Mitogenome characteristics of two Tetragonula stingless bees (Hymenoptera: Apidae) from Hainan, China.

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Learn more: PMC Disclaimer | PMC Copyright Notice J Insect Sci . 2026 Jun 4;26(3):ieag045. doi: 10.1093/jisesa/ieag045 Mitogenome characteristics of two Tetragonula stingless bees (Hymenoptera: Apidae) from Hainan, China Shi-Jie Wang 1 , Jiao Wu 2 , Xi-Zhe Yang 3, 4 , Min Zhao 5, 6 , Cheng-Ye Wang 7, 8, ✉ Editor: Louis Hesler Author information Article notes Copyright and License information 1 Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China 2 Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China 3 Institute of Highland Forest Science, Chinese Academy of Forestry, Kunming, China 4 Key Laboratory of Breeding and Utilization of Resource Insects, National Forestry and Grassland Administration, Kunming, China 5 Institute of Highland Forest Science, Chinese Academy of Forestry, Kunming, China 6 Key Laboratory of Breeding and Utilization of Resource Insects, National Forestry and Grassland Administration, Kunming, China 7 Institute of Highland Forest Science, Chinese Academy of Forestry, Kunming, China 8 Key Laboratory of Breeding and Utilization of Resource Insects, National Forestry and Grassland Administration, Kunming, China ✉ Corresponding author. (Email: cywang11@126.com ). Roles Shi-Jie Wang : Conceptualization, Resources, Writing - original draft, Writing - review & editing Jiao Wu : Formal analysis Xi-Zhe Yang : Methodology, Software Min Zhao : Conceptualization Cheng-Ye Wang : Conceptualization, Data curation, Formal analysis, Methodology, Software, Validation, Writing - original draft, Writing - review & editing Louis Hesler : Subject Editor Received 2025 Nov 24; Revised 2026 May 6; Accepted 2026 May 12; Collection date 2026 May. © The Author(s) 2026. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( https://creativecommons.org/licenses/by-nc/4.0/ ), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. PMC Copyright notice PMCID: PMC13236724  PMID: 42242709 Abstract The complete mitochondrial genomes (mitogenomes) of two ecologically important stingless bees, Tetragonula gressitti (Sakagami) and T. laeviceps (Smith), were sequenced and analyzed. Both species display the typical insect mitogenome structures but exhibit species-specific structural rearrangements and sequence variation. Their mitogenomes are AT-rich (81.5% and 75.2%, respectively), significantly influencing codon usage patterns. We identified novel features in tRNA and rRNA secondary structures, and observed gene rearrangements that exceed the typical conservatism in Hymenoptera, likely reflecting adaptation to distinct ecological pressures. Phylogenomic analyses robustly place both species within a monophyletic Meliponini clade, distinct from other Apoidea ( Apis and Bombus ), supporting their current taxonomic placement. These mitogenomes provide valuable resources for Meliponini phylogenetics and may inform conservation and utilization strategies for these species. Keywords: comparative genomics, high-throughput sequencing, genetic diversity Introduction Stingless bees (Hymenoptera: Meliponini) are key pollinators in tropical and subtropical ecosystems ( Bueno et al. 2023 ), supporting plant diversity, crop yields, and local livelihoods through meliponiculture and honey production ( Lourino et al. 2006 ). Approximately 600 stingless bee species have been described across 61 genera ( Lepeco et al. 2024 ). Their high species diversity and adaptability make them important for both wild and agricultural systems ( Engel et al. 2023 ), but the same diversity increases the chance of cryptic species and complicates species delimitation. Traditional morphological identification relies on detailed morphometrics, but these characters often overlap among closely related taxa ( Quezada-Euán et al. 2015 , Geue and Thomassen 2020 , Williams et