Figure 1.
Global oceanic distribution of sharpnose sharks.
Species geographical ranges were based on data available at Florida Museum of Natural History (http://www.flmnh.ufl.edu/fish/) and Fishbase (http://www.fishbase.org/) websites. World map in raw version was taken from wikipedia.
Table 1.
Inventory of sharpnose sharks showing the number of individuals investigated by species and their geographic ocean basin origins.
Table 2.
Size of ITS2 locus of sharpnose sharks excluding 5.8S and 28S rRNA gene flanking regions.
Figure 2.
Triplex scheme of ITS2 species-diagnostic primers.
Representation of the shark nuclear 5.8S and 28S ribosomal RNA genes and ITS2 locus showing relative annealing sites and orientation of the shark universal ITS2 primers (Fish 5.8SF and 28SR indicated by gray irregular pentagons). The Brazilian sharpnose (R. lalandei) Rlal293F primer is an example of a species-specific primer used in this study and is shown as a dark gray irregular pentagon. Also represented are the positive control and species-specific amplicons expected to be produced using this combination of three primers when tested against the target species, R.lalandei, DNA (Figure adapted from Shivji et al. 2002).
Figure 3.
Nonaplex scheme of ITS2 species-diagnostic primers.
Representation of the shark nuclear 5.8S and 28S ribosomal RNA genes and ITS2 locus showing relative annealing sites and orientation of primers used in the nonaplex-PCR assay. Shark universal primers (Fish 5.8SF and Fish 28SR) are shown as gray irregular pentagons, while the seven sharpnose species-specific primers are shown by dark gray irregular pentagons. Rlal: Rhizoprionodon lalandei; Rtay: R. taylori; Rolig: R. oligolinx; Rter: R. terranovae; Rlong: R. longurio; Rpor: R. porosus; Racut: R. acutus.
Table 3.
Genetic distances within and between sharpnose sharks calculated as pairwise Tamura-Nei for the nuclear ITS2 locus.
Table 4.
Species-specific primers designed along with their sequences and expected amplicon sizes for each species.
Figure 4.
Triplex primer amplication gel profile.
Results of amplification reactions utilizing the triplex primer combination of two shark ITS2 universal primers and the R. lalandei species-specific primer Rlal293F (Lanes 1 to 7). Lane 1 shows the target species-specific (arrow) and positive control (arrowhead) amplicons. Lanes 2–7 show amplification products from non-target Rhizoprionodon species tested for Rlal293F primer cross-amplification: 2, R. porosus; 3, R. terranovae; 4, R. acutus; 5, R. longurio; 6, R. oligolinx; 7, R. taylori; 8, Negative control (no shark DNA in the PCR). Lanes labeled M contain the molecular size-standard 1 kb plus.
Figure 5.
Nonaplex primer amplification gel profile.
Results of amplification reactions utilizing the nine-primer nonaplex: two shark universal primers and all the seven species-specific primers. Lanes 1–7 show nonaplex-PCR amplification products of target Rhizoprionodon samples: 1- R. acutus; 2- R. porosus; 3- R. longurio; 4- R. terranovae; 5- R. oligolinx; 6- R. taylori; 7- R. lalandei. Lanes 8–17 show nonaplex-PCR amplification products from non-target species: 8- Galeocerdo cuvier; 9- Negaprion acutidens; 10- Carcharhinus porosus; 11- Prionace glauca; 12- Isurus paucus; 13- Alopias superciliosus; 14- Squalus acantias; 15- Nebrius ferrugineus; 16- Squatina californica; 17- Hexanchus griseus. Lines 8–11, Carcharhiniformes; Line 12, Orectolobiformes; Line 13, Squaliformes; Lines 14–15, Lamniformes; Line 16, Squatiformes; Line 17, Hexanchiformes; Line 18 is the negative control. Lanes labeled “M” contain the molecular size-standard 1kb plus. Faint non-specific bands likely correspond to pseudogenes or uncommon variant copies of ribosomal genes rarely amplified by universal and species-specific primers.