
Corresponding editor: Jose Lopez
Abstract The population genetic structure of 251 bonnethead sharks, Sphyrna tiburo, from estuarine and nearshore ocean waters of the Western North Atlantic Ocean (WNA), was assessed using sequences of the mitochondrial DNA-control region. Highly significant genetic differences were observed among bonnetheads from 3 WNA regions; Atlantic coast of Florida, Gulf coast of Florida, and southwestern Gulf of Mexico (analysis of molecular variance, ΦCT = 0.137; P=0.001). Within the Gulf coast of Florida region, small but significant genetic differences were observed between bonnetheads from neighboring estuaries. These overall patterns were consistent with known latitudinal and inshore-offshore movements that occur seasonally for this species within US waters, and with the residency patterns and high site fidelity to feeding/nursery grounds reported in estuaries along the Atlantic coast of Florida and South Carolina. Historical demography also supported the occurrence of past population expansions occurring during Pleistocene glacial-interglacial cycles that caused drastic reductions in bonnethead population size, as a consequence of the eustatic processes that affected the Florida peninsula. This is the first population genetics study for bonnetheads to report genetic divergence among core abundance areas in US and Mexican waters of the WNA. These results, coupled with recent advances in knowledge regarding regional differences in life-history parameters of this species, are critical for defining management units to guide future management strategies for bonnetheads within US waters and across international boundaries into Mexico. Subject areas: Population structure and phylogeography; Conservation genetics and biodiversity Key words: bonnethead shark, nursery areas, philopatry, population structure
Conservation of genetic resources of elasmobranchs is especially important as many shark species have exhibited significant population declines or range contraction as a consequence of increased fishing pressure, habitat loss, or other co-occurring factors (Shepherd
and Myers 2005; Cortes et al. 2007; Baum and Blanchard 2010). Elasmobranchs are typically characterized by slow growth, long life span, late maturity, and low fecundity compared with teleost fishes, which result in low intrinsic rates of increase and low resilience to
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markers is valuable to address these issues (Portnoy and Heist 2012). The present study aims to use mitochondrial DNA-control region (mtDNA-CR) sequences, to evaluate the genetic population structure of bonnetheads from 3 major regions: 1) the Gulf of Mexico coast of Florida (hereafter referred as Gulf coast of Florida); 2) Atlantic coast of Florida; and 3) Mexican waters of the Gulf of Mexico (hereafter the southwestern Gulf of Mexico), and generate information to address important questions regarding the population genetic structure and the philopatric behavior of this coastal shark species.
Materials and Methods Sample Collection and DNA Isolation Bonnetheads were collected from 1992 to 2013 by the Florida Fish and Wildlife Conservation Commission-Fish and Wildlife Research Institute’s Fisheries-Independent Monitoring Program and cooperative research cruises operating in estuarine systems and adjacent coastal waters. Otherwise, samples were obtained from recreational and commercial fisheries in the nearshore and offshore waters of Florida in the Atlantic and Gulf regions, and from commercial landings in the southwestern Gulf of Mexico (Figure 1). Dorsal muscle and fin clips from each specimen were collected and preserved in nondenatured ethanol. Atlantic region study areas ranged from waters near the GeorgiaFlorida border south to the Florida Keys (FK) area. Specific estuarine study areas within the Atlantic region included the St. Marys River system, the Nassau Sound - Nassau River system, the lower St. Johns River system, all of which were combined into a north Atlantic Florida group (NAFL). The Indian River Lagoon and adjacent coastal waters representing the central portion of the Atlantic coast of Florida were combined into the central Atlantic Florida group (CAFL). Within Florida Gulf of Mexico waters, bonnetheads were collected from the northwestern Florida coast near Cedar Key (CK), the southwestern Florida coast including Charlotte Harbor (CH) and Tampa Bay (collectively referred to as CH-TB). Additional samples were collected from adjacent nearshore and offshore waters within the West Florida Shelf (WFS) and FK. The samples collected in the southwestern Gulf of Mexico were directly from commercial fisheries operating within inshore (estuarine) waters from Champoton Campeche Mexico (CAM) and Frontera Tabasco (TAB) during 2012 and 2013, respectively.
PCR Amplification and Sequencing Total genomic DNA was isolated using Wizard Genomic® Promega kit and resuspended in 50–100 μL of TE buffer. A fragment of 940 base pairs (bp) of the mtDNA-CR of bonnetheads was amplified in 251 samples by using the primers ElasmoCR15642F (5′-TTGGCTCCCAAAGCC-3′) and ElasmoCR16638R (5′-CCCTCGTTTTWGGGGTTTTTCGAG-3′; Stoner et al. 2003). Reactions for sequencing were done in a total volume of 50 μL containing 50–100 ng DNA in buffer 10 mM TRIS-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.1 mM of each primer, and 2.5 units of platinum Taq DNA polymerase. PCR amplifications consisted of an initial step of 5 min at 94 °C for denaturation, 35 cycles of 1 min at 95 °C for denaturation, 1 min at 59 °C for annealing, and 1 min at 65 °C for extension, and a final extension step at 65 °C for 3 min. PCR products were sequenced in the forward direction on an ABI 3730xl automated sequencer applying the dye-termination method (Applied Biosystems). Only high quality sequences were used for the analyses and quality control consisted on the review of every polymorphic site in the chromatogram using
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fishing mortality (Hoenig and Gruber 1990; Stevens et al. 2000; Reynolds et al. 2001). Recent conservation and management focus has been placed on delineation of nursery areas for early life stages of sharks (Heithaus 2007) as they may contain evolutionary significant units, and since early stages can be particularly vulnerable to fisheries. Coastal and estuarine systems are used by many shark species as primary and secondary nursery areas, which potentially affects species survival because these waters frequently suffer from significant anthropogenic alteration and habitat loss or degradation. Critical nursery areas have been identified on both the Gulf of Mexico and Atlantic coasts of Florida for multiple shark species (Castro 1993; Heupel et al. 2007; Reyier et al. 2008; Curtis et al. 2011). Bonnetheads, Sphyrna tiburo, are commonly distributed in the western Atlantic Ocean from North Carolina, United States to southern Brazil, including the Gulf of Mexico and the Caribbean, and are seasonally found within estuarine, coastal, and continental shelf waters (Compagno 1984). They are also found in the Eastern Pacific Ocean, ranging from southern California to Ecuador (Compagno 1984). Although the continuous distribution of individuals across the species range in Atlantic US waters may suggest the existence of a single panmictic population, regional differences in life-history parameters of bonnetheads have been observed which suggest there may be multiple populations within the Western North Atlantic (WNA). For example, latitudinal differences in maximum adult size, size at parturition, and size and age at maturity were found among 3 different areas of the Gulf coast of Florida (northwest Florida, [Tampa Bay], Florida Bay) (Parsons 1993; Lombardi-Carlson et al. 2003). Additionally, significant differences in age and growth parameters were detected between bonnetheads from the southeastern US Atlantic coast and the Gulf of Mexico (Frazier et al. 2014). Significant differences have also been noted in the concentration of thyroidal hormone from maternal serum and embryo yolk tissue between Florida Bay and Tampa Bay (TB) estuaries (McComb et al. 2005). These regional/ latitudinal differences may be environmentally controlled or physiologically influenced, but similar to other fish species (Conover and Present 1990), genetic factors may also play a role. There have been also no observations of bonnetheads mixing or moving between US waters of the Gulf of Mexico and the south Atlantic Ocean (non-Gulf waters) (Kohler and Turner 2007; Driggers et al. 2013) and preliminary genetic data for this species suggests the existence of multiple genetically distinct populations (Diaz-Jaimes et al. 2013). In addition, some studies based on acoustic tagging in estuarine waters of the Gulf of Mexico coast of Florida have suggested that bonnetheads are long-term residents within a specific estuary, with low dispersal among different estuaries, and do not appear to make long coastal migrations (Heupel et al. 2006; Bethea and Grace 2013). However, evidence of repeated use and seasonal site fidelity, with associated significant coastal migrations on a seasonal basis, has been provided for South Carolina estuaries in the WNA, where extensive conventional tagging data also revealed significant group cohesion of bonnetheads over yearly scales (Driggers et al. 2014). The proclivity of individuals to remain or return for extended periods to areas where they were born is one of the main criteria for philopatry (Feldheim et al. 2014). These areas are critical for protection of neonates and young juveniles and for subsequent recruitment into the adult population. Bonnetheads in US waters are currently managed as one population and expanded genetic information is critical to effectively assess the number of management units for conservation (Moritz 1994) of bonnetheads within its range in the WNA and also to assess potential genetic differences or similarities among nursery grounds of both Atlantic Ocean and Gulf of Mexico waters. The use of molecular
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the sequence of S. tiburo from the Genbank as reference (accession number GU385313.1) and also the sequence for the most common haplotype. Every chromatogram was checked by 2 independent people and disagreements between reviewers, if any, were verified thoroughly or sequence repeated in order to determine if substitutions were potential sources of sequencing errors.