al. 2022 ). Within Meliponini, the genus Tetragonula comprises two subgenera: Tetragonilla (four described species) and Tetragonula (about 40 species) ( Engel et al. 2023 ). Species in Tetragonula are morphologically similar but vary in nesting architecture and foraging behavior, and have become favored in meliponiculture due to ease of management and reliable production. Two Tetragonula species, Tetragonula gressitti (Sakagami) and T. laeviceps (Smith), have been recorded in China and are important for biodiversity conservation and economic and nutritional value ( Wu et al. 2020 , Mubin et al. 2022 ). Nevertheless, quantitative data on their population dynamics and island-wide distributions on Hainan Island are scarce. Hainan Island, a continental tropical island off southern China, encompasses extensive lowland rainforests, montane forests, and agricultural mosaics. These heterogeneous habitats sustain high biodiversity and numerous insular taxa that are particularly susceptible to habitat loss and fragmentation driven by urbanization, tourism development, and land-use change. These stressors, together with predation and other anthropogenic disturbances, have created significant knowledge gaps in our understanding of stingless bee ecology ( Toledo-Hernández et al. 2022 ). Because oceanic isolation and recent human-mediated landscape alterations can limit natural gene flow on islands, and because commercial stingless bee colony movements can redistribute genotypes and pathogens across regions, documenting population structure on Hainan is essential both for local conservation planning and for robust phylogeographic comparisons with mainland samples such as those from Yunnan Province ( Li et al. 2024 ). Conservation is hindered by a lack of genetic baseline data. For T. gressitti and T. laeviceps on Hainan Island, few assessments address population structure, genetic diversity, gene flow, or adaptive potential. This constrains evidence-based conservation, complicates the definition of evolutionary significant units, and limits predictions of resilience to environmental stressors and disease. Integrative genetic studies combining genome-wide markers, high-quality mitogenomes, and targeted functional analyses are urgently needed to guide conservation and sustainable meliponiculture on Hainan Island. Complete mitogenomes are valuable for species delimitation, phylogenetics, and biodiversity assessment ( Galtier et al. 2009 ). Recent mitogenomic studies in Meliponini, including our earlier characterizations of several stingless bee mitogenomes ( Wang et al. 2020 , 2021 , 2022 , 2025 ), have improved resolution of interspecific relationships. To date, mitogenomes for 11 Tetragonula species have been reported ( Wang et al. 2022 , Françoso et al. 2023 , Karuppaiah et al. 2023 , Li et al. 2024 , Yong et al. 2024 ), but taxon- and region-level coverage remains uneven. In particular, robust mitogenomic data are lacking for T. gressitti and many T. laeviceps populations, limiting phylogenetic and taxonomic inference. Mitogenomic studies in Tetragonula and related Meliponini have uncovered pronounced structural features, including high rates of gene rearrangement, long inverted repeats (LIRs), whole-genome duplications, and heteroplasmy, that are uncommon in other bees ( Wang et al. 2022 , Françoso et al. 2023 , Li et al. 2024 , Yong et al. 2024 ). For example, some T. laeviceps populations exhibit heteroplasmy with mitogenomic variants containing LIRs and gene inversions ( Yong et al. 2024 ), while species such as T. carbonaria and T. hockingsi show whole-mitogenome duplications ( Françoso et al. 2023 , Li et al. 2024 ). These rearrangements yield strong phylogenetic signals but complicate assembly and annotation ( Wang et al. 2021 , Karuppaiah et al. 2023 , Li et al. 2024 ). Morphological diagnosis in Tetragonula is further complicated by subtle interspecific differences and high