Species identification and genetic diversity of stingless bee tetragonilla collina in Thailand using AFLP and PCR-SSCP analyses

Abstract

A molecular maker for authenticating species origin of the stingless bee (Tetragonilla collina) was developed. Initially, amplified fragment length polymorphism analysis was made of 11 stingless bee species using 64 primer combinations. A 316-bp band found only in T. collina was cloned and sequenced. A primer pair (CUTc1-F/R) was designed and tested for specificity in 15 stingless bee species (239 nests). The expected 259-bp fragment was consistently amplified in all T.collina individuals (134/134 nests, 100%). Cross-species amplification was observed in T.pagdeni (43/51 nests; 84.3%), but not in other species. SSCP analysis of CUTc1 unambiguously differentiated T. collina from T.pagdeni. CUTc1 generated three genotypes in Thai T.collina (134 nests). An AA (259/259 bp) genotype was found in all stingless bees from the north (21 nests) and northeast (32 nests), and 23/28 nests from the Central region, whereas a BB (253/253 bp) genotype was observed in most samples from peninsular Thailand (42/53 nests). Heterozygotes exhibiting the AB (259/253 bp) genotype were observed in 5 of 28 nests from Prachuap Khiri Khan located slightly above the Kra ecotone and 11 of 53 nests originated further south of the Kra ecotone. Genotype distribution patterns of CUTc1 clearly indicated intraspecific population differentiation of Thai T.collina. The regional genetic variation of Thai T.collina was investigated using DNA fingerprinting technique, three-enzyme amplified fragment length polymorphisms (TE-AFLPs) and Analysis of Molecular Variance (AMOVA). High levels of genetic variation among individuals in each population were detected. AMOVA analysis indicated significant genetic differentiation among the four geographic regions; North, Central, Northeast, and Peninsular Thailand (Φ PT = 0.258, P = 0.001). The smaller but significant differentiation between samples from North and South of Isthmus of Kra was also detected (Φ PT = 0.207, P = 0.001). The genetic differentiation in T.collina was also analyzed by TE-AFLP derived SCAR marker (TECU marker) using SSCP analysis. The greatest differentiation was detected among 4 populations; North+Central+Northeast, Prachuap Khiri Khan, Chumphon, and Peninsular Thailand (Φ PT = 0.903, P = 0.001). The SSCP analysis of the 16S rRNA, COI, and cytb genes was also used to clarify the mtDNA diversity of T. collina. High levels of genetic variation among individuals within each population were observed. AMOVA analysis of the 16S rRNA, COI, and cytb genes showed high genetic differentiation among 6 populations; North, Central, Northeast, Prachuap Khiri Khan, Chumphon, and Peninsular Thailand (Φ PT = 0.563, Φ PT = 0.204, and Φ PT = 0.294, P = 0.001, respectively) while the significant genetic differentiations between samples from North and South of Isthmus of Kra were also found (Φ PT = 0.334, Φ PT = 0.106, and Φ PT = 0.133, P = 0.001, respectively).