Data Analyses Multiple alignment was performed with Clustal X ver. 1.8 (Thompson et al. 1997) as well as by optimizing the gap penalties in order to minimize artificial homologies between haplotypes (homoplasy) during the alignment. The hierarchical likelihood ratio method implemented in jModelTest 2.0 (Darriba et al. 2012) was applied to define the most appropriate substitution model of sequence evolution for the segment of the mtDNA-CR analyzed. Additionally, a minimum spanning tree (MST) was constructed using pairwise number of sequence differences with Arlequin. Haplotype (h) and nucleotide (π) diversities were estimated using Arlequin 3.5 (Excoffier and Lischer 2010). Pairwise ΦST between sample locations were obtained to identify genetically differentiated localities. Similarly, the molecular analogue of the unbiased Wright’s F-statistics (Φ-statistics) was computed with the application of the TPM3uf + I + G substitution model (with a gamma of 0.124) in a hierarchical analyses of molecular variance (AMOVA). Sex-biased dispersal may result in differences of mtDNA haplotype frequencies between nurseries after several generations (Hueter et al. 2004). For this reason a measure of genetic differences based on the proportion of allele frequencies rather than that based on genetic distances between alleles is more appropriate (Daly-Engel et al. 2012). The conventional pairwise FST estimates using haplotype frequencies were obtained in order to assess genetic differences due to philopatry between potential nursery grounds. Both approaches, based on distances between haplotypes (ΦST) and haplotype frequencies (FST), were used to assess the AMOVA with samples grouped into Atlantic coast of Florida (NAFL and CAFL), Gulf coast of Florida (CK, CH-TB, and WFS), the transition zone between
Gulf and Atlantic coasts of Florida (FK) and southwestern Gulf of Mexico (CAM and TAB). To confirm the existence of boundaries to gene flow among the main sample groups assessed in the hierarchical AMOVA, the BARRIER v.2.2 program was used. This software implements a Monmonier’s algorithm that finds the edges associated with genetic differences and the geographical localization of samples to identify genetic boundaries (Manni et al. 2004) and delivers graphical representations of discontinuities in gene flow that can be visualized in a geographical context. As no specific criteria to select the optimal number of boundaries is described in Barrier and because the program defines the potential barriers in a sequential fashion according to its importance, 4 boundaries were selected corresponding to the number of groups tested in the AMOVA. The demographic parameters τ, θ0, and θ1 were obtained from nucleotide mismatch distributions with the Arlequin software to examine the impact of demographic fluctuations related to past glaciations on the molecular architecture of bonnetheads. The parameters included Tau (τ) and the θ-estimates θ0 = 2N0μ and θ1 = 2N1μ, where μ is the mutation rate, and N0 and N1 are the female effective population sizes before time 0 and after time 1; expansions were translated into parameter estimations using as range, the calibrated mutation rates of 0.67–1.2% of Keeney and Heist (2006) and Nance et al. (2011) for blacktip shark Carcharhinus limbatus and scalloped hammerhead S. lewini, respectively. Fu’s Fs, as implemented in Arlequin was used to test for departures from neutrality due to recent population expansions or to selection (Fu 1997). Bayesian sampling coalescence-based and Markov chain Monte Carlo (MCMC) methods implemented in IMa2 (Hey and Nielsen 2004) were used to estimate the “isolation with migration” parameters for the most divergent populations (CK, CH-TB, NAFL, CAFL, TAB, and CAM). IMa2 uses a multipopulation approach to obtain simultaneously estimates of population size, θ or 4Nu (where N is the effective population size), as well as a mutational scaled migration to each population with which it coexists in time m = M/u (M is the migration rate per generation per gene copy). In addition to
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Figure 1. Sampling locations where genetic tissue samples of bonnetheads were collected. North Atlantic Florida group (NAFL); central Atlantic Florida group (CAFL); Florida Keys (FK); Cedar Key (CK); Tampa Bay (TB); Charlotte Harbor (CH); West Florida Shelf (WFS); Champoton Campeche, Mexico (CAM); Frontera Tabasco, Mexico (TAB).
