Ancestral Gene Order Research Articles

The first complete mitochondrial genome of Bdelloidea (Trombidiformes, Eupodina) and comparative genomics provide insights into gene rearrangement and evolution of trombidiform mites

In this study, we reported the first complete mitogenome of Bdelloidea, Spinibdella lignicola, and combined with other 26 trombidiform mitogenomes, we conducted gene order (GO) comparative analyses of trombidiform mites. The mitogenome of S. lignicola, having a full set of 37 coding genes, exhibits a novel gene cluster of “trnA-trnQ-trnS1-trnR” among trombidiform mites. Phylogenetic reconstruction revealed that S. lignicola is closely related to Riccardoella reaumuri, supporting its position within Eupodides. Like most previous studies, the monophyly of Trombidiformes is rejected for Eriophyoidea separates from Trombidiformes. Shared gene blocks and derived gene boundary analysis concluded that four blocks (cox1∼cox3; nad6∼trnS2; nad1-trnL2; trnM ∼ trnC) are the dominant arrangements of trombidiform mite mitogenomes and evolution synapomorphies occurred in trombidiform mites have phylogenetic relatedness. Furthermore, we propose possible ancestral GO of Trombidiformes and provide GO evidence for eriophyoid mites evolving independently from non-eriophyoid trombidiform mites. Our study confirms that comparative analysis of GO is a valuable information tool that improves our understanding of mitogenome evolution and phylogeny of trombidiform mites.

The complete mitochondrial genomes and phylogenetic analysis of two Nycteribiidae bat flies (Diptera: Hippoboscoidea)

We sequenced the complete mitochondrial genomes of two bat fly species within the Nycteribiidae (Diptera: Hippoboscoidea) – Dipseliopoda setosa (Cyclopodiinae) and Basilia ansifera (Nycteribiinae). Both mitogenomes were complete and contained 13 protein-coding genes, 22 tRNAs, and two rRNAs. Relative to the inferred ancestral gene order of dipteran mitochondrial genomes, no rearrangements were identified in either species. There were large differences in size between the two genomes, with D. setosa having a larger genome (19,164 bp) than B. ansifera (16,964 bp); both species had larger genomes than two previously published Streblidae bat fly species (e.g., Paradyschiria parvula and Paratrichobius longicrus). The increased genome sizes were due to expansions in the control region and the non-coding region downstream of the light-strand origin of replication. Additional differences between the two mitogenomes included a significantly longer cox3 gene in B. ansifera and a longer nad1 gene in D. setosa. Interestingly, both genomes also had the lowest GC content (D. setosa – 15.9%; B. ansifera – 17.0%) of any available Hippoboscoidea mitochondrial genome (18.8–23.9%). These mitogenomes represent the first sequences from species within the bat fly family Nycteribiidae. The sequence data here will provide a foundation for continued studies of genome evolution more generally within obligate blood-feeding ectoparasites, and specifically for the bat flies as vectors of significant ‘bat-associated’ viruses and microorganisms.

The slow-evolving Acorus tatarinowii genome sheds light on ancestral monocot evolution

Monocots are one of the most diverse groups of flowering plants, and tracing the evolution of their ancestral genome into modern species is essential for understanding their evolutionary success. Here, we report a high-quality assembly of the Acorus tatarinowii genome, a species that diverged early from all the other monocots. Genome-wide comparisons with a range of representative monocots characterized Acorus as a slowly evolved genome with one whole-genome duplication. Our inference of the ancestral monocot karyotypes provides new insights into the chromosomal evolutionary history assigned to modern species and reveals the probable molecular functions and processes related to the early adaptation of monocots to wetland or aquatic habitats (that is, low levels of inorganic phosphate, parallel leaf venation and ephemeral primary roots). The evolution of ancestral gene order in monocots is constrained by gene structural and functional features. The newly obtained Acorus genome offers crucial evidence for delineating the origin and diversification of monocots, including grasses.

Complete mitochondrial genomes and phylogenetic analysis of the Hawaiian planthoppers Iolania perkinsi and Oliarus cf. filicicola (Hemiptera: Cixiidae)

We sequenced the complete mitochondrial genomes of one Iolania perkinsi (Kirkaldy 1902) and one Oliarus cf. filicicola (Kirkaldy 1909) planthopper (Hemiptera: Fulgoroidea: Cixiidae) from Volcano Village, located on the eastern flank of Mauna Loa. The I. perkinsi complete mitogenome is 14,949 bp in length and contains 13 protein-coding genes, 22 tRNAs and 2 rRNAs. The O. cf. filicicola nearly complete mitogenome is 15,196 bp in length due to an expanded (but incomplete) control region, and contains all of the typical metazoan genes: 13 protein-coding genes, 22 tRNAs and two rRNAs. Relative to the inferred ancestral gene order of insect mitochondrial genomes, no rearrangements were identified in either species. In addition to the shorter control region in I. perkinsi, the differences between the two mitogenomes consist of longer cox1, cox2, cob, nad1, nad5, nad6 genes but shorter nad4 gene in I. perkinsi relative to O. cf. filicicola. These mitogenomes represent the