Worldwide phylogeography of the blacktip shark (Carcharhinus limbatus) inferred from mitochondrial DNA reveals isolation of western Atlantic populations coupled with recent Pacific dispersal

Although many coastal shark species have widespread distributions, the genetic relatedness of worldwide populations has been examined for few species. The blacktip shark, (Carcharhinus limbatus), inhabits tropical and subtropical coastal waters throughout the world. In this study, we examined the genetic relationships of blacktip shark populations (n = 364 sharks) throughout the majority of the species' range using the entire mitochondrial control region (1067-1070 nucleotides). Two geographically distinct maternal lineages (western Atlantic, Gulf of Mexico, and Caribbean Sea clades, and eastern Atlantic, Indian, and Pacific Ocean clades) were identified and shallow population structure was detected throughout their geographic ranges. These findings indicate that a major population subdivision exists across the Atlantic Ocean, but not the Pacific Ocean. The historical dispersal of this widespread, coastal species may have been interrupted by the rise of the Isthmus of Panama. This scenario implies historical dispersal across the Pacific Ocean (supported by the recovery of the same common haplotype from the Philippines, Hawaii, and the Gulf of California reflecting recent/contemporary dispersal abilities) and an oceanic barrier to recent migration across the Atlantic. Genetic structure within the eastern Atlantic/Indo-Pacific (Phi(ST) = 0.612, P < 0.001) supports maternal philopatry throughout this area, expanding previous western Atlantic findings. Eastern Atlantic/Indo-Pacific C. limbatus control region haplotypes were paraphyletic to Carcharhinus tilstoni haplotypes in our maximum-parsimony analysis. The greater divergence of western Atlantic C. limbatus than C. tilstoni from eastern Atlantic/Indo-Pacific C. limbatus reflects the taxonomic uncertainty of western Atlantic C. limbatus.     

Blacktip shark Carcharhinus limbatus presence at fishing piers in South Carolina: association and environmental drivers

We tagged 12 Carcharhinus limbatus with acoustic transmitters and monitored their presence at five piers along the north-east coast of South Carolina, USA in 2016 and four piers in 2017 using acoustic receivers. Data were analysed with pier association indices (PAI), mixed models and fast Fourier transformation analyses to identify potential factors related to residence time and presence at piers and any cyclical patterns in visits to piers. While the majority of monitored C. limbatus were infrequently detected at piers, three (25.0%) were highly associated with piers (PAI ≥ 0.50). Of the C. limbatus that were detected after initial capture, three (25.0%) recorded detection events only at the pier where they were tagged and two individuals (16.7%) recorded at least one detection event at all monitored piers. The best-fit model explaining C. limbatus residence time at piers included terms for pier location and diel cycle (w_i = 0.88), whereas the best fit model explaining presence–absence of C. limbatus at piers included terms for tidal height, diel cycle, barometric pressure and angler count (w_i = 0.98). Carcharhinus limbatus did not appear to display cyclical patterns in their visits to piers. Along the north-east coast of South Carolina, association of C. limbatus with piers is a phenomenon for a proportion of mature individuals, but continued research is necessary to understand if this behaviour is driven by attraction to and feeding on angler discards or increased foraging opportunities resulting from the attraction of potential prey to the physical structure provided by piers.     

Characterization of microsatellite loci isolated from the blacktip shark and their utility in requiem and hammerhead sharks

We isolated and characterized 16 microsatellite loci from the blacktip shark, Carcharhinus limbatus, and tested cross‐species amplification in 11 Carcharhinus species and five additional shark genera. Thirty‐six (1.6%) and 180 (48%) colonies were positive for dinucleotide repeat motifs from unenriched and enriched libraries, respectively. Heterozygosities of polymorphic loci ranged from 0.04 to 0.96 with two to 22 alleles per locus. Amplification products were observed at nine to 13 loci (five to 11 of which where polymorphic) in 10 Carcharhinus species. Several loci were also polymorphic in each of the additional genera examined.     

