Genetic diversity and connectivity of the megamouth shark (Megachasma pelagios)

Amplification of genetic markers

The partial mitochondrial DNA gene cox1 was amplified with the primer pair F1/R1 described by Ward et al. (2005). An additional microsatellite locus (Loc6) that has been successfully cross-amplified in lamniform sharks was also amplified, since it showed a high variation in not only repeat number but also flanking regions (Martin et al., 2002). PCRs were run in 30 μL reactions containing 10–40 ng template DNA, 3 μL 10X buffer, 0.2 mM dNTPs, 1.5 mM MgCl₂, 10 mM of each primer, and 0.2 units of Taq polymerase (MDbio, Taipei, Taiwan). The thermocycling profile consisted of initial denaturation at 94 °C for 2 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 40 s, and a final extension at 72 °C for 2 min. This program was used to amplify the cox1 gene and Loc6. The nucleotide sequences of PCR products of both loci were determined using an ABI 377 automated sequencer (Carlsbad, CA, USA). Nucleotide sequences were assembled and edited using Geneious 9.1.2 (Biomatters, Auckland, New Zealand).

Genetic analyses

Two cox1 gene sequences of individuals from Indonesia (EU3938905) and Puerto Rico (KY392958.1) were downloaded from GenBank. In addition, a Loc6 sequence derived from a Japanese specimen was downloaded (AF423063) (Fig. 1). Arlequin 3.5 (Excoffier & Lischer, 2010) was used to analyze genetic diversity indexes, including haplotype diversity (h) and nucleotide diversity (π). Sequences were aligned and exported to MEGA 7 (Tamura et al., 2013) to visually inspect all alignments. Phylogenetic analyses were used to reveal potential genetic divergences among specimens from different geographic locations, with maximum likelihood (ML) and Bayesian inference assessments being performed on the CIPRES Science Gateway (Miller et al., 2015) and MrBayes (MB) version 3.2.2 (Ronquist et al., 2012), respectively. The latter implemented two parallel runs of four simultaneous Markov chains for 10 million generations, sampling every 1,000 generations and using default parameters. The first million generations (10%) were discarded as burn-in, based on the stationarity of log-likelihood tree scores. ML analyses were conducted in RAxML version 8.1.24 (Stamatakis, 2014) using the HKY substitution model chosen by MEGA 7. Supporting values on the branch were evaluated by non-parametric bootstrapping with 1,000 replicates performed with RAxML (ML) and by posterior probabilities (MB). Moreover, median-joining haplotype networks were generated based on cox1 and Loc6 sequence datasets by using Popart 1.7 (Leigh & Bryant, 2015).

Catch information

Basic catch information showed that megamouth sharks were mainly caught between April and August, with total weights ranging 210–1,147 kg and total lengths ranging 341–710 cm. The sex ratio (female:male) was 16:11, which was not significantly different from 1:1. Five of the 27 individuals were determined to be adults and the others were sub-adults (Table 1).

Genetic information

The cox1 gene (623 bp) and Loc6 microsatellite sequence (592 bp) were amplified and analyzed for 29 individuals obtained from Taiwan and Mexico. Three individuals failed to amplify on both loci, including MP3, MP16, and MP21, due to low DNA quality. There were two parsimony informative sites, and the nucleotide diversity (p) and haplotype diversity (h) of the cox1 gene was 0.000616 ± 0.000695 (mean ± SD) and 0.3305 ± 0.1083, respectively. Twenty-seven cox1 sequences were composed of three unique haplotypes, and the sequences from Taiwan, Mexico, Indonesia, and Puerto Rico shared a dominant haplotype (Fig. 2A haplotype network). The phylogenetic analyses showed that the sequences we used in the present study formed a monophyletic clade and that there were two nodes with substantial support, including one composed of MP2, MP7, and MP26, and the other composed of MP11 and MP18 (Fig. 2A). On the other hand, MP7 and MP24 failed to amplify for Loc6 from a sequence downloaded from GenBank derived from a Japanese specimen; therefore, a total of 25 sequences were obtained for further genetic analyses. Our results showed that the 23 sequences from Taiwan and 2 from the Mexico were identical. The haplotype derived from the Japanese coast specimen had one singleton and formed a unique haplotype separate from the dominant one. No parsimony informative sites were found, and in addition, phylogenetic analyses showed that those sequences were clustered as a single clade in the topology of the cox1 gene tree.