intraspecific variation, promoting misidentification and masking cryptic diversity. In T. laeviceps , genetic distances exceeding 10% among populations from Malaysia, China, Indonesia, and India suggest a potential species complex rather than a single panmictic taxon ( Ador et al. 2023 , Yong et al. 2024 ). The uneven geographic sampling of existing mitogenomes—concentrated primarily in India, Southeast Asia, and Australia (eg T. iridipennis , T. pagdeni , T. carbonaria , T. hockingsi )—further limits our capacity to delineate geographic lineages and evolutionary history ( Wang et al. 2022 , Françoso et al. 2023 , Karuppaiah et al. 2023 ). In this study, we sequenced the complete mitogenomes of T. gressitti and T. laeviceps sampled from Hainan Island, China. We analyzed gene order, nucleotide composition, and secondary structures, and reconstructed phylogenetic relationships within Apoidea to shed light on their evolutionary history. The newly obtained data could contribute to the development of effective management and conservation strategies for these species. Materials and Methods Sample Collection and DNA Extraction Tetragonula gressitti and T. laeviceps workers were collected in March 2023 from Jianfengling National Nature Reserves on Hainan Island (108°49′16.89″E, 18°42′46.22″N). After collecting, the fresh materials were immediately preserved in 100% ethanol and stored at −80 °C until DNA extraction. The total DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, United States), following the manufacturer’s protocol. Sequencing and Assembly The library was constructed with 1.0 μg total DNA using KAPA HyperPrep Kits (KAPA Biosystems). Sequencing was performed on an Illumina NextSeq 500, with a total data volume of 4 Gb (150-bp paired-end reads) for each species. Raw reads were quality-checked with FastQC (v0.12.0) ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) and processed using Trimmomatic (v0.35) ( Bolger et al. 2014 ) to remove adapters, short reads, and low-quality bases. High-quality reads were assembled de novo with IDBA-UD (v1.1.3) and SPAdes (v3.15.2) ( Gurevich et al. 2013 ). The resulting assemblies obtained from the two assemblers were evaluated using standard assembly quality metrics, including total assembly size, number of contigs (>500 bp), N50 value, maximum contig length, GC content consistency, read mapping rate back to the assembly, assembly completeness, and contamination estimates. The final assembly was selected based on higher N50 and longer maximum contig length, lower number of contigs, higher percentage of reads mapping back to the assembly, greater completeness, and lower contamination estimates. If trade-offs between metrics were observed, priority was given to assembly completeness and structural contiguity. The assembler that produced the best overall balance of these metrics was selected for downstream analyses. Protein-coding genes (PCGs) were identified via BLAST searches against NCBI ( https://www.ncbi.nlm.nih.gov ), and transfer RNA (tRNA) genes were detected using tRNAscan-SE ( Schattner et al. 2005 ). tRNA secondary structures were predicted with MITOS ( Bernt et al. 2013 ). Mitogenome maps were generated with CGView ( Grant and Stothard 2008 ). Phylogeny Analysis To analyze the taxonomic relationship of T. gressitti and T. laeviceps with other Apidae species, concatenated PCG sequences were aligned using ClustalW in MEGA 7.0 ( Kumar et al. 2016 ). Mitogenome sequences of representative Apidae species were retrieved from GenBank ( https://www.ncbi.nlm.nih.gov/ ). The combination and subsequent procedures of mitogenomes were performed in PhyloSuite (v1.2.3) ( Zhang et al. 2020 ). Maximum likelihood (ML) trees were inferred with IQ-TREE (v1.6.12) ( Nguyen et al. 2015 ), with partitioning by ModelFinder and General Time Reversible substitution model. Ultrafast bootstrap support was estimated with 10,000 replicates. Bayesian inference (BI) was conducted in MrBayes ( Ronquist and Huelsenbeck 2003 ) for 2 × 10 6 generations, sampling every 100 generations; the first 25% of samples were discarded as burn-in, and a 50% majority-rule consensus tree was constructed. Bayesian posterior probabilities were calculated for each node. Trees were visualized and refined using FigTree (v1.4.2). Mitogenome Composition Analysis and Gene Rearrangement Evaluation Base composition and codon numbers were analyzed in MEGA 7.0 ( Kumar et al. 2016 ). Relative synonymous codon usage (RSCU) was calculated in PhyloSuite ( Zhang et al. 2020 ); the mitochondrial codon table was used (UGA coding for tryptophan). Incomplete stop codons were excluded from RSCU analyses. AT- and GC-skews were calculated as AT-skew = (A − T)/(A + T) and GC-skew = (G − C)/(G + C) ( Perna and Kocher 1995 ). Gene order was compared with sequences retrieved from GenBank, and rearrangement events relative to a reference or inferred ancestral state were assessed in PhyloSuite (v1.2.2) ( Zhang et al. 2020 ). Rearrangement patterns were visualized on the phylogenetic framework using iTOL v6 ( Letunic and Bork 2019 ). Results and Discussion Mitogenome Composition and Architecture The mitogenomes of T. gressitti and T. laeviceps were 15,907 base pairs (bp) and 15,738 bp in size, respectively, each containing 13 PCGs, two ribosomal RNA (rRNA) genes, a noncoding control region (D-loop), and 22 tRNA genes ( Fig. 1 , Supplementary Tables S1 and S2 ). The two assembled mitogenomes displayed the compact, canonical gene organization typical of bees and other Hymenoptera. The circular configurations of both mitogenomes were confirmed by analyzing read-depth profiles, which revealed consistent high-depth coverage (average sequencing depth 326×, coverage range 172× to 363×) devoid of significant gaps or breakpoints at the closure sites. This circularity was further substantiated by identifying terminal overlapping motifs (>150 bp) between the flanking regions of the assembly intermediates, thereby ensuring the completeness and accuracy of the final circular molecules. Fig. 1. Open in a new tab The circular map of complete mitogenomes of Tetragonula gressitti and T. laeviceps . The outer features represent annotated genes, and the inner ring indicates the GC content variation. Note that Yong et al. (2024) reported two mitogenome sequences for T. laeviceps (GenBank accessions PP351837 and PP351838 ), both are 29,084 bp and described as heteroplasmic variants with LIRs. The size discrepancy between the T. laeviceps mitogenome obtained in this study and that reported by Yong et al. (2024) is primarily attributable to the presence or absence of LIRs. This variation putatively stems from intraspecific polymorphism within the species. Consistent with the evolutionary framework postulated by Li et al. (2024) , LIRs are found exclusively in the Indo-Malay/Australian clade of Meliponini, with a predicted single origin in their common ancestor approximately 48.36 million years ago. In T. gressitti , we observed duplication of the trnW with one copy on each strand and absence of trnE ( Supplementary Table S1 ). Such strand-specific duplications and gene losses reflect mitochondrial plasticity in Hymenoptera ( Dowton et al. 2009 , Mao et al. 2014 , Chen et al. 2018 , Xiong et al. 2023 ). These rearrangements may arise from slipped-strand mispairing or intramolecular recombination, potentially affecting transcriptional efficiency and codon–anticodon interactions ( Conant and Wolfe 2008 ). The duplicated trnW in T. gressitti might compensate for the loss of trnE via tRNA remodeling ( Cameron et al. 2008 ). Intergenic regions in T. gressitti ranged from −43 bp (overlap between trnQ and rrnS ) to 150 bp (between trnS2 and nad6 ) ( Supplementary Table S1 ). Similar compact organization and gene overlaps were observed in T. laeviceps (eg −1 bp between nad4 and trnH ; −3 bp between nad1 and