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these parameters and on the basis of a predefined population tree topology, it is possible to obtain the splitting time, t = T/u where T is the time in generations. We assess convergence by running IMa in M-mode and by sampling autocorrelation and estimated sample sizes (ESS) values each 6.0 h monitoring the trend line plots and verifying that ESS reached values over 50. Runs were repeated 3 times in order to get an exhaustive sampling of the posterior distribution. All runs consisted of at least 100 000 000 generations and 1 000 000 generations discarded as burn-in. After M-mode runs we ran IMa2 in L-mode to obtain likelihood ratio tests to compare the fit of the models implemented in IMa2 to the full model (i.e., m1 ≠ m2; θ1≠θ2≠θA). In fulfillment of data archiving guidelines, we have deposited the primary data underlying these analyses in Genbank (accessions KM987020 through 987112).
mtDNA-CR Variability and Phylogeny We sequenced a fragment of 940 bp of the mtDNA-CR for a total of 251 bonnetheads that resulted in 98 haplotypes. Sixty-three segregating sites were observed, featuring 44 transitions and 15 transversions. Mean haplotype diversity was high (h = 0.932), yet within the range for the reported values for other shark species, with values ranging from 0.826 for CH-TB samples to 1.0 for bonnetheads from the FK and samples collected from the WFS. The average number of nucleotide differences between haplotypes was 2.0 and the mean nucleotide diversity π was 0.32% which means that differences between haplotypes were based on variation at 3 variable sites. Estimates of nucleotide diversity per sample ranged from 0.16% for CH-TB to 0.43% for TAB (Table 1). The MST resulted in 2 main haplotypes separated by one mutation; the haplotype St-1 was most abundant (20.3%) and contained mostly sequences from the Gulf coast of Florida (CK, CH-TB, and WFS) and Florida Atlantic coast (NAFL, CAFL) plus one from the southwestern Gulf of Mexico (TAB). Although the second most abundant haplotype St-9 (12.9%), contained individuals from all areas, they were mainly from the Gulf coast of Florida (CK, CH-TB, FK, and WFS) and the southwestern Gulf of Mexico (TAB and
Population Genetic Divergence Estimates of pairwise sample ΦST and FST are shown in Table 2. In general, ΦST showed higher values as compared with FST estimates; however, both estimators displayed consistent patterns of genetic differences among locations from the main sampled regions. Highly significant genetic differences of bonnetheads were observed when we compared estuaries from both Florida coasts; Gulf (CK and CH-TB) versus Atlantic (NAFL and CAFL). Significant differences were observed for comparisons between CK and NAFL (ΦST = 0.059; P NAFL = 0.06; mCAM>CAFL = 0.1; mNAFL>CAM = 0.05). Estimates of time for population divergence were different from zero for comparisons among Florida and Mexican areas (which ranged from 0.98 to 1.16) corresponding to a divergence estimated to occur some 87 000–103 000 years ago. In contrast, time for population divergence for comparisons between locations from the Gulf and Atlantic coasts of Florida, was nearly zero (range: 0.17–0.38), which corresponds to a divergence that occurred between 15 000 and 34 000 years ago.
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Likewise, sampling efforts should be directed to collect young-ofthe-year individuals, juveniles, and/or postpartum females from nursery areas.
Historical Demography and Bonnethead Shark Population History In Florida, climate change during Pleistocene glacial-interglacial cycles has played a pivotal role in defining the species’ genetic architecture as consequence of fluctuations in population size originating from habitat loss by sea-level changes at key distribution areas (Avise 2000). This is likely the main cause of the unimodal distribution of mismatches observed in bonnethead populations, which is consistent with a relatively recent population expansion that was estimated to have occurred approximately 100 000–160 000 years ago during the Illinois-Wisconsin interglacial period (136 000–115 000 years; Muhs et al. 2002). During glacial events some 150 000 years ago, sea level dropped as much as 120 m, significantly increasing the land area coverage (Lambeck and Chappell 2001; Muhs et al. 2002). During these sea-level changes, many estuaries might have disappeared, reducing habitable areas for bonnetheads. Food sources for bonnetheads may have been limited or depleted as habitat loss progressed during glacial maxima, reducing their populations drastically. Once the glaciers retracted and sea level increased again, estuaries were reflooded providing optimal conditions for population expansion, including a main prey of bonnetheads, the blue crab, C. sapidus (Cortés et al. 1996), and other prey species. Sudden population expansion has been detected also for the blue crab from similar locations at both the Gulf and Atlantic coasts of Florida which has been associated also to population reductions during Pleistocene glaciations followed by expansion at interglacial periods (McMillen-Jackson and Bert 2004). Moreover, the phylogeographic pattern reported in the blue crab and that for bonnetheads was similar in supporting the existence of gene flow between adjacent estuaries related to seasonal northward migrations reported for both species (Steele, 1991; Bethea and Grace 2013; Kohler et al. 2013; Driggers et al. 2014). It is important to note that this similarity is consistent with the findings of Driggers et al. (2014) with regard to site fidelity of bonnetheads populations to feeding grounds. Phylogeographic patterns associated with population reductions and expansions have been identified for other invertebrate and/or fish species along coastlines of Florida from the Atlantic and Gulf of Mexico by Avise (2000), especially for coastal species which were pushed southward during cooling sea temperatures, toward warmer and more suitable conditions. Analyses from IMa appeared to support this expansion scenario, because all comparisons between samples from the main estuaries coincided with small ancestral population sizes, suggesting that current populations originated after a genetic bottleneck. The reduction of the ancestral populations may have stemmed from the eustatic events that occurred during glacial periods. The estimated time for population divergence between the Gulf and Atlantic coasts of Florida, was relatively recent (between 23 000 and 50 000 years ago) suggesting that populations from both sides of Florida mixed in the past during glacial periods when populations were pushed southward. The past opportunities of contact between populations from the Gulf and Atlantic coast of Florida have occurred also for other coastal marine species (Avise 2000). Contrastingly, the time for population divergence between all Florida locations (especially those from the Gulf coast of Florida) and the southwestern Gulf of Mexico region was earlier (146 000–140 000 years ago) and seems to coincide with the interglacial Illinois-Wisconsin supporting the expansion of populations.
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However, evidence of philopatry for the scalloped hammerhead shark was later supported by Daly-Engel et al. (2012) based on the strong discrepancies in the genetic signal of differentiation between uniparental (mtDNA) and bi-parental (nDNA) markers. Moreover, whereas locations from the same marginal coastline showed highly significant genetic differences with mtDNA, the low or nonexistent genetic differentiation observed between locations from different continental margins, was concluded as evidence of philopatry (DalyEngel et al. 2012). Evidence for philopatry over small spatial scales has also been reported for other shark species within the WNA, such as the lemon shark (Feldheim et al. 2014), blacktip shark (Keeney et al. 2005), and bull shark (Karl et al. 2011). Both, mtDNA and nuclear data were examined in each of these previous studies, and the genetic differences occurred between adjacent or nearby estuarine systems. Considering that potential shark nursery grounds for both Gulf and Atlantic coasts of Florida have been reported (Carlson 2002; McCandless et al. 2007), that include CK and CH, and due to the small scale differences explaining philopatry for other co-occurring shark species, one could expect similar differences for bonnetheads. This was not the case for bonnetheads for 2 possible nursery grounds on the Gulf coast of Florida (CK and CH-TB), which showed weakly significant differences, while no differences were detected between estuaries from the Atlantic coast of Florida. Moreover, it is not possible to fully distinguish between population structure and philopatry with the single use of maternally inherited mtDNA and we are limited on fully addressing the existence of natal philopatry. However, a general conclusion about philopatry can be drawn based on evidence provided by tagging data. Although high residency of bonnetheads to estuaries has been reported (Heupel et al. 2006), seasonal migrations southward from South Carolina waters to coastal Florida waters have been also observed (Driggers et al. 2014). Furthermore, observations of postpartum females and neonates in estuaries from the US Atlantic coast are limited (Ulrich et al. 2007; Driggers et al. 2014), suggesting that mating and parturition of bonnetheads may occur outside of estuaries in this part of their range. With these points in mind, there is currently not enough data to support or refute the existence of natal or reproductive philopatry for bonnetheads. The high residence in estuaries might be related to the use of these estuarine areas as feeding grounds or serve as a combination of both nursery and feeding functions for bonnetheads on the southeastern US Atlantic coast (Driggers et al. 2014). The latitudinal migrations of bonnetheads during late fall, winter, and spring, has been hypothesized as evidence of bonnetheads using South Carolina estuaries as feeding grounds concurrent with high abundance peaks of preferred prey (mature female blue crabs Callinectes sapidus) during spring and summer months, a behavior that may be socially transmitted to young sharks from experienced older adults. In this case, limited or no genetic differences between closely adjacent estuaries would be expected. The highly significant differences among bonnetheads from the 3 major regions we examined and the lack of consistent differences within areas points for now, to the limited dispersal of bonnetheads between spatially separated regions as the most plausible explanation of the differences observed. Estuarine or nearshore nursery areas are critical for protection of neonates and young juveniles and for subsequent recruitment into the adult population. Although there is a lack of clear evidence of philopatry for bonnetheads, it should be explored in more detail in future studies by using a wider set of molecular markers, especially those based on nuclear DNA.
