The nuclear and mitochondrial genome assemblies of Tetragonisca angustula (Apidae: Meliponini), a tiny yet remarkable pollinator in the Neotropics.

BACKGROUND: The field of bee genomics has considerably advanced in recent years, however, the most diverse group of honey producers on the planet, the stingless bees, are still largely neglected. In fact, only eleven of the ~ 600 described stingless bee species have been sequenced, and only three using a long-read (LR) sequencing technology. Here, we sequenced the nuclear and mitochondrial genomes of the most common, widespread and broadly reared stingless bee in Brazil and other neotropical countries-Tetragonisca angustula (popularly known in Brazil as jataí). RESULTS: A total of 48.01 Gb of DNA data were generated, including 2.31 Gb of Pacific Bioscience HiFi reads and 45.70 Gb of Illumina short reads (SRs). Our preferred assembly comprised 683 contigs encompassing 284.49 Mb, 62.84 Mb of which (22.09%) corresponded to 445,793 repetitive elements. N50, L50 and complete BUSCOs reached 1.02 Mb, 91 contigs and 97.1%, respectively. We predicted that the genome of T. angustula comprises 17,459 protein-coding genes and 4,108 non-coding RNAs. The mitogenome consisted of 17,410 bp, and all 37 genes were found to be on the positive strand, an unusual feature among bees. A phylogenomic analysis of 26 hymenopteran species revealed that six odorant receptor orthogroups of T. angustula were found to be experiencing rapid evolution, four of them undergoing significant contractions. CONCLUSIONS: Here, we provided the first nuclear and mitochondrial genome assemblies for the ecologically and economically important T. angustula, the fourth stingless bee species to be sequenced with LR technology thus far. We demonstrated that even relatively small amounts of LR data in combination with sufficient SR data can yield high-quality genome assemblies for bees.

Decoding bee cleptoparasitism through comparative transcriptomics of Coelioxoides waltheriae and its host Tetrapedia diversipes.

Cleptoparasitism, also known as brood parasitism, is a widespread strategy among bee species in which the parasite lays eggs into the nests of the host species. Even though this behavior has significant ecological implications for the dynamics of several species, little is known about the molecular pathways associated with cleptoparasitism. To shed some light on this issue, we used gene expression data to perform a comparative analysis between two solitary neotropical bees: Coelioxoides waltheriae, an obligate parasite, and their specific host Tetrapedia diversipes. We found that ortholog genes involved in signal transduction, sensory perception, learning, and memory formation were differentially expressed between the cleptoparasite and the host. We hypothesize that these genes and their associated molecular pathways are engaged in cleptoparasitism-related processes and, hence, are appealing subjects for further investigation into functional and evolutionary aspects of cleptoparasitism in bees.

The complete mitochondrial genome of Trigonisca nataliae (Hymenoptera, Apidae) assemblage reveals heteroplasmy in the control region.

The evolution of mitochondrial genomes in the stingless bees is surprisingly dynamic, making them a model system to understand mitogenome structure, function, and evolution. Out of the seven mitogenomes available in this group, five exhibit atypical characteristics, including extreme rearrangements, rapid evolution and complete mitogenome duplication. To further explore the mitogenome diversity in these bees, we utilized isolated mtDNA and Illumina sequencing to assemble the complete mitogenome of Trigonisca nataliae, a species found in Northern Brazil. The mitogenome of T. nataliae was highly conserved in gene content and structure when compared to Melipona species but diverged in the control region (CR). Using PCR amplification, cloning and Sanger sequencing, six different CR haplotypes, varying in size and content, were recovery. These findings indicate that heteroplasmy, where different mitochondrial haplotypes coexist within individuals, occurs in T. nataliae. Consequently, we argue that heteroplasmy might indeed be a common phenomenon in bees that could be associated with variations in mitogenome size and challenges encountered during the assembly process.

Rapid evolution, rearrangements and whole mitogenome duplication in the Australian stingless bees Tetragonula (Hymenoptera: Apidae): A steppingstone towards understanding mitochondrial function and evolution.