trnL1 ) ( Supplementary Table S2 ). Protein-coding genes used various start codons, including ATG, ATA, ATC, ATT, and TTG, reflecting flexible initiation in mitochondrial translation. Stop codons were largely complete (TAA and TAG) but included truncated forms (eg TA for nad4 ) ( Supplementary Tables S1 and S2 ). This is a common feature among invertebrate mitogenomes, such as insects (eg Lepidoptera and Diptera) and mites ( Fenn et al. 2007 , Donath et al. 2019 , Yang et al. 2019 , Liang et al. 2023 ). The mitogenome of T. gressitti was highly A + T-rich (A + T = 81.5%), with tRNAs showing the highest A + T content (83.9%) ( Table 1 ). Positive AT-skew values in tRNAs (+0.029) and rRNAs (+0.042) indicated an A bias, and GC-skews were also positive in these regions, indicating a G bias. T. laeviceps showed a lower overall A + T content (75.2%), with tRNAs (79.6%), rRNAs (77.9%), and PCGs (73.5%), reflecting similar trends. Its genome displayed a slight T bias (AT-skew = −0.046) and near-neutral GC-skew (−0.006). Table 1. Base composition and skewness of the Tetragonula gressitti and T. laeviceps mitogenomes a Length (bp) A (bp) C (bp) G (bp) T (bp) A (%) C (%) G (%) T (%) A + T (%) AT-skew GC-skew T. gressitti Whole mitogenome 15,907 6,291 1,541 1,401 6,674 39.5 9.7 8.8 42 81.5 −0.03 −0.048 PCGs 11,108 3,974 1,161 1,005 4,968 35.8 10.5 9 44.7 80.5 −0.111 −0.072 tRNAs 1,445 624 94 138 589 43.2 6.5 9.6 40.8 83.9 0.029 0.19 rRNAs 2,182 906 174 269 833 41.5 8 12.3 38.2 79.7 0.042 0.214 rrnL 1,364 550 109 163 542 40.3 8 12 39.7 80.1 0.007 0.198 rrnS 818 356 65 106 291 43.5 7.9 13 35.6 79.1 0.1 0.24 T. laeviceps Whole mitogenome 15,738 5,643 1,963 1,940 6,192 35.9 12.5 12.3 39.3 75.2 −0.046 −0.006 PCGs 10,895 3,562 1,589 1,301 4,443 32.7 14.6 11.9 40.8 73.5 −0.11 −0.1 tRNAs 1,488 620 136 167 565 41.7 9.1 11.2 38 79.6 0.046 0.102 rRNAs 2,201 890 188 299 824 40.4 8.5 13.6 37.4 77.9 0.038 0.228 rrnL 1,382 555 116 177 534 40.2 8.4 12.8 38.6 78.8 0.019 0.208 rrnS 819 335 72 122 290 40.9 8.8 14.9 35.4 76.3 0.072 0.258 Open in a new tab a The parameters in the table were calculated using J-chain (Forward direction) except rrnL and rrnS which were calculated using N-chain (Reverse direction). D-loop Features The D-loop contains repetitive elements and length-variable tandem repeats. These features are difficult to resolve with short-read sequencing due to repeat collapse and ambiguous mapping ( Treangen and Salzberg 2012 , Nagarajan and Pop 2013 ). It regulates mitochondrial replication and transcription, and its high AT content may facilitate helix unwinding ( Rajewska et al. 2012 ). Structural comparisons among T. gressitti , T. laeviceps , and representative Apinae species ( Melipona bicolor , Lepidotrigona flavibasis , T. pagdeni , Apis nuluensis ) reveal species-specific architectural variations in the D-loop ( Supplementary Fig. S1 ). Conserved regulatory motifs (TATA and CAAT boxes) and repeat units appear across species; for example, A. nuluensis additionally contains trinucleotide repeats. The D-loops of T. gressitti and T. laeviceps were shorter and structurally simpler than those of several other species, showing reduced TATA/CAAT box density while retaining (A) 6 repeats common to Tetragonula . Reduced complexity may indicate alternative mechanisms of replication initiation or factor binding, but functional implications warrant further study. Additionally, repetitive regions can artificially inflate sequencing coverage, as reads may align to multiple repeat units ( Li 2014 ). Thus, high read depth does not necessarily guarantee full structural resolution of the control region. Future validation using targeted PCR amplification or long-read sequencing ( Rang et al. 2018 , Wenger et al. 2019 ) is needed to confirm the control region’s structural integrity and length. Codon Usage Codon usage reflects the pronounced A + T bias, favoring A/T-rich codons such as TTA (Leu) and ATA (Met), while G/C-rich codons are underrepresented or absent ( Fig. 2 ). RSCU analyses confirm a strong preference for A/T at third codon positions; UUA (Leu), AUU (Ile), and UUU (Phe) display high RSCU values ( Fig. 3 ). Notable interspecific differences include a higher RSCU for UUA in T. gressitti (3.71) compared to T. laeviceps (1.88) ( Supplementary Tables S3 and S4 ), possibly reflecting species-specific translational or ecological adaptations. Fig. 2. Open in a new tab Amino acids distribution in mitogenome of Tetragonula gressitti (A) and T. laeviceps (B). The number on the left is the total number of each amino acid. The 20 amino acid types are represented on the x axis. Fig. 3. Open in a new tab The RSCU in the mitogenome of Tetragonula gressitti (A) and T. laeviceps (B). The figure plots codon types on the x axis and their corresponding RSCU values on the y axis. tRNAs and rRNAs Mitochondrial tRNAs often deviate from canonical cloverleaf structures, sometimes lacking entire arms. Nevertheless, they can adopt alternative stable folds that preserve essential spatial relationships ( Biela et al. 2023 ). Predicted tRNA secondary structures for T. gressitti and T. laeviceps were shown in Figs. 4 and 5 , respectively. Most tRNAs retain the cloverleaf, but deviations occur, such as trnS1 in T. laeviceps lacks the DHU arm, a common insect feature that may be compensated post-transcriptionally ( Wolstenholme 1992 , Cameron 2014 ). G-U mismatches are frequent in tRNA secondary structures; though less stable than Watson–Crick pairs, they are often tolerated and stabilized by surrounding structure. Fig. 4. Open in a new tab Predicted secondary structures of tRNAs in Tetragonula gressitti mitogenome. ‘-’ indicates Watson–Crick base pairing and ‘ · ’ indicates GU base pairing. Fig. 5. Open in a new tab Predicted secondary structures of tRNAs in Tetragonula laeviceps mitogenome. ‘-’ indicates Watson–Crick base pairing and ‘ · ’ indicates GU base pairing. The 16S ( rrnL ) and 12S ( rrnS ) rRNA secondary structures were predicted for both species ( Supplementary Figs. S2–S5 ). Subtle differences between species may reflect species-specific adaptations linked to metabolic or ecological requirements. G-U mismatches also occur in rRNAs, contributing to structural flexibility. Phylogenetic Analysis Phylogenetic trees inferred by ML and BI were congruent, placing both species within a strongly supported Meliponini clade distinct from honey bees ( Apis spp.) and bumble bees ( Bombus spp.) ( Fig. 6 ), consistent with their taxonomic assignment. Although taxon sampling is smaller than that in Lepeco et al. (2024) , our mitogenome-based topology broadly agrees with higher-level separations among stingless bees, bumble bees, and honey bees. Within Apidae, stingless bees cluster separately and show closer affinity to bumble bees than to honey bees ( Fig. 6 ). Within Meliponini, T. gressitti and T. laeviceps group with other Tetragonula species and then with two Lepidotrigona species, forming a clade supported by high bootstrap values ( Fig. 6 ). Broader sampling suggests that Lepidotrigona may not be monophyletic across the tribe ( Lepeco et al. 2024 ). Fig. 6. Open in a new tab Phylogenetic tree constructed using ML and BI. Node labels show BI posterior probabilities and ML bootstrap support. Notably, stingless bees generally exhibit limited dispersal capacity and strong population genetic structure. In conjunction with the accelerated evolutionary rate of mitochondrial DNA, profound intraspecific divergence is a well-documented phenomenon within the Meliponini ( Li et al. 2024 ). In the present study, the genetic distance between our T. laeviceps mitogenome and those reported by Yong et al. (2024) reached 10.22%. This value dramatically exceeds the conventional 2% threshold typically recognized for intraspecific variation in animal mitochondrial genes, thereby underscoring the existence of