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This is the first population genetic study of bonnetheads to report genetic divergence between core abundance areas from US waters on Florida’s coasts and Mexican waters of the southwestern Gulf of Mexico. These results are critical for defining future management strategies for bonnetheads populations. Bonnetheads in US waters have been managed as one population. Our results, coupled with recent advances in knowledge regarding differences in life-history parameters of this species are important considerations for effective species management within US waters and across international boundaries into Mexico. However, it is also clear that the divergence pattern observed for bonnetheads needs to be further investigated using nuclear markers in order to better define the roles of specific habitat use and dispersal capability, and this research is currently in progress.
Supplementary Material
Funding Programa de Apoyo a Proyectos de Investigación e Inovación Tecnológica PAPIIT at DGAPA-UNAM (IN208112). This project was supported in part by proceeds from State of Florida saltwater recreational fishing licenses, and by funding from the U.S. Department of the Interior, U.S. Fish and Wildlife Service, Federal Aid for Sportfish Restoration (Project Number F14AF00328).
Acknowledgments We thank N. S. Laurrabaquio and G. Martínez for sample processing, and N. Bayona for data analysis. The efforts of the FWC-FWRI FisheriesIndependent Monitoring program in collecting sharks for this study are greatly appreciated. We also appreciate additional sharks provided by Eric Reyier of NASA’s Kennedy Space Center Ecological Program, as well as Cape Canaveral Scientific, Inc. Thanks to the 3 anonymous reviewers for their comments which notably improved the manuscript.
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Supplementary material can be found at http://www.jhered.oxfordjournals.org/.
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Research. Paper 179. Available from: http://digitalcommons.unl.edu/ usgsstaffpub/179 Moritz C. 1994. Defining ‘Evolutionarily Significant Units’ for conservation. Trends Ecol Evol. 9:373–375. Nance HA, Klimley P, Galván-Magaña F, Martínez-Ortíz J, Marko PB. 2011. Demographic processes underlying subtle patterns of population structure in the scalloped hammerhead shark, Sphyrna lewini. PLoS One. 6:e21459. Nielsen R, Wakeley J. 2001. Distinguishing migration from isolation: a Markov chain Monte Carlo approach. Genetics. 158:885–896. Parsons GR. 1993. Geographic variation in reproduction between two populations of the bonnethead shark, Sphyrna tiburo. Environm Biol Fish. 38:25–35. Portnoy DS, McDowell JR, Heist EJ, Musick JA, Graves JE. 2010. World phylogeography and male-mediated gene flow in the sandbar shark, Carcharhinus plumbeus. Mol Ecol. 19:1994–2010. Portnoy DS, Heist EJ. 2012. Molecular markers: progress and prospects for understanding reproductive ecology in elasmobranchs. J Fish Biol. 80:1120–1140. Reyier EA, Adams DH, Lowers RH. 2008. First evidence of a high density nursery ground for the lemon shark, Negaprion brevirostris, near Cape Canaveral, Florida. Florida Scientist. 71:134–148. Reynolds JD, Jennings S, Dulvy NK. 2001. Life histories of fishes and population responses to exploitation. In: Reynolds JD, Mace GM, Redford KH, Robinson JG, editors. Conservation of exploited species, conservation biology 6. Cambridge: Cambridge University Press. p. 47–168. Shepherd TD, Myers RA. 2005. Direct and indirect fishery effects on small coastal elasmobranchs in the northern Gulf of Mexico. Ecol Lett. 8:1095– 1104. Steele P. 1991. Population dynamics and migration of the blue crab, Callinectes sapidus (Rathbun), in the eastern Gulf of Mexico. Proc Gulf Caribb Fish Inst. 40:241–244. Stevens JD, Bonfil R, Dulvy NK, Walker PA. 2000. The effects of fishing on sharks, rays, and chimaeras (Chondrichthyans), and the implications for marine ecosystems. ICES J Mar Sci. 57:476–494. Stoner DS, Grady JM, Priede KA, Quattro JM. 2003. Amplification primers for the mitochondrial control region and sixth intron of the nuclearencoded lactate dehydrogenase A gene in elasmobranch fishes. Cons Gen. 4:805–808. Taguchi M, Kitamura T, Yokawa K. 2011. Genetic population structure of shortfin mako (Isurus oxyrinchus) inferred from mitochondrial DNA on inter-oceanic scale. ISC Shark Working Group Workshop, ISC/11/ SHARKWG-1/02. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876– 4882. Tyminski JP, Hueter RE, Morris J. 2013. Tag-recapture results of bonnethead (Sphyrna tiburo) and Atlantic sharpnose (Rhizoprionodon terraenovae) sharks in the Gulf of Mexico and Florida Coastal Waters. SEDAR34WP-31. North Charleston (SC): SEDAR. p. 12. Ulrich GF, Jones CM, Driggers III WB, Drymon JM, Oakley D, Riley C. 2003. Habitat utilization, relative abundance, and seasonality of sharks in the estuarine and nearshore waters of South Carolina. Am Fish Soc Symp. 50:125–139.