Inclusion of South American samples reveals new population structuring of the blacktip shark (Carcharhinus limbatus) in the western Atlantic

Carcharhinus limbatus has a cosmopolitan distribution and marked genetic structuring, mainly because of its philopatric behavior. However, analysis of this structuring has not previously included South American populations. In the present study, we analyzed a sample of adult individuals collected on the northern coast of Brazil and compared the sequences of the mitochondrial control region with those of populations already genotyped. Relatively high haplotype diversity (12 haplotypes, genetic diversity of 0.796) was observed, similar to that in other populations but with a much larger number of private alleles. In contrast to populations studied previously, which were represented by neonates, the pronounced allelic variability found in the South American individuals may have resulted from migrations from other populations in the region that have yet to be genotyped. This population was also genetically distinct from the other Atlantic populations (F_st > 0.8), probably because of female philopatry, and apparently separated from the northwestern Atlantic group 1.39 million years ago. These findings indicate that the C. limbatus population from northern Brazil is genetically distinct from all other populations and should be considered as a different management unit for the protection of stocks.     

Isolation and characterization of five dinucleotide microsatellite loci in the sandbar shark,Carcharhinus plumbeus

Five dinucleotide markers were isolated and optimized from a microsatellite‐enriched genomic library obtained from the sandbar shark, Carcharhinus plumbeus. Genotypic distributions of all markers were found to be in conformance with the expectations of Hardy–Weinberg equilibrium with four to 39 alleles present per locus. We amplified these loci in two female sharks and their litters. A maternal allele was recovered at each locus in all progeny indicating reliable amplification. More than two paternal alleles were recovered across both litters indicating genetic polyandry. Additionally, these markers were amplified across 10 carcharhiniform species to examine their utility in other studies.     

Microsatellite and mitochondrial DNA analyses of Atlantic bluefin tuna (Thunnus thynnus thynnus) population structure in the Mediterranean Sea

Genetic variation was surveyed at nine microsatellite loci and the mitochondrial control region (868 bp) to test for the presence of genetic stock structure in young-of-the-year Atlantic bluefin tuna (Thunnus thynnus thynnus) from the Mediterranean Sea. Bluefin tuna were sampled over a period of 5 years from the Balearic and Tyrrhenian seas in the western basin of the Mediterranean Sea, and from the southern Ionian Sea in the eastern basin of the Mediterranean Sea. Analyses of multilocus microsatellite genotypes and mitochondrial control region sequences revealed no significant heterogeneity among collections taken from the same location in different years; however, significant spatial genetic heterogeneity was observed across all samples for both microsatellite markers and mitochondrial control region sequences (FST=0.0023, P=0.038 and PhiST=0.0233, P=0.000, respectively). Significant genetic differentiation between the Tyrrhenian and Ionian collections was found for both microsatellite and mitochondrial markers (FST=0.0087, P=0.015 and PhiST=0.0367, P=0.030, respectively). These results suggest the possibility of a genetically discrete population in the eastern basin of the Mediterranean Sea.     

Population structure of the dusky pipefish (Syngnathus floridae) from the Atlantic and Gulf of Mexico,as revealed by mitochondrial DNA and microsatellite analyses