significant cryptic lineages or substantial intraspecific differentiation within this stingless bee species. Gene Order and Rearrangement Gene order (ie the arrangement of tRNAs, rRNAs, and PCGs) is informative for comparative mitogenomics because rearrangements are relatively rare. Our comparisons reveal a mosaic of conserved blocks and lineage-specific rearrangements ( Fig. 7 ). Rearrangements are concentrated in tRNA loci and, in some lineages, extend to adjacent PCGs, consistent with recent reports in Meliponini ( Karuppaiah et al. 2023 , Li et al. 2024 ). Fig. 7. Open in a new tab Gene order comparison of the Tetragonula gressitti and T. laeviceps mitogenomes with 11 other species in Apinae. tRNA genes for distinct amino acids are denoted by their corresponding single-letter amino acid code. Genes marked with an underline indicate that they are absent in the mitogenome. Notable features include structural polymorphisms and gene block inversions, demonstrating that intrapopulation variability can produce diagnosable rearrangements ( Li et al. 2024 , Yong et al. 2024 ). For example, distinct gene order differences exist involving the nad4 locus block when comparing our T. laeviceps sequence ( PX939156 ) with the two conspecific sequences from Yong et al. (2024) . Specifically, the block nad4-nad4L-trnP(tgg)-trnS1(tct) in TL1-1 ( PP351837 ) is inverted in TL1-2 ( PP351838 ), appearing as trnS1(tct)-trnP(tgg)-nad4L-nad4 . Relative to TL1-1, our assembly ( PX939156 ) exhibits an inversion event involving a broader block nad5-trnH(gtg)-nad4-nad4L-trnP(tgg)-trnS1(tct) , which is rearranged into trnS1(tct)-trnP(tgg)-nad4L-nad4-trnH(gtg)-nad5 . This configuration demonstrates a higher degree of syntenic conservation with TL1-2 ( PP351838 ), except for the additional inversion involving trnH(gtg)-nad5 . Beyond these block inversions, comparative mitogenomic analysis revealed other significant structural polymorphisms. The present assembly lacks LIRs identified in the sequences from Yong et al. (2024) . In addition, the position corresponding to trnG(tcc) (located between cox3 and nad3 ) in the canonical segment of the two sequences from Yong et al. (2024) is occupied by trnE in our mitogenome. Conversely, trnE is absent from the canonical segment in their sequences, instead being localized within the LIR at the trnG(tcc) position. In our T. laeviceps sequence ( PX939156 ), trnG was found to be translocated to the junction between rrnL and trnL1 . In our samples, nad1-trnL1-rrnL-trnV-rrnS cluster is inverted in T. gressitti and a trnG is inserted into this cluster in T. laeviceps ( Fig. 7 ). Variability in trnK positioning was also observed relative to honey bees and bumble bees ( Wang et al. 2022 ). In T. gressitti , the trnK block was organized as trnW1-D-loop-trnK-trnA-trnI-trnM , while in T. laeviceps , it was arranged as nad5-trnK-trnA-trnI-trnM . The super-conserved atp8-atp6-cox3-trnG block of Hymenoptera mitogenomes was altered in T. laeviceps , where an unusual trnG translocation indicates a more dynamic genome compared to T. gressitti . In T. gressitti , dramatic positional changes in rRNA and tRNA genes were observed, including duplication of the trnW gene and the absence of the trnE gene ( Fig. 7 ). Both species showed ambiguous positioning of trnG or trnE between cox3 and nad3 , consistent with previous findings in Tetragonula ( Li et al. 2024 ). Rearrangements in Meliponini are documented in several species, for example, Frieseomelitta varia , Lepidotrigona flavibasis , L. terminata , T. iridipennis , and T. pagdeni ( de Paula Freitas et al. 2020 , Wang et al. 2021 , 2022 , Françoso et al. 2023 , Karuppaiah et al. 2023 ). The Indo-Malay/Australian Tetragonula clade exhibits LIRs and multigene rearrangements that contribute to size variation of the mitogenomes and unique architectures ( Françoso et al. 2023 , Li et al. 2024 ). Specifically, Yong et al. (2024) reported