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Hoenig JM, Gruber SH. 1990. Life-history patterns in the elasmobranchs: implications or fisheries management. In: Pratt HL, Gruber SH Jr, Taniuchi T, editors. Elasmobranchs as living resources: advances in the biology, ecology, systematics, and the status of fisheries. U.S. Department of Commerce, NOAA Technical Report NMFS 90 (National Marine Fisheries Service) p. 1–16. Hueter RE, Heupel MR, Heist EJ, Keeney DB. 2002. The implications of philopatry in sharks for the management of shark fisheries. North West Atlantic Fisheries Organization, Scientific Council Meeting-September 2002. NAFO SCR Doc. 02/122. Hueter RE, Heupel MR, Heist EJ, Keeney DB. 2004. Evidence of philopatry in sharks and implications for the management of shark fisheries. J Northwest At Fish Sci. 35:239–247. Karl SA, Castro ALF, Lopez JA, Charvet P, Burgess GH. 2011. Phylogeography and conservation of the bull shark (Carcharhinus leucas) inferred from mitochondrial and microsatellite DNA. Con Gen. 12:371–382. Keeney DB, Heupel MR, Hueter RE, Heist E J. 2005. The genetic structure of blacktip shark (Carcharhinus limbatus) nurseries in the western Atlantic, Gulf of Mexico, and Caribbean Sea inferred from control region sequences and microsatellites. Mol Ecol. 14:1911–1923. Keeney DB, Heist EJ. 2006. Worldwide phylogeography of the blacktip shark (Carcharhinus limbatus) inferred from mitochondrial DNA reveals isolation of western Atlantic populations coupled with recent Pacific dispersal. Mol Ecol. 15:3669–3679. Kohler NE, Turner PA. 2007. Small Coastal Shark 07 SEDAR Data Workshop Document: preliminary mark/recapture data for four species of small coastal sharks in the western North Atlantic. North Charleston (SC): SEDAR 13-DW-23. Kohler NE, Sawicki E, Turner PA, McCandless C. 2013. Mark/recapture data for the bonnethead (Sphyrna tiburo), in the Western North Atlantic from the NEFSC Cooperative Shark Tagging Program. North Charleston (SC): SEDAR. p. 15. Lambeck K, Chappell J. 2001. Sea level change through the last glacial cycle. Science. 292:679–686. Lombardi-Carlson LA, Cortés E, Parsons GR, Manire CA. 2003. Latitudinal variation in life-history traits of bonnethead sharks, Sphyrna tiburo, (Carcharhiniformes: Sphyrnidae) from the eastern Gulf of Mexico. Mar Fresh Res. 54:875–883. Manni F, Guérard E, Heyer E. 2004. Geographic patterns of (genetic, morphologic, linguistic) variation: how barriers can be detected by using Monmonier’s algorithm. Hum Biol. 76:173–190. McCandless CT, Kohler NE, Pratt HL Jr. (eds.) 2007. Shark nursery grounds of the Gulf of Mexico and the east coast waters of the United States. Am Fish Soc Symp. 50. Bethesda (MD). p. 390. McComb DM, Gelsleichter J, Manire CA, Brinn R, Brown CL. 2005. Comparative thyroid hormone concentration in maternal serum and yolk of the bonnethead shark (Sphyrna tiburo) from two sites along the coast of Florida. Gen Comp Endocrinol. 144:167–173. McMillen-Jackson AL, Bert TM. 2004. Mitochondrial DNA variation and population genetic structure of the blue crab Callinectes sapidus in the eastern United States. Mar Biol. 145:769–777. Muhs DR, Simmons KR, Steinke B. 2002. Timing and warmth of the Last Interglacial period: new U-series evidence from Hawaii and Bermuda and a new fossil compilation for North America. USGS Staff-Published
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Original Article
Population Genetic Structure of the Bonnethead Shark, Sphyrna tiburo, from the Western North Atlantic Ocean Based on mtDNA Sequences Downloaded from http://jhered.oxfordjournals.org/ at New York University on June 15, 2015
Elena Escatel-Luna, Douglas H. Adams, Manuel Uribe-Alcocer, Valentina Islas-Villanueva, and Píndaro Díaz-Jaimes From the Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Apdo. Postal 70–305 Ciudad Universitaria, México D.F. 04510, Mexico (Escatel-Luna); Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 1220 Prospect Avenue, Suite 285, Melbourne, FL 32901 (Adams); and Laboratorio de Genética de Organismos Acuáticos, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Apdo. Postal 70–305, México D.F. 04510, México (Uribe-Alcocer, Islas-Villanueva, and Díaz-Jaimes). Address correspondence to Píndaro Díaz-Jaimes at the address above, or e-mail: [email protected]. Received September 5, 2014; First decision October 30, 2015; Accepted April 23, 2015.
Corresponding editor: Jose Lopez
Abstract The population genetic structure of 251 bonnethead sharks, Sphyrna tiburo, from estuarine and nearshore ocean waters of the Western North Atlantic Ocean (WNA), was assessed using sequences of the mitochondrial DNA-control region. Highly significant genetic differences were observed among bonnetheads from 3 WNA regions; Atlantic coast of Florida, Gulf coast of Florida, and southwestern Gulf of Mexico (analysis of molecular variance, ΦCT = 0.137; P=0.001). Within the Gulf coast of Florida region, small but significant genetic differences were observed between bonnetheads from neighboring estuaries. These overall patterns were consistent with known latitudinal and inshore-offshore movements that occur seasonally for this species within US waters, and with the residency patterns and high site fidelity to feeding/nursery grounds reported in estuaries along the Atlantic coast of Florida and South Carolina. Historical demography also supported the occurrence of past population expansions occurring during Pleistocene glacial-interglacial cycles that caused drastic reductions in bonnethead population size, as a consequence of the eustatic processes that affected the Florida peninsula. This is the first population genetics study for bonnetheads to report genetic divergence among core abundance areas in US and Mexican waters of the WNA. These results, coupled with recent advances in knowledge regarding regional differences in life-history parameters of this species, are critical for defining management units to guide future management strategies for bonnetheads within US waters and across international boundaries into Mexico. Subject areas: Population structure and phylogeography; Conservation genetics and biodiversity Key words: bonnethead shark, nursery areas, philopatry, population structure
Conservation of genetic resources of elasmobranchs is especially important as many shark species have exhibited significant population declines or range contraction as a consequence of increased fishing pressure, habitat loss, or other co-occurring factors (Shepherd
and Myers 2005; Cortes et al. 2007; Baum and Blanchard 2010). Elasmobranchs are typically characterized by slow growth, long life span, late maturity, and low fecundity compared with teleost fishes, which result in low intrinsic rates of increase and low resilience to
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markers is valuable to address these issues (Portnoy and Heist 2012). The present study aims to use mitochondrial DNA-control region (mtDNA-CR) sequences, to evaluate the genetic population structure of bonnetheads from 3 major regions: 1) the Gulf of Mexico coast of Florida (hereafter referred as Gulf coast of Florida); 2) Atlantic coast of Florida; and 3) Mexican waters of the Gulf of Mexico (hereafter the southwestern Gulf of Mexico), and generate information to address important questions regarding the population genetic structure and the philopatric behavior of this coastal shark species.