Kuroshio as the passage to feeding grounds

More than 74% (74/99) of sighting records were from countries along the Kuroshio Current, including the Philippines, Taiwan, and Japan. Therefore, this region is likely a hotspot for the occurrence of the megamouth shark. Along the east coast of Taiwan particularly, different sizes of megamouth sharks were caught mainly from April to August off the Hualien coast (Table 1). The stomach contents of a megamouth shark caught off Ibaraki Prefecture (Japan) suggested that it fed almost exclusively on Euphausia pacifica (Sawamoto & Matsumoto, 2012). Euphausia pacifica is the dominant species of euphausiid in the North Pacific (Boden, Johnson & Brinton, 1955; Brinton, 1975) and dominates the zooplankton community in the East Sea (Sea of Japan) (Mauchline, 1980) and Yellow Sea (Yoon et al., 2000). Endo (1981) reported that the eggs and larvae of this species occur throughout the year in Sanriku waters, but are most abundant in April–June. In the Yellow Sea, E. pacifica was the most dominant euphausiid species in both summer and winter (Yoon et al., 2000). Therefore, we propose that the Kuroshio Current may be the lower latitude passage for the megamouth shark to reach its feeding grounds in higher latitudes such as the Yellow Sea and Sanriku waters where E. pacifica is abundant. Seasonal movements between productive high-latitude feeding grounds and low-latitude breeding grounds have been commonly used to explain the migration of baleen whales (e.g., Norris, 1967), and we suggest this may also explain the seasonal migration of the megamouth shark. However, a future satellite tagging study is needed to track the movement and habitat use of the megamouth shark to verify this hypothesis.

Genetic diversity and connectivity in the megamouth shark

Although the megamouth shark appears to be very rarely encountered throughout its range, IUCN assessed its population status as Least Concern based on its wide distribution (Simpfendorfer & Compagno, 2015). This rarity may lead to intrinsic sensitivity to overexploitation since the effects of genetic drift are stronger in smaller populations, which ultimately leads to a substantial loss of genetic variation (Allendorf et al., 2008) and consequently increases the probability of the fixation of deleterious alleles and reduces the resilience of overfished species (Hare et al., 2011). Genetic diversity is also one of the important indexes to be considered in shark management and conservation polices because the long-term survival of a species is strongly dependent on the levels of genetic diversity within and between populations (Domingues, Hilsdorf & Gadig, 2017). In the present study, the increasing number of captures in the Kuroshio region (Table S1), particularly Taiwan, may indicate increasing fishing pressure on megamouth sharks. Comparing its cox1 genetic diversity with other sharks (Alopias pelagicus, Scyliorhinus canicula, Squalus blainville, and R. typus; Table 2), the megamouth shark has the lowest nucleotide diversity (0.000616), and relatively lower haplotype diversity (0.3305). Among these sharks, the pelagic thresher shark (A. pelagicus) is one of the most abundant open ocean sharks and one of the most over-exploited shark species in the Pacific (Tsai, Liu & Joung, 2010; Caballero et al., 2011). Even under great fishing pressure, its nucleotide diversity was higher than that of the megamouth shark. With its rarity, increasing capture in the Kuroshio region and potentially low genetic diversity found in the present study, establishing species-specific regulations or management schemes for the megamouth shark is urgently needed.

On the other hand, information regarding population connectivity is an important consideration when establishing conservation strategies to manage threatened species. In sharks, habitat usage could be one of the major factors influencing the connectivity pattern. For example, pelagic sharks (e.g., the basking shark Cetorhinus maximus, whale shark R. typus, and blue shark Prionace glauca) that undergo long oceanic movements showed less genetic structure either within-ocean or between-ocean scales compared to coastal sharks, except that the whale shark showed a genetic break between the Pacific and Atlantic Oceans (Table 3). In the present study, neither the mitochondrial cox1 gene nor Loc6 sequence revealed any genetic structure. While a cox1 gene sequence from a specimen caught in the Caribbean was included in the analysis, it was identical to the dominant cox1 gene haplotype found in the Pacific. This suggests the megamouth shark might travel across the world’s oceans, which corresponds to its pelagic-oceanic life. Therefore, the careful tracking of fisheries captures and the implementation of a long-term global monitoring program are needed to reassess its population status and ensure that this species does not become threatened in the near future.