heteroplasmy, inversions, and inversion repeats in the mitogenome of T. laeviceps from Peninsular Malaysia. Atypical features, including inversion repeats of entire mitogenomes, have also been reported in T. carbonaria , T. davenporti , and T. hockingsi ( Françoso et al. 2023 , Li et al. 2024 ). Such deviations may be tolerated in regions that do not critically impair mitochondrial function and may relate to species-specific metabolic or reproductive adaptations. The mitogenomes of T. gressitti and T. laeviceps combine conserved elements with lineage-specific rearrangements, particularly in tRNAs and selected gene blocks. These patterns are informative for phylogenetics and enhance our understanding of mitochondrial evolution in stingless bees. Given the mitogenome’s role in metabolism and adaptation ( Aw et al. 2018 ), expanded taxon sampling and integration with functional genomic data will clarify the adaptive significance of these variations and support conservation and sustainable use of stingless bee resources. Supplementary Material ieag045_Supplementary_Data ieag045_supplementary_data.doc (7.7MB, doc) Acknowledgements We sincerely appreciate Mr. Pan Xu from Diggers (Wuhan) Biotechnology Co., Ltd. for the technical support. Contributor Information Shi-Jie Wang, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China. Jiao Wu, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China. Xi-Zhe Yang, Institute of Highland Forest Science, Chinese Academy of Forestry, Kunming, China; Key Laboratory of Breeding and Utilization of Resource Insects, National Forestry and Grassland Administration, Kunming, China. Min Zhao, Institute of Highland Forest Science, Chinese Academy of Forestry, Kunming, China; Key Laboratory of Breeding and Utilization of Resource Insects, National Forestry and Grassland Administration, Kunming, China. Cheng-Ye Wang, Institute of Highland Forest Science, Chinese Academy of Forestry, Kunming, China; Key Laboratory of Breeding and Utilization of Resource Insects, National Forestry and Grassland Administration, Kunming, China. Author Contributions Shi-Jie Wang (Conceptualization [equal], Resources [lead], Writing—original draft [lead], Writing—review & editing [equal]), Jiao Wu (Formal analysis [equal]), Xi-Zhe Yang (Methodology [equal], Software [equal]), Min Zhao (Conceptualization [equal]), and Cheng-Ye Wang (Conceptualization [equal], Data curation [lead], Formal analysis [equal], Methodology [equal], Software [equal], Validation [lead], Writing—original draft [equal], Writing—review & editing [equal]) Supplementary Material Supplementary material is available at Journal of Insect Science online. Funding This work was supported by Hainan Province Science and Technology Special Fund (ZDYF2023RDYL01), the Hainan Institute of National Park (KY-24ZK02), China Agriculture Research System of MOF and MARA (Grant No. CARS-44-SYZ-11), and Chinese Academy of Tropical Agricultural Sciences for Science and Technology Innovation Team of National Tropical Agricultural Science Center (Grant No. CATASCXTD202522). Conflicts of Interest None declared. Data Availability Raw sequence data and the assembled mitogenome sequences of T. gressitti and T. laeviceps described in this study have been deposited in GenBank. The raw data are available under BioProject accessions PRJNA1429588 and PRJNA1429792, and mitogenome sequences are available in GenBank under accession numbers PX939156 and PX956992 . References Ador K, Gobilik J, Benedick S.  2023. Phylogenetic and morphological characteristics reveal cryptic speciation in stingless bee, Tetragonula laeviceps s.l. Smith 1857 (Hymenoptera; Meliponinae). Insects  14:438. 10.3390/insects14050438 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Aw WC, Towarnicki SG, Melvin RG, et al.  2018. Genotype to phenotype: diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness. 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