Materials and Methods Sample Collection and DNA Isolation Bonnetheads were collected from 1992 to 2013 by the Florida Fish and Wildlife Conservation Commission-Fish and Wildlife Research Institute’s Fisheries-Independent Monitoring Program and cooperative research cruises operating in estuarine systems and adjacent coastal waters. Otherwise, samples were obtained from recreational and commercial fisheries in the nearshore and offshore waters of Florida in the Atlantic and Gulf regions, and from commercial landings in the southwestern Gulf of Mexico (Figure 1). Dorsal muscle and fin clips from each specimen were collected and preserved in nondenatured ethanol. Atlantic region study areas ranged from waters near the GeorgiaFlorida border south to the Florida Keys (FK) area. Specific estuarine study areas within the Atlantic region included the St. Marys River system, the Nassau Sound - Nassau River system, the lower St. Johns River system, all of which were combined into a north Atlantic Florida group (NAFL). The Indian River Lagoon and adjacent coastal waters representing the central portion of the Atlantic coast of Florida were combined into the central Atlantic Florida group (CAFL). Within Florida Gulf of Mexico waters, bonnetheads were collected from the northwestern Florida coast near Cedar Key (CK), the southwestern Florida coast including Charlotte Harbor (CH) and Tampa Bay (collectively referred to as CH-TB). Additional samples were collected from adjacent nearshore and offshore waters within the West Florida Shelf (WFS) and FK. The samples collected in the southwestern Gulf of Mexico were directly from commercial fisheries operating within inshore (estuarine) waters from Champoton Campeche Mexico (CAM) and Frontera Tabasco (TAB) during 2012 and 2013, respectively.
PCR Amplification and Sequencing Total genomic DNA was isolated using Wizard Genomic® Promega kit and resuspended in 50–100 μL of TE buffer. A fragment of 940 base pairs (bp) of the mtDNA-CR of bonnetheads was amplified in 251 samples by using the primers ElasmoCR15642F (5′-TTGGCTCCCAAAGCC-3′) and ElasmoCR16638R (5′-CCCTCGTTTTWGGGGTTTTTCGAG-3′; Stoner et al. 2003). Reactions for sequencing were done in a total volume of 50 μL containing 50–100 ng DNA in buffer 10 mM TRIS-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.1 mM of each primer, and 2.5 units of platinum Taq DNA polymerase. PCR amplifications consisted of an initial step of 5 min at 94 °C for denaturation, 35 cycles of 1 min at 95 °C for denaturation, 1 min at 59 °C for annealing, and 1 min at 65 °C for extension, and a final extension step at 65 °C for 3 min. PCR products were sequenced in the forward direction on an ABI 3730xl automated sequencer applying the dye-termination method (Applied Biosystems). Only high quality sequences were used for the analyses and quality control consisted on the review of every polymorphic site in the chromatogram using
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fishing mortality (Hoenig and Gruber 1990; Stevens et al. 2000; Reynolds et al. 2001). Recent conservation and management focus has been placed on delineation of nursery areas for early life stages of sharks (Heithaus 2007) as they may contain evolutionary significant units, and since early stages can be particularly vulnerable to fisheries. Coastal and estuarine systems are used by many shark species as primary and secondary nursery areas, which potentially affects species survival because these waters frequently suffer from significant anthropogenic alteration and habitat loss or degradation. Critical nursery areas have been identified on both the Gulf of Mexico and Atlantic coasts of Florida for multiple shark species (Castro 1993; Heupel et al. 2007; Reyier et al. 2008; Curtis et al. 2011). Bonnetheads, Sphyrna tiburo, are commonly distributed in the western Atlantic Ocean from North Carolina, United States to southern Brazil, including the Gulf of Mexico and the Caribbean, and are seasonally found within estuarine, coastal, and continental shelf waters (Compagno 1984). They are also found in the Eastern Pacific Ocean, ranging from southern California to Ecuador (Compagno 1984). Although the continuous distribution of individuals across the species range in Atlantic US waters may suggest the existence of a single panmictic population, regional differences in life-history parameters of bonnetheads have been observed which suggest there may be multiple populations within the Western North Atlantic (WNA). For example, latitudinal differences in maximum adult size, size at parturition, and size and age at maturity were found among 3 different areas of the Gulf coast of Florida (northwest Florida, [Tampa Bay], Florida Bay) (Parsons 1993; Lombardi-Carlson et al. 2003). Additionally, significant differences in age and growth parameters were detected between bonnetheads from the southeastern US Atlantic coast and the Gulf of Mexico (Frazier et al. 2014). Significant differences have also been noted in the concentration of thyroidal hormone from maternal serum and embryo yolk tissue between Florida Bay and Tampa Bay (TB) estuaries (McComb et al. 2005). These regional/ latitudinal differences may be environmentally controlled or physiologically influenced, but similar to other fish species (Conover and Present 1990), genetic factors may also play a role. There have been also no observations of bonnetheads mixing or moving between US waters of the Gulf of Mexico and the south Atlantic Ocean (non-Gulf waters) (Kohler and Turner 2007; Driggers et al. 2013) and preliminary genetic data for this species suggests the existence of multiple genetically distinct populations (Diaz-Jaimes et al. 2013). In addition, some studies based on acoustic tagging in estuarine waters of the Gulf of Mexico coast of Florida have suggested that bonnetheads are long-term residents within a specific estuary, with low dispersal among different estuaries, and do not appear to make long coastal migrations (Heupel et al. 2006; Bethea and Grace 2013). However, evidence of repeated use and seasonal site fidelity, with associated significant coastal migrations on a seasonal basis, has been provided for South Carolina estuaries in the WNA, where extensive conventional tagging data also revealed significant group cohesion of bonnetheads over yearly scales (Driggers et al. 2014). The proclivity of individuals to remain or return for extended periods to areas where they were born is one of the main criteria for philopatry (Feldheim et al. 2014). These areas are critical for protection of neonates and young juveniles and for subsequent recruitment into the adult population. Bonnetheads in US waters are currently managed as one population and expanded genetic information is critical to effectively assess the number of management units for conservation (Moritz 1994) of bonnetheads within its range in the WNA and also to assess potential genetic differences or similarities among nursery grounds of both Atlantic Ocean and Gulf of Mexico waters. The use of molecular
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the sequence of S. tiburo from the Genbank as reference (accession number GU385313.1) and also the sequence for the most common haplotype. Every chromatogram was checked by 2 independent people and disagreements between reviewers, if any, were verified thoroughly or sequence repeated in order to determine if substitutions were potential sources of sequencing errors.
