Species identification and genetic diversity of stingless bees trigona pagdeni in thailand using aflp analysis and mitochondrial DNA sequence

Abstract

Species diagnostic marker for identification of the stingless bees (Trigona pagdeni) was successfully developed. Initially, amplified fragment length polymorphism (AFLP) analysis was carried out across representatives of 12 stingless bees species using 64 primer combinations. A 284 bp band restrictively found in T. pagdeni was cloned and sequenced. A primer pair (CUTPTP1-F/R) was designed and tested for specificity. In total, 100% (129/129 individuals) of T. pagdeni provided a 163 bp expected product. Nevertheless, the amplified CUTPTP was also found in T. fimbriata (1/3, 33.3%), T. collina (11/112, 9.8%), T. laeviceps (1/12, 8.3%) and T. fuscobalteata (15/15, 100%) but not the remaining species. SSCP analysis, single strand conformational polymorphism, of the CUTPTP fragment could successfully differentiate T. fuscobalteata and T. collina from T. pagdeni. Although, no successful discrimination of T. laeviceps and T. fimbriata from T. pagdeni was obtained by using polymorphisms of the CUTPTP fragment, T. pagdeni, T. laeviceps and T. fimbriata can be easily differentiated using their morphology. Genetic variation and population structure of T. pagdeni in Thailand were further investigated using TE-AFLP, and Analysis of Molecular Variance (AMOVA). We found high levels of genetic variation among individuals in each population. We used AMOVA to calculate ΦPT to compare genetic differentiation among populations. This revealed significant genetic differentiation among populations (ΦPT = 0.18, p = 0.001). We also detected differentiation (ΦPT = 0.13, p = 0.001) between samples from north and south of Isthmus of Kra. The greatest differentiation was detected between samples from the northeast and the other locations combined (ΦPT = 0.21, p = 0.001). The SSCP analysis of the cytb, ATPase(6, 8) and 16S rRNA genes, was also used to clarify the genetic diversity and population structure of T. pagdeni. High levels of genetic variation among individuals in each population were detected. AMOVA analysis of the cytb, ATPase(6, 8) and 16S rRNA genes indicated high genetic differentiation among populations (ΦPT = 0.35, ΦPT = 0.27 and ΦPT = 0.28, P = 0.001, respectively), and between samples from north and south of Isthmus of Kra (ΦPT = 0.15, 0.20 and 0.18, P = 0.001 respectively). Likewise, about 69% of the mitochondrial genome of T. pagdeni was determined by long PCR technique. It included the 12 mitochondrial protein-coding genes (ND4L, ND4, ND5, ND6, cytb, ND1, ND3, COIII, ATPase 6, ATPase 8, COII and partial sequence of COI), 12 of 22 tRNA genes, and both rRNA genes. The protein-coding genes had a bias towards AT-rich codons. The protein-encoding genes were initiated with ATT, ATA, ATC and ATG codons and ended with TAA or TAG. Both mtDNA strands contained coding regions. The sequenced mtDNA of T. pagdeni lacked the COI-COII intergenic region, and the mitochondrial gene order in T. pagdeni differed significantly from other stingless bees Melipona bicolor and honey bees Apis mellifera.