Tests of hybridisation in Tetragonula stingless bees using multiple genetic markers.

Discrepancies in mitochondrial and nuclear genetic data are often interpreted as evidence of hybridisation. We re-examined reports of hybridisation in three cryptic stingless bee species in the genus Tetragonula in South East Queensland, Australia (T. carbonaria, T. davenporti, and T. hockingsi). Previous studies on this group using microsatellite markers proposed that occasional hybrids are found. In contrast, we find that allele frequencies at neutral regions of the nuclear genome, both microsatellites and random snps, reliably separated the three species, and thus do not support hybridisation. We found no inter-species variation in PCR amplicons of the nuclear gene EF1alpha, but low and moderate species-specific polymorphisms in the nuclear gene Opsin and the mitochondrial 16S respectively, with no cases of mito-nuclear discordance at these genes. We confirm that nuclear divergence between these species is low, based on 10-26kb of non-coding sequence flanking EF1alpha and Opsin (0.7-1% pairwise difference between species). However, we find mitogenomes to be far more diverged than nuclear genomes (21.6-23.6% pairwise difference between species). Based on these comprehensive analyses of multiple marker types, we conclude that there is no ongoing gene flow in the Tetragonula species of South East Queensland, despite their high morphological similarity to one another and the low nuclear divergence among them. The mitogenomes and draft nuclear genomes provided for these species will be a resource for further molecular studies on this group, which are important pollinators in Australian natural and agroecosystems.