Mitochondrial polymorphism in tetragonisca angustula and tetragonisca weyrauchi (Apidae) in northern Brazil

Abstract

Bees are important pollinating agents and are an integral part of food production and plant maintenance. It is useful to investigate mitochondrial polymorphisms in bees to obtain information that may be relevant to conservation strategies. We examined mitochondrial haplotypes and evaluated genetic diversity and differentiation in the native stingless bees Tetragonisca angustula and Tetragonisca weyrauchi using PCR-restriction fragment length polymorphism. Worker bees were collected from nests in Rondônia state. After isolating DNA, analyses were performed using 10 pairs of heterologous primers for T. weyrauchi, amplifying various regions of mitochondrial DNA (mtDNA). Primers and restriction enzymes were used for the first time for T. weyrauchi but had already been used in other studies with T. angustula. Only four of the tested primers (primer 1-ND2 and COI; primer 2-COI; primer 8-16S and 12S, and primer 9-COII) were used for further analyses. For restriction analysis of the amplified regions, 13 enzymes were tested. Primer 1-(ND2, COI) allowed the identification of a fragment approximately 2,400 bp in size in T. angustula and T. weyrauchi. Fragment cleavage was accomplished using EcoRI and EcoRV enzymes. An approximately 1,850 bp fragment was amplified in the two species using region (COI) primer 2. Using the EcoRV enzyme, cleavage was confirmed only in T. angustula individuals; however, it was observed in both species using the HinfI enzyme. Amplification of the region, using 16S and 12S-primer 8, generated two fragments (1,850 and 350 bp in size), and cleavage was observed in both species using EcoRV, RsaI, and PstI enzymes; however, the XbaI enzyme cleaved in T. weyrauchi alone. Amplification of DNA, using (COII) primer 9, generated a 1,000 bp fragment. The cleavage was performed using ClaI and HinfI enzymes in T. angustula. Bayesian inference analysis showed that mtDNA of T. angustula has a greater genetic variability than that of T. weyrauchi. The variations observed by the analysis of mtDNA using PCR-RFLP showed differences in mtDNA in the two species.