Inter-specific variation of the mitochondrial r16S gene among silversides, “Peces Blancos”, (Atherinopsidae: Menidiinae) and its utilization for species identification

Analysis of 570 nucleotide sequences of the mitochondrial r16S gene of four species of silversides “peces blancos” (Chirostoma sp.) and of two euryhaline related species Membras martinica and Menidia menidia indicated variation that aided in development of a DNA-based method for species identification. The nucleotide sequence data showed levels of inter-generic genetic variation that ranged from 3.3-7.9% and from 1.06 to 1.6% at the inter-specific level. The variation observed allowed the identification of 18 diagnostic restriction enzymes that in any combination of two or three could aid in the diagnosis of species of “peces blancos”; six of these enzymes could also discriminate between the genera Membras and Menidia; the enzymes AseI, CviAII and FatI discriminate the genus Membras from Chirostoma; and the combination of any of the enzymes BsaAI, CviAII, FatI or HpyCH4IV discriminate Menidia from the “peces blancos”. PCR-RFLP analysis of amplified mitochondrial r16S fragments of 90 un-identified “peces blancos” from Lake Patzcuaro utilizing the restriction enzymes HpyCH41V, TaqI, BsmF1 and BbvI allowed us to diagnose unambiguously cultivated Chirostoma estor and C. humboldtianum in juvenile and adult stages. The use of a non-invasive method to PCR-amplify specific DNA markers from live brood stocks suggests use of this tool for identification of endangered or threatened species.