The extreme conservation of mitochondrial genomes in metazoans poses a significant challenge to understanding mitogenome evolution. However, the presence of variation in gene order or genome structure, found in a small number of taxa, can provide unique insights into this evolution. Previous work on two stingless bees in the genus Tetragonula (T. carbonaria and T. hockingsi) revealed highly divergent CO1 regions between them and when compared to the bees from the same tribe (Meliponini), indicating rapid evolution. Using mtDNA isolation and Illumina sequencing, we elucidated the mitogenomes of both species. In both species, there has been a duplication of the whole mitogenome to give a total genome size of 30,666 bp in T. carbonaria; and 30,662 bp in T. hockingsi. These duplicated genomes present a circular structure with two identical and mirrored copies of all 13 protein coding genes and 22 tRNAs, with the exception of a few tRNAs that are present as single copies. In addition, the mitogenomes are characterized by rearrangements of two block of genes. We believe that rapid evolution is present in the whole Indo-Malay/Australasian group of Meliponini but is extraordinarily elevated in T. carbonaria and T. hockingsi, probably due to founder effect, low effective population size and the mitogenome duplication. All these features - rapid evolution, rearrangements, and duplication - deviate significantly from the vast majority of the mitogenomes described so far, making the mitogenomes of Tetragonula unique opportunities to address fundamental questions of mitogenome function and evolution.

Mitochondrial DNA intra-individual variation in a bumblebee species: A challenge for evolutionary studies and molecular identification.

Mitochondrial DNA (mtDNA) regions have been widely used as molecular markers in evolutionary studies and species identification. However, the presence of heteroplasmy and NUMTs may represent obstacles. Heteroplasmy is a state where an organism has different mitochondrial haplotypes. NUMTs are nuclear pseudogenes originating from mtDNA sequences transferred to nuclear DNA. Evidences of heteroplasmy were already verified in the bumblebee Bombus morio in an earlier study. The present work investigated in more detail the presence of intra-individual haplotypes variation in this species. Heteroplasmy was detected in individuals from all the ten sampled locations, with an average of six heteroplasmic haplotypes per individual. In addition, some of these heteroplasmic haplotypes were shared among individuals from different locations, suggesting the existence of stable heteroplasmy in B. morio. These results demonstrated that heteroplasmy is likely to affect inferences based on mtDNA analysis, especially in phylogenetic, phylogeographic and population genetics studies. In addition, NUMTs were also detected. These sequences showed divergence of 2.7% to 12% in relation to the mitochondrial haplotypes. These levels of divergence could mislead conclusions in evolutionary studies and affect species identification through DNA barcoding.

Conserved numts mask a highly divergent mitochondrial-COI gene in a species complex of Australian stingless bees Tetragonula (Hymenoptera: Apidae).

Tetragonula carbonaria, Tetragonula davenporti, Tetragonula hockingsi and Tetragonula mellipes comprise a species complex of Australian stingless bee species known as the 'Carbonaria' group. The species are difficult to distinguish morphologically and the major species-defining characters relate to comb architecture and nest entrance ornamentation. The taxonomy of the group is further complicated by likely nuclear mitochondrial pseudogenes (numts) and inter-specific hybrids. Here we demonstrate the existence of COI numts and isolate and characterize the 'true' mt-COI gene in T. carbonaria and T. hockingsi. Numts were isolated from enriched-nuclear DNA extraction followed by PCR amplification and Sanger sequencing, and were recognized by the presence of deletions and/or premature stop codons in the translated sequences. The mt-COI sequences were obtained from NGS sequencing using purified mtDNA. In T. carbonaria, two numts (numt1 and numt2) were identified and a third (numt3) was identified in T. hockingsi. Numt2 and numt3 are similar (1.2% sequence divergence), indicating a recent common origin. The genetic distance between the mt-COI of the two Tetragonula species was higher than might be expected for closely related species, 16.5%, corroborating previous studies in which T. carbonaria and T. hockingsi were regarded as separate species. The three numts are more similar to the COI of other stingless bee species, including Australian Austroplebia australis and South American Melipona bicolor (81.7-83.9%) than to the mt-COI of their own species (70-71.4%). This is because the mt-COI of T. carbonaria and T. hockingsi differ greatly from other Meliponinae. Our findings explain some formerly puzzling aspects of Carbonaria biogeography, and misinterpreted amplifications.

Fidelity of DNA polymerases in the detection of intraindividual variation of mitochondrial DNA.

Here we investigated the consequences of PCR amplification errors in the identification of intraindividual mtDNA variation. The bumblebee Bombus morio was chosen as model for the COI gene amplification tests with two DNA polymerases (Taq and Q5) presenting different error rates. The amplifications using Taq resulted in a significant increase of singleton haplotypes per individual in comparison to Q5. The sequence characteristics indicated that Taq resulted haplotypes are mostly due to amplification errors. Studies focusing on intraindividual variability should address special attention to the DNA polymerase fidelity to avoid overestimation of heteroplasmic haplotypes.