Aim To elucidate the historical phylogeography of the dusky pipefish (Syngnathus floridae) in the North American Atlantic and Gulf of Mexico ocean basins. Location Southern Atlantic Ocean and northern Gulf of Mexico within the continental United States. Methods A 394‐bp fragment of the mitochondrial cytochrome b gene and a 235‐bp fragment of the mitochondrial control region were analysed from individuals from 10 locations. Phylogenetic reconstruction, haplotype network, mismatch distributions and analysis of molecular variance were used to infer population structure between ocean basins and time from population expansion within ocean basins. Six microsatellite loci were also analysed to estimate population structure and gene flow among five populations using genetic distance methods (F_ST, Nei’s genetic distance), isolation by distance (Mantel’s test), coalescent‐based estimates of genetic diversity and migration patterns, Bayesian cluster analysis and bottleneck simulations. Results Mitochondrial analyses revealed significant structuring between ocean basins in both cytochrome b (Φ_ST = 0.361, P < 0.0001; Φ_CT = 0.312, P < 0.02) and control region (Φ_ST = 0.166, P < 0.0001; Φ_CT = 0.128, P < 0.03) sequences. However, phylogenetic reconstructions failed to show reciprocal monophyly in populations between ocean basins. Microsatellite analyses revealed significant population substructuring between all locations sampled except for the two locations that were in closest proximity to each other (global F_ST value = 0.026). Bayesian analysis of microsatellite data also revealed significant population structuring between ocean basins. Coalescent‐based analyses of microsatellite data revealed low migration rates among all sites. Mismatch distribution analysis of mitochondrial loci supports a sudden population expansion in both ocean basins in the late Pleistocene, with the expansion of Atlantic populations occurring more recently. Main conclusions Present‐day populations of S. floridae do not bear the mitochondrial DNA signature of the strong phylogenetic discontinuity between the Atlantic and Gulf coasts of North America commonly observed in other species. Rather, our results suggest that Atlantic and Gulf of Mexico populations of S. floridae are closely related but nevertheless exhibit local and regional population structure. We conclude that the present‐day phylogeographic pattern is the result of a recent population expansion into the Atlantic in the late Pleistocene, and that life‐history traits and ecology may play a pivotal role in shaping the realized geographical distribution pattern of this species.     

A novel field method to distinguish between cryptic carcharhinid sharks,Australian blacktip shark Carcharhinus tilstoni and common blacktip shark C. limbatus,despite the presence of hybrids

Multivariate and machine‐learning methods were used to develop field identification techniques for two species of cryptic blacktip shark. From 112 specimens, precaudal vertebrae (PCV) counts and molecular analysis identified 95 Australian blacktip sharks Carcharhinus tilstoni and 17 common blacktip sharks Carcharhinus limbatus. Molecular analysis also revealed 27 of the 112 were C. tilstoni × C. limbatus hybrids, of which 23 had C. tilstoni PCV counts and four had C. limbatus PCV counts. In the absence of further information about hybrid phenotypes, hybrids were assigned as either C. limbatus or C. tilstoni based on PCV counts. Discriminant analysis achieved 80% successful identification, but machine‐learning models were better, achieving 100% successful identification, using six key measurements (fork length, caudal‐fin peduncle height, interdorsal space, second dorsal‐fin height, pelvic‐fin length and pelvic‐fin midpoint to first dorsal‐fin insertion). Furthermore, pelvic‐fin markings could be used for identification: C. limbatus has a distinct black mark >3% of the total pelvic‐fin area, while C. tilstoni has markings with diffuse edges, or has smaller or no markings. Machine learning and pelvic‐fin marking identification methods were field tested achieving 87 and 90% successful identification, respectively. With further refinement, the techniques developed here will form an important part of a multi‐faceted approach to identification of C. tilstoni and C. limbatus and have a clear management and conservation application to these commercially important sharks. The methods developed here are broadly applicable and can be used to resolve species identities in many fisheries where cryptic species exist.     

Population genetic structure of the lemon shark (Negaprion brevirostris) in the western Atlantic: DNA microsatellite variation

DNA microsatellite markers were used to characterize the population genetic structure of the lemon shark, Negaprion brevirostris, in the western Atlantic. This study demonstrates for the first time the usefulness of microsatellites to study population genetic structure and mating systems in the Chondricthyes. Lemon sharks (mostly juveniles) were sampled non-destructively from four locations, Gullivan Bay and Marquesas Key in Florida, Bimini, Bahamas, and Atol das Rocas, Brazil. At least 545 individuals were genotyped at each of four dinucleotide loci. The number of alleles per locus ranged from 19 to 43, and expected heterozygosities ranged from 0.69 to 0.90. Relatively little genetic structure was found in the western Atlantic, with small but significant values for estimators of F(ST) and R(ST) among populations, theta (0.016) and rho (0.026), respectively. No sharp discontinuities were found between the Caribbean sites and Brazil, and most alleles were found at all four sites, indicating that gene flow occurs throughout the western Atlantic with no evidence for distinct stocks.     