Data Analyses Multiple alignment was performed with Clustal X ver. 1.8 (Thompson et al. 1997) as well as by optimizing the gap penalties in order to minimize artificial homologies between haplotypes (homoplasy) during the alignment. The hierarchical likelihood ratio method implemented in jModelTest 2.0 (Darriba et al. 2012) was applied to define the most appropriate substitution model of sequence evolution for the segment of the mtDNA-CR analyzed. Additionally, a minimum spanning tree (MST) was constructed using pairwise number of sequence differences with Arlequin. Haplotype (h) and nucleotide (π) diversities were estimated using Arlequin 3.5 (Excoffier and Lischer 2010). Pairwise ΦST between sample locations were obtained to identify genetically differentiated localities. Similarly, the molecular analogue of the unbiased Wright’s F-statistics (Φ-statistics) was computed with the application of the TPM3uf + I + G substitution model (with a gamma of 0.124) in a hierarchical analyses of molecular variance (AMOVA). Sex-biased dispersal may result in differences of mtDNA haplotype frequencies between nurseries after several generations (Hueter et al. 2004). For this reason a measure of genetic differences based on the proportion of allele frequencies rather than that based on genetic distances between alleles is more appropriate (Daly-Engel et al. 2012). The conventional pairwise FST estimates using haplotype frequencies were obtained in order to assess genetic differences due to philopatry between potential nursery grounds. Both approaches, based on distances between haplotypes (ΦST) and haplotype frequencies (FST), were used to assess the AMOVA with samples grouped into Atlantic coast of Florida (NAFL and CAFL), Gulf coast of Florida (CK, CH-TB, and WFS), the transition zone between
Gulf and Atlantic coasts of Florida (FK) and southwestern Gulf of Mexico (CAM and TAB). To confirm the existence of boundaries to gene flow among the main sample groups assessed in the hierarchical AMOVA, the BARRIER v.2.2 program was used. This software implements a Monmonier’s algorithm that finds the edges associated with genetic differences and the geographical localization of samples to identify genetic boundaries (Manni et al. 2004) and delivers graphical representations of discontinuities in gene flow that can be visualized in a geographical context. As no specific criteria to select the optimal number of boundaries is described in Barrier and because the program defines the potential barriers in a sequential fashion according to its importance, 4 boundaries were selected corresponding to the number of groups tested in the AMOVA. The demographic parameters τ, θ0, and θ1 were obtained from nucleotide mismatch distributions with the Arlequin software to examine the impact of demographic fluctuations related to past glaciations on the molecular architecture of bonnetheads. The parameters included Tau (τ) and the θ-estimates θ0 = 2N0μ and θ1 = 2N1μ, where μ is the mutation rate, and N0 and N1 are the female effective population sizes before time 0 and after time 1; expansions were translated into parameter estimations using as range, the calibrated mutation rates of 0.67–1.2% of Keeney and Heist (2006) and Nance et al. (2011) for blacktip shark Carcharhinus limbatus and scalloped hammerhead S. lewini, respectively. Fu’s Fs, as implemented in Arlequin was used to test for departures from neutrality due to recent population expansions or to selection (Fu 1997). Bayesian sampling coalescence-based and Markov chain Monte Carlo (MCMC) methods implemented in IMa2 (Hey and Nielsen 2004) were used to estimate the “isolation with migration” parameters for the most divergent populations (CK, CH-TB, NAFL, CAFL, TAB, and CAM). IMa2 uses a multipopulation approach to obtain simultaneously estimates of population size, θ or 4Nu (where N is the effective population size), as well as a mutational scaled migration to each population with which it coexists in time m = M/u (M is the migration rate per generation per gene copy). In addition to
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Figure 1. Sampling locations where genetic tissue samples of bonnetheads were collected. North Atlantic Florida group (NAFL); central Atlantic Florida group (CAFL); Florida Keys (FK); Cedar Key (CK); Tampa Bay (TB); Charlotte Harbor (CH); West Florida Shelf (WFS); Champoton Campeche, Mexico (CAM); Frontera Tabasco, Mexico (TAB).
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these parameters and on the basis of a predefined population tree topology, it is possible to obtain the splitting time, t = T/u where T is the time in generations. We assess convergence by running IMa in M-mode and by sampling autocorrelation and estimated sample sizes (ESS) values each 6.0 h monitoring the trend line plots and verifying that ESS reached values over 50. Runs were repeated 3 times in order to get an exhaustive sampling of the posterior distribution. All runs consisted of at least 100 000 000 generations and 1 000 000 generations discarded as burn-in. After M-mode runs we ran IMa2 in L-mode to obtain likelihood ratio tests to compare the fit of the models implemented in IMa2 to the full model (i.e., m1 ≠ m2; θ1≠θ2≠θA). In fulfillment of data archiving guidelines, we have deposited the primary data underlying these analyses in Genbank (accessions KM987020 through 987112).
mtDNA-CR Variability and Phylogeny We sequenced a fragment of 940 bp of the mtDNA-CR for a total of 251 bonnetheads that resulted in 98 haplotypes. Sixty-three segregating sites were observed, featuring 44 transitions and 15 transversions. Mean haplotype diversity was high (h = 0.932), yet within the range for the reported values for other shark species, with values ranging from 0.826 for CH-TB samples to 1.0 for bonnetheads from the FK and samples collected from the WFS. The average number of nucleotide differences between haplotypes was 2.0 and the mean nucleotide diversity π was 0.32% which means that differences between haplotypes were based on variation at 3 variable sites. Estimates of nucleotide diversity per sample ranged from 0.16% for CH-TB to 0.43% for TAB (Table 1). The MST resulted in 2 main haplotypes separated by one mutation; the haplotype St-1 was most abundant (20.3%) and contained mostly sequences from the Gulf coast of Florida (CK, CH-TB, and WFS) and Florida Atlantic coast (NAFL, CAFL) plus one from the southwestern Gulf of Mexico (TAB). Although the second most abundant haplotype St-9 (12.9%), contained individuals from all areas, they were mainly from the Gulf coast of Florida (CK, CH-TB, FK, and WFS) and the southwestern Gulf of Mexico (TAB and
Population Genetic Divergence Estimates of pairwise sample ΦST and FST are shown in Table 2. In general, ΦST showed higher values as compared with FST estimates; however, both estimators displayed consistent patterns of genetic differences among locations from the main sampled regions. Highly significant genetic differences of bonnetheads were observed when we compared estuaries from both Florida coasts; Gulf (CK and CH-TB) versus Atlantic (NAFL and CAFL). Significant differences were observed for comparisons between CK and NAFL (ΦST = 0.059; P NAFL = 0.06; mCAM>CAFL = 0.1; mNAFL>CAM = 0.05). Estimates of time for population divergence were different from zero for comparisons among Florida and Mexican areas (which ranged from 0.98 to 1.16) corresponding to a divergence estimated to occur some 87 000–103 000 years ago. In contrast, time for population divergence for comparisons between locations from the Gulf and Atlantic coasts of Florida, was nearly zero (range: 0.17–0.38), which corresponds to a divergence that occurred between 15 000 and 34 000 years ago.
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Likewise, sampling efforts should be directed to collect young-ofthe-year individuals, juveniles, and/or postpartum females from nursery areas.
