Reassessing Hybridisation in Australian Tetragonula Stingless Bees Using Multiple Genetic Markers.

Area of Science:

• Zoology
• Genetics
• Evolutionary Biology

Background:

• Cryptic stingless bee species in the genus *Tetragonula* in South East Queensland, Australia (*T. carbonaria*, *T. davenporti*, and *T. hockingsi*) have been previously reported to hybridize.
• Previous studies utilizing microsatellite markers suggested occasional hybridization events among these species.

Purpose of the Study:

• To re-examine hybridization reports in three cryptic *Tetragonula* stingless bee species.
• To determine the extent of contemporary gene flow among these closely related species using advanced genetic markers.

Main Methods:

• Analysis of 1745 single nucleotide polymorphisms (SNPs) for species discrimination.
• Sequencing of nuclear genes (*EF1alpha*, *Opsin*) and mitochondrial 16S rRNA gene.
• Assessment of nuclear and mitochondrial genome divergence.

Main Results:

• SNPs reliably distinguished the three *Tetragonula* species, showing no evidence of contemporary hybridization.
• Low nuclear divergence (0.7%-1%) was observed, with conserved *EF1alpha* sequences but species-specific polymorphisms in *Opsin*.
• Mitochondrial genomes showed significantly higher divergence (21.6%-23.6%) compared to nuclear genomes, with no mito-nuclear discordance.
• Identical amplicon sequences of the nuclear gene *EF1alpha* were found across most individuals of the three species.

Conclusions:

• There is no ongoing gene flow among the *Tetragonula* species in South East Queensland.
• Despite morphological similarities and low nuclear divergence, these species remain reproductively isolated.
• High-resolution SNP markers provide greater accuracy in estimating contemporary hybridization, potentially leading to lower reported rates in other studies.