Evidence of reproductive isolation among closely related sympatric species of Serrasalmus (Ostariophysii, Characidae) from the Upper Madeira River, Amazon, Bolivia

The delimitation of the Serrasalmus species (Characiformes, Serrasalminae) from the Bolivian Amazon (Amazonas, Madeira) was examined using intron length polymorphism assessed by EPIC-PCR. The six pairs of primers provided 16 polymorphic loci across the species of the region and the allelic diversity ranged between two and 10 alleles per locus. For each locus, the lack of departure from Hardy-Weinberg expectations in a large number of the populations analysed and the homogenous distribution of linkage disequilibrium between paralogous loci and loci belonging to different intronic systems provided strong lines of evidence that the 16 amplified loci constituted independent neutral markers. Furthermore, allelic diversity was size-dependent, thereby indicating that insertion-deletions occurred frequently but randomly in introns, and that intron length polymorphism was a valid marker for investigating the systematics of piranhas. EPIC-PCR demonstrated that eight of the nine nominal species of piranha of the Upper Madeira were reproductively isolated and identified a new species that differed from its closest morphological and genetic relatives by seven diagnostic or semi-diagnostic loci. By contrast, no diagnostic or semi-diagnostic locus was found between S. spilopleura and S. eigenmanni , nor were their allelic frequencies different, thereby questioning the validity of their biological species status, at least in the Upper Madeira. This study, which was one of the first applications of EPIC-PCR to a large-scale molecular systematic purpose, demonstrates that it is a rapid, reliable and cost-effective tool for elucidating issues pertaining to fish systematics.     

Genetic relationships of populations and the origins of new infestations of the Mediterranean fruit fly

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach based on the variation in intron sequences of the glucose-6-phosphate dehydrogenase (Zw) gene was used to assess genetic variability in 26 populations and infestations of the Mediterranean fruit fly (medfly), Ceratitis capitata. Beginning with the exon-primed intron-crossing PCR (EPIC-PCR) method to amplify introns of this gene, five alleles were identified on the basis of DNA sequence variants. Several of these variants affect recognition sites for the restriction enzymes RsaI and TaqI. Using these enzymes in successive digestions of the EPIC-PCR products, each of these alleles can be identified directly from individuals. From this, surveys were conducted to document genotypes and allele frequencies in these samples. The relationships of existing populations and the invasion process represented by new infestations of the medfly were analysed using a principal coordinate analysis and the amova method to quantify the distribution of genetic diversity at different levels in a hierarchical manner. From these results, a framework of genetic relationships among the populations and infestations is presented. In addition, for at least some of the infestations, populations that are probably acting as sources of origin have been identified.     

Development of Exon-Primed Intron-Crossing (EPIC) PCR primers for the malaria vector Anopheles pseudopunctipennis (Diptera: Culicidae)

Using the Anopheles gambiae Giles genome as a template, we designed, screened and identified 14 novel Exon-Primed Intron-Crossing (EPIC) PCR primer pairs for Anopheles pseudopunctipennis Theobald 1901, a major vector of human Plasmodium sp. in South America. These primers were designed to target the conserved regions flanking consecutive exons of different genes and enabled the amplification of 17 loci of which nine were polymorphic. Polymorphisms at these loci ranged from two to four alleles. Intron length polymorphism analysis is a useful tool, which will allow the study of the population structure of this mosquito species, which remains poorly understood.     

Intraspecific Genetic Diversity in the Marine Shrimp Penaeus vannamei: Multiple Polymorphic Elongation Factor-1α Loci Revealed by Intron Sequencing

Intron sequences from the elongation factor-1α (EF1α) gene from the marine shrimp Penaeus vannamei reveal extensive variation even among inbred populations of hatchery-raised shrimp. Among 44 individuals analyzed, we found 13 alleles varying by up to 7.5% sequence differences, and including several allele-diagnostic insertions and deletions. High heterozygosity contrasts with low genetic variation at allozyme loci, but we observed up to four alleles per individual, suggesting that we have identified two separate, polymorphic loci. We partitioned the observed alleles into two groups representing hypothetical duplicated loci. However, the alleles are so similar to one another that a phylogenetic analysis does not cluster them into monophyletic groupings. A possible explanation is that concerted evolution is acting to homogenize genetic variation among these two putative loci. Received July 17, 1998; accepted November 23, 1998.     

Direct Evidence for Mendelian Inheritance of the Variations in the Ribosomal Protein Gene Introns in Yellowfin Tuna (Thunnus albacares)

Restriction fragment length polymorphism found in the S7 ribosomal protein gene introns of yellowfin tuna (Thunnus albacares) was compared between a single pair of parents and their offspring. The sizes of the first intron (RP1) and second intron (RP2) amplified by polymerase chain reaction were 810 bp and 1400 bp, respectively. The dam and sire had different restriction types from one another in HhaI and RsaI digestions for RP1 and in DdeI, HhaI, and ScrFI digestions for RP2. Putative genotypes in both introns of 64 larvae were found to be segregated in Mendelian proportions. Genotype distributions in a wild yellowfin tuna sample (n= 34) were in Hardy-Weinberg proportions, and observed heterozygosity ranged from 0.149 to 0.388. This study presents novel Mendelian markers, which are feasible for tuna population genetic study and pedigree analysis. Received December 8, 1999; accepted April 11, 2000.