Historical Demography and Bonnethead Shark Population History In Florida, climate change during Pleistocene glacial-interglacial cycles has played a pivotal role in defining the species’ genetic architecture as consequence of fluctuations in population size originating from habitat loss by sea-level changes at key distribution areas (Avise 2000). This is likely the main cause of the unimodal distribution of mismatches observed in bonnethead populations, which is consistent with a relatively recent population expansion that was estimated to have occurred approximately 100 000–160 000 years ago during the Illinois-Wisconsin interglacial period (136 000–115 000 years; Muhs et al. 2002). During glacial events some 150 000 years ago, sea level dropped as much as 120 m, significantly increasing the land area coverage (Lambeck and Chappell 2001; Muhs et al. 2002). During these sea-level changes, many estuaries might have disappeared, reducing habitable areas for bonnetheads. Food sources for bonnetheads may have been limited or depleted as habitat loss progressed during glacial maxima, reducing their populations drastically. Once the glaciers retracted and sea level increased again, estuaries were reflooded providing optimal conditions for population expansion, including a main prey of bonnetheads, the blue crab, C. sapidus (Cortés et al. 1996), and other prey species. Sudden population expansion has been detected also for the blue crab from similar locations at both the Gulf and Atlantic coasts of Florida which has been associated also to population reductions during Pleistocene glaciations followed by expansion at interglacial periods (McMillen-Jackson and Bert 2004). Moreover, the phylogeographic pattern reported in the blue crab and that for bonnetheads was similar in supporting the existence of gene flow between adjacent estuaries related to seasonal northward migrations reported for both species (Steele, 1991; Bethea and Grace 2013; Kohler et al. 2013; Driggers et al. 2014). It is important to note that this similarity is consistent with the findings of Driggers et al. (2014) with regard to site fidelity of bonnetheads populations to feeding grounds. Phylogeographic patterns associated with population reductions and expansions have been identified for other invertebrate and/or fish species along coastlines of Florida from the Atlantic and Gulf of Mexico by Avise (2000), especially for coastal species which were pushed southward during cooling sea temperatures, toward warmer and more suitable conditions. Analyses from IMa appeared to support this expansion scenario, because all comparisons between samples from the main estuaries coincided with small ancestral population sizes, suggesting that current populations originated after a genetic bottleneck. The reduction of the ancestral populations may have stemmed from the eustatic events that occurred during glacial periods. The estimated time for population divergence between the Gulf and Atlantic coasts of Florida, was relatively recent (between 23 000 and 50 000 years ago) suggesting that populations from both sides of Florida mixed in the past during glacial periods when populations were pushed southward. The past opportunities of contact between populations from the Gulf and Atlantic coast of Florida have occurred also for other coastal marine species (Avise 2000). Contrastingly, the time for population divergence between all Florida locations (especially those from the Gulf coast of Florida) and the southwestern Gulf of Mexico region was earlier (146 000–140 000 years ago) and seems to coincide with the interglacial Illinois-Wisconsin supporting the expansion of populations.
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However, evidence of philopatry for the scalloped hammerhead shark was later supported by Daly-Engel et al. (2012) based on the strong discrepancies in the genetic signal of differentiation between uniparental (mtDNA) and bi-parental (nDNA) markers. Moreover, whereas locations from the same marginal coastline showed highly significant genetic differences with mtDNA, the low or nonexistent genetic differentiation observed between locations from different continental margins, was concluded as evidence of philopatry (DalyEngel et al. 2012). Evidence for philopatry over small spatial scales has also been reported for other shark species within the WNA, such as the lemon shark (Feldheim et al. 2014), blacktip shark (Keeney et al. 2005), and bull shark (Karl et al. 2011). Both, mtDNA and nuclear data were examined in each of these previous studies, and the genetic differences occurred between adjacent or nearby estuarine systems. Considering that potential shark nursery grounds for both Gulf and Atlantic coasts of Florida have been reported (Carlson 2002; McCandless et al. 2007), that include CK and CH, and due to the small scale differences explaining philopatry for other co-occurring shark species, one could expect similar differences for bonnetheads. This was not the case for bonnetheads for 2 possible nursery grounds on the Gulf coast of Florida (CK and CH-TB), which showed weakly significant differences, while no differences were detected between estuaries from the Atlantic coast of Florida. Moreover, it is not possible to fully distinguish between population structure and philopatry with the single use of maternally inherited mtDNA and we are limited on fully addressing the existence of natal philopatry. However, a general conclusion about philopatry can be drawn based on evidence provided by tagging data. Although high residency of bonnetheads to estuaries has been reported (Heupel et al. 2006), seasonal migrations southward from South Carolina waters to coastal Florida waters have been also observed (Driggers et al. 2014). Furthermore, observations of postpartum females and neonates in estuaries from the US Atlantic coast are limited (Ulrich et al. 2007; Driggers et al. 2014), suggesting that mating and parturition of bonnetheads may occur outside of estuaries in this part of their range. With these points in mind, there is currently not enough data to support or refute the existence of natal or reproductive philopatry for bonnetheads. The high residence in estuaries might be related to the use of these estuarine areas as feeding grounds or serve as a combination of both nursery and feeding functions for bonnetheads on the southeastern US Atlantic coast (Driggers et al. 2014). The latitudinal migrations of bonnetheads during late fall, winter, and spring, has been hypothesized as evidence of bonnetheads using South Carolina estuaries as feeding grounds concurrent with high abundance peaks of preferred prey (mature female blue crabs Callinectes sapidus) during spring and summer months, a behavior that may be socially transmitted to young sharks from experienced older adults. In this case, limited or no genetic differences between closely adjacent estuaries would be expected. The highly significant differences among bonnetheads from the 3 major regions we examined and the lack of consistent differences within areas points for now, to the limited dispersal of bonnetheads between spatially separated regions as the most plausible explanation of the differences observed. Estuarine or nearshore nursery areas are critical for protection of neonates and young juveniles and for subsequent recruitment into the adult population. Although there is a lack of clear evidence of philopatry for bonnetheads, it should be explored in more detail in future studies by using a wider set of molecular markers, especially those based on nuclear DNA.
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This is the first population genetic study of bonnetheads to report genetic divergence between core abundance areas from US waters on Florida’s coasts and Mexican waters of the southwestern Gulf of Mexico. These results are critical for defining future management strategies for bonnetheads populations. Bonnetheads in US waters have been managed as one population. Our results, coupled with recent advances in knowledge regarding differences in life-history parameters of this species are important considerations for effective species management within US waters and across international boundaries into Mexico. However, it is also clear that the divergence pattern observed for bonnetheads needs to be further investigated using nuclear markers in order to better define the roles of specific habitat use and dispersal capability, and this research is currently in progress.
Supplementary Material
Funding Programa de Apoyo a Proyectos de Investigación e Inovación Tecnológica PAPIIT at DGAPA-UNAM (IN208112). This project was supported in part by proceeds from State of Florida saltwater recreational fishing licenses, and by funding from the U.S. Department of the Interior, U.S. Fish and Wildlife Service, Federal Aid for Sportfish Restoration (Project Number F14AF00328).
Acknowledgments We thank N. S. Laurrabaquio and G. Martínez for sample processing, and N. Bayona for data analysis. The efforts of the FWC-FWRI FisheriesIndependent Monitoring program in collecting sharks for this study are greatly appreciated. We also appreciate additional sharks provided by Eric Reyier of NASA’s Kennedy Space Center Ecological Program, as well as Cape Canaveral Scientific, Inc. Thanks to the 3 anonymous reviewers for their comments which notably improved the manuscript.
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