Sample collection

The samples of the P. kamoharai were collected by fishery observers placed onboard commercial pelagic longline vessels from Portuguese, Brazilian and French fleets that operate in several regions of the Atlantic and southwest Indian Oceans (Fig. 1). For the French fleet based in Reunion Island, samples were also taken from crocodile sharks donated by vessels’ captains, and during a research cruise of the Institut Français de Recherche pour l'Exploitation de la Mer. The samples were collected opportunistically during the normal fishing operations of the vessels. Whenever a dead crocodile shark was captured in the longline, the fishery observer took a small muscle fragment (<1cm³) which was stored frozen in 96% ethanol. No specific permissions are required for collecting samples of crocodile sharks, a species currently not protected, from those locations/activities.

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Fig 1. Geographic distribution of samples of P. kamoharai, with the network of haplotypes analyzed and compiled from the sequences of the mitochondrial DNA control region.

Intermediate points in the network of haplotypes represent hypothetical haplotypes not sampled. All haplotypes differ by only a single mutation in the network, and the sizes of the circles are proportional to their frequencies.

https://doi.org/10.1371/journal.pone.0117549.g001

In total of 255 specimens were sampled, with 217 collected from different regions of the Atlantic Ocean, namely 48 from the western equatorial Atlantic, 72 from the northeast tropical Atlantic, 68 from the eastern equatorial Atlantic, 14 from the southeast Atlantic and 15 from the southwest Atlantic (Fig. 1). Another 38 samples were collected from the southwest Indian Ocean region. In each case, a small tissue sample (< 1 cm²) was collected from each specimen and preserved in 96% ethanol.

DNA extraction, amplification and sequencing

Genomic DNA was extracted using the NucleoSpin Tissue XS Kit (Macherey & Nagel, Düren, Germany). Partial sequences of the mitochondrial control region were obtained according to the methodology used by [10]. Cycle sequencing was performed with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Individual reactions were performed with approximately 30 ng template PCR product, 3.2 pmol primer, 1 μl terminator mix, and 5 μl Better Buffer (The Gel Co.) in a total volume of 15 μl. PCR sequencing profiles consisted of an initial denaturation step of 4min at 96°C, followed by 30 cycles of 30s at 96°C, 15s at 50°C, and 4 min at 60°C. Sequencing was carried out on an automated sequencer ABI 3130 Applied Biosystems, edited using BioEdit [11] and aligned using ClustalW [12]. The haplotype sequences were deposited in GenBank under accession numbers KF952742 to KF952772.

Population analysis

The relative nucleotide composition, number of polymorphic sites, haplotype diversity (h), nucleotide diversity (π), and number of pairwise nucleotide differences among populations were calculated using ARLEQUIN 3.5.1.3 software [13]. To estimate the levels of genetic divergence among populations of P. kamoharai, the diversity measure Φ_ST was calculated using analysis of molecular variance (AMOVA) [14] under the parameters of the Tamura-Nei [15] nucleotide substitution model. Φ_ST estimates were non-parametrically tested (1000 bootstrapped replicates) using the ARLEQUIN 3.5.1.3 software, and adjusted for simultaneous pairwise comparisons using the sequential Bonferroni procedure [16]. A minimum-spanning haplotype network was estimated using the Network 4.611 program [17]. Evidence of population expansion was tested using Fu’s F test [18] and Tajima’s D [19] in ARLEQUIN 3.5.1.3. The mismatch analysis in ARLEQUIN 3.5.1.3 included a raggedness index to determine goodness-of-fit to a unimodal distribution [20].

Population genetics

In recent years, with the use of molecular tools, there has been an increase in the availability of genetics data for many species, and information such as variability, population structure, gene flow and historical events have became widely considered in the conservation of threatened species and populations, and in the development of more efficient conservation plans.

In the present study the P. kamoharai indexes of nucleotide diversity were similar to reported for other pelagic shark with a single population as Cetorhinus maximus [21] and Rhincodon typus [22, 23]. When compared the indexes to other pelagic and coastal sharks many populations have haplotype and nucleotide diversities similar to or lower than that found in crocodile shark [24, 25, 26, 27, 28]. With the analysis of molecular variance, possible signals of structured populations in the Atlantic were not detected, even when considering various simulations in terms of hypothetical scenarios of differentiation. Thus, it can be assumed that the species P. kamoharai constitutes a single genetic stock with high gene flow throughout its distribution range in the Atlantic Ocean. However, there are some features that should be highlighted. While it is clear that there is a sharing of haplotypes by the various sampled regions, a higher nucleotide and haplotype diversity seems to be associated with the eastern equatorial region, closer to the Gulf of Guinea. Considering that there is no population structure among the overall sampled region, the slightly higher diversity in the eastern equatorial sample may just be due to variation among finite samples drawn from a single population. When the samples from the Indian Ocean were included in the AMOVA analysis similar results were obtained, with an absence of genetic structure differentiation between the Indian and all other groups of the Atlantic. Although the sampled area in the Indian Ocean is only representative of a relatively small area in the southwest Indian Ocean, the pattern of a high gene flow observed in almost all areas of occurrence of the species in the Atlantic, as well as the gene flow between the two Oceans, seems to point to the possibility of a single genetic stock of P. kamoharai distributed between these two Oceans basins, configuring a single population.

The negative indices found for the Tajima’s D test can mean an excess of low frequency polymorphisms in relation to expectations, observed in the high number of haplotypes with single representative (20), indicating an event of expansion of population size, that probably occurred after a bottleneck event or purification selection. However, the occurrence of shared haplotypes among the Atlantic and Indian Oceans may indicate a recent or contemporary dispersal, suggesting that these sharks cross waters off the southern tip of the African Continent, and support the continuous gene flow between these two Oceans.

In the haplotype network a configuration related to the absence of population genetic structure with three haplotypes represented in all sampled groups was observed. Considering the distribution of the sampled groups in four major areas, North Atlantic, Equatorial Atlantic, South Atlantic and Indian Ocean, the presence of four shared haplotypes was observed. Other 6 haplotypes are shared by at least two major areas. Haplotype 1, with the highest relative frequency (60.4%) was represented by individuals on all sampled areas, including the Indian Ocean, being found in greater numbers in the North Atlantic (n = 45). However, the frequency of this haplotype was observed in their majority in the South Atlantic (71.4%). The haplotype 1 had many derived less frequent haplotypes, setting a star configuration that suggests a recent process of population expansion after a bottleneck event.

Conservation

In the last years sharks have gained some relative importance in terms of commercial value, mainly due to the increasing value of their meat and fins. In the particular case of the crocodile shark, and even though this species has no commercial value and is therefore usually discarded, there is still fishing mortality taking place and impacting the populations, as part of the catch is captured and discarded already dead [9]. Therefore, this shark constitutes a particular species where no conservation and/or management initiatives are usually considered or implemented.

Genetic factors influence the risk of extinction mainly because endangered species usually have small and/or declining populations, and in such populations inbreeding and loss of genetic diversity will inevitably put the populations at risk, with evolutionary potential lost even before they may eventually become effectively extinct. These results for P. kamoharai suggest the occurrence of a single stock, without a strong differentiation in haplotype frequencies, forming one basic unit of management in the Atlantic and possibly, in the Indian Ocean. With an absence of population genetic structure, genetic diversity similar or higher than that found in many shark species, although still without consideration of conservation measures, attention with a continuing monitoring through time and an evaluation of the effects caused by fishing pressure are recommended.