Subtle population genetic structure in the Hawaiian grouper, Epinephelus quernus (Serranidae) as revealed by mitochondrial DNA analyses

The endemic Hawaiian grouper, Epinephelus quernus , is a commercially important species experiencing intense fishing pressure in part of its distributional range. We examined population genetic structure with 398 base pairs of the mitochondrial control region across a large portion of the range of E. quernus , spanning approximately 2000 km of the Hawaiian archipelago. Examination of genetic diversity shows that Gardner Island, situated midway along the island chain, harbours the most diverse haplotypes. F -statistics and Bayesian estimates of migration also reveal the mid-archipelago as genetically differentiated, where the first significant break among adjacent pairs of populations lies between the islands of Nihoa and Necker. Most island comparisons beyond Necker and Gardner to the north-west and among the lower five islands to the south-east show little to no genetic differences. Evidence of historical population expansion across the islands was also found by Maximum Likelihood analyses. The results suggest that management should be structured to reflect the genetic differentiation and diversity in the mid-archipelago, the patterns of which may be associated with oceanic current patterns.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 81 , 449–468.     

Selachohemecus benzi n. sp. (Digenea: Sanguinicolidae) from the blacktip shark Carcharhinus limbatus (Carcharhinidae) in the northern Gulf of Mexico

Selachohemecus benzi Bullard & Overstreet n. sp. infects the heart and kidney of the blacktip shark Carcharhinus limbatus in the northern Gulf of Mexico off Florida and Mississippi, USA. Specimens of S.␣olsoni Short, 1954, the only congener and only other named blood fluke reported from a chondrichthyan in the Gulf of Mexico, were collected from the heart of the Atlantic sharpnose shark Rhizoprionodon terraenovae from two new localities, Apalachicola Bay, Florida, and Mississippi Sound, Mississippi, USA. The new species differs from S. olsoni by having a larger body (1.4–3.8 mm long), robust tegumental body spines numbering 51–63 along each lateral body margin, a testis extending from the posterior caeca to the ovary, and a medial ovary with lobes. We amend the diagnosis of Selachohemecus Short, 1954 to accommodate it and provide a diagnostic key for all named chondrichthyan blood flukes.     

Selachohemecus benzi n. sp. (Digenea: Sanguinicolidae) from the blacktip shark Carcharhinus limbatus (Carcharhinidae) in the northern Gulf of Mexico

Selachohemecus benzi Bullard & Overstreet n. sp. infects the heart and kidney of the blacktip shark Carcharhinus limbatus in the northern Gulf of Mexico off Florida and Mississippi, USA. Specimens of S. olsoni Short, 1954, the only congener and only other named blood fluke reported from a chondrichthyan in the Gulf of Mexico, were collected from the heart of the Atlantic sharpnose shark Rhizoprionodon terraenovae from two new localities, Apalachicola Bay, Florida, and Mississippi Sound, Mississippi, USA. The new species differs from S. olsoni by having a larger body (1.4-3.8 mm long), robust tegumental body spines numbering 51-63 along each lateral body margin, a testis extending from the posterior caeca to the ovary, and a medial ovary with lobes. We amend the diagnosis of Selachohemecus Short, 1954 to accommodate it and provide a diagnostic key for all named chondrichthyan blood flukes.     

Spatial and temporal analysis of juvenile blacktip reef shark (Carcharhinus melanopterus) demographies identifies critical habitats

Reef shark species have undergone sharp declines in recent decades, as they inhabit coastal areas, making them an easy target in fisheries (i.e., sharks are exploited globally for their fins, meat, and liver oil) and exposing them to other threats (e.g., being part of by-catch, pollution, and climate change). Reef sharks play a critical role in coral reef ecosystems, where they control populations of smaller predators and herbivorous fishes either directly via predation or indirectly via behavior, thus protecting biodiversity and preventing potential overgrazing of corals. The urgent need to conserve reef shark populations necessitates a multifaceted approach to policy at local, federal, and global levels. However, monitoring programmes to evaluate the efficiency of such policies are lacking due to the difficulty in repeatedly sampling free-ranging, wild shark populations. Over nine consecutive years, we monitored juveniles of the blacktip reef shark (Carcharhinus melanopterus) population around Moorea, French Polynesia, and within the largest shark sanctuary globally, to date. We investigated the roles of spatial (i.e., sampling sites) and temporal variables (i.e., sampling year, season, and month), water temperature, and interspecific competition on shark density across 10 coastal nursery areas. Juvenile C. melanopterus density was found to be stable over 9 years, which may highlight the effectiveness of local and likely federal policies. Two of the 10 nursery areas exhibited higher juvenile shark densities over time, which may have been related to changes in female reproductive behavior or changes in habitat type and resources. Water temperatures did not affect juvenile shark density over time as extreme temperatures proven lethal (i.e., 33°C) in juvenile C. melanopterus might have been tempered by daily variation. The proven efficiency of time-series datasets for reef sharks to identify critical habitats (having the highest juvenile shark densities over time) should be extended to other populations to significantly contribute to the conservation of reef shark species.     

Population genetic structure of North Atlantic, Mediterranean Sea and Sea of Cortez fin whales, Balaenoptera physalus (Linnaeus 1758): analysis of mitochondrial and nuclear loci

Samples were collected from 407 fin whales, Balaenoptera physalus , at four North Atlantic and one Mediterranean Sea summer feeding area as well as the Sea of Cortez in the Pacific Ocean. For each sample, the sex, the sequence of the first 288 nucleotides of the mitochondrial (mt) control region and the genotype at six microsatellite loci were determined. A significant degree of divergence was detected at all nuclear and mt loci between North Atlantic/Mediterranean Sea and the Sea of Cortez. However, the divergence time estimated from the mt sequences was substantially lower than the time elapsed since the rise of the Panama Isthmus, suggesting occasional gene flow between the North Pacific and North Atlantic ocean after the separation of the two oceans. Within the North Atlantic and Mediterranean Sea, significant levels of heterogeneity were observed in the mtDNA between the Mediterranean Sea, the eastern (Spain) and the western (the Gulf of Maine and the Gulf of St Lawrence) North Atlantic. Samples collected off West Greenland and Iceland could not be unequivocally assigned to either of the two areas. The homogeneity tests performed using the nuclear data revealed significant levels of divergence only between the Mediterranean Sea and the Gulf of St Lawrence or West Greenland. In conclusion, our results suggest the existence of several recently diverged populations in the North Atlantic and Mediterranean Sea, possibly with some limited gene flow between adjacent populations, a population structure which is consistent with earlier population models proposed by Kellogg, Ingebrigtsen, and Sergeant.     

Blacktip reef sharks,Carcharhinus melanopterus,have high genetic structure and varying demographic histories in their Indo‐Pacific range

For free‐swimming marine species like sharks, only population genetics and demographic history analyses can be used to assess population health/status as baseline population numbers are usually unknown. We investigated the population genetics of blacktip reef sharks, Carcharhinus melanopterus; one of the most abundant reef‐associated sharks and the apex predator of many shallow water reefs of the Indian and Pacific Oceans. Our sampling includes 4 widely separated locations in the Indo‐Pacific and 11 islands in French Polynesia with different levels of coastal development. Four‐teen microsatellite loci were analysed for samples from all locations and two mitochondrial DNA fragments, the control region and cytochrome b, were examined for 10 locations. For microsatellites, genetic diversity is higher for the locations in the large open systems of the Red Sea and Australia than for the fragmented habitat of the smaller islands of French Polynesia. Strong significant structure was found for distant locations with F_ST values as high as ~0.3, and a smaller but still significant structure is found within French Polynesia. Both mitochondrial genes show only a few mutations across the sequences with a dominant shared haplotype in French Polynesia and New Caledonia suggesting a common lineage different to that of East Australia. Demographic history analyses indicate population expansions in the Red Sea and Australia that may coincide with sea level changes after climatic events. Expansions and flat signals are indicated for French Polynesia as well as a significant recent bottleneck for Moorea, the most human‐impacted lagoon of the locations in French Polynesia.     

Microsatellite Variation Among Red Snapper (Lutjanus campechanus) from the Gulf of Mexico

Allelic variation at a total of 20 nuclear-encoded microsatellites was examined among adult red snapper (Lutjanus campechanus) sampled from 4 offshore localities in the Gulf of Mexico. The number of alleles at the 20 microsatellites ranged from 5 to 20; average (± SE) direct count heterozygosity values ranged from 0.148 ± 0.025 to 0.902 ± 0.008. No significant departures from expectations of Hardy-Weinberg equilibrium were found for any locus within samples, and genotypes at pairs of microsatellites appeared to be randomly associated, i.e., in genotypic equilibrium. Tests of homogeneity in allele distributions among the 4 localities were nonsignificant for 19 of the microsatellites. Allele distribution at microsatellite Lca 43 was heterogeneous among localities before (but not after) Bonferroni corrections for multiple tests executed simultaneously. Tests of homogeneity in the distribution of individual alleles at Lca 43 gave similar results: one low frequency allele was distributed heterogeneously among samples before, but not after, Bonferroni correction. Molecular analysis of variance indicated that more than 99% of variation at each microsatellite was distributed within sample localities. These results generally are consistent with the hypothesis of a single population (stock) of red snapper in the northern Gulf of Mexico. Received September 25, 2000; accepted January 16, 2001     

Direct genetic evidence for reproductive philopatry and associated fine‐scale migrations in female blacktip reef sharks (Carcharhinus melanopterus) in French Polynesia

Conservation of top predators has been emphasized as essential in an ecosystem due to their role in trophic chain regulation. Optimizing conservation strategies for these endangered marine top predators requires direct estimates of breeding patterns and connectivity as these are essential to understanding the population dynamics. There have been some attempts to investigate breeding patterns of reef sharks from litter reconstruction using molecular analyses. However, direct fine‐scale migrations of female sharks for parturition as well as connectivity at a medium scale like between islands remain mostly unknown. We used microsatellite DNA markers and a likelihood‐based parentage analysis to determine breeding patterns of female blacktip reef sharks in Moorea (Society Islands, French Polynesia). Most females gave birth at their home island but some migrated to specific nursery areas outside the area they are attached to, sometimes going to another island 50 km away across deep ocean. Our analysis also revealed that females migrated to the same nursery for every birthing event. Many offspring showed a high level of inbreeding indicating an overall reduced population size, restricted movements and dispersal, or specific mating behaviour. Females represent the vectors that transport the genes at nursery grounds, and their fidelity should thus define reproductive units. As females seem to be philopatric, males could be the ones dispersing genes between populations. These results highlight the need to conserve coastal zones where female reef sharks seem to exhibit philopatry during the breeding season.     

New insights into the trophic ecology of blacktip sharks (Carcharhinus limbatus) from a subtropical estuary in the western Gulf of Mexico

As environmental change persists, understanding resource use patterns is of value to predict the consequences of shifting trophic structures. While many sharks are opportunistic predators, some exhibit prey selectivity, putting them at higher risk compared to species with greater trophic plasticity. In the Gulf of Mexico (GOM), Clupeids and Sciaenids comprise 69% of blacktip shark (Carcharhinus limbatus) diets, which is consequential considering potential responses of these prey groups to disturbance and over harvesting. We assessed if blacktips exhibit selectivity for Clupeids and Sciaenids in the western GOM based on stomach contents from sharks in coastal Texas. Clupeids comprised <2% of diets, while striped mullet (Mugil cephalus) and red drum (Sciaenops ocellatus) comprised >70% of identifiable prey. Ontogenetic shifts from smaller (Clupeids, small Sciaenids) to larger, higher trophic level (Ariidae, Elasmobranchii) prey fits our understanding of foraging among coastal sharks, and suggests our regional understanding of blacktip trophic ecology may be limited by the sizes of sampled sharks. Observed increases in blacktip densities coupled with declines in prey (Mugilids, Sciaenids) is concerning if blacktips have limited diet plasticity. Yet GOM blacktips may be more generalized than previously thought, which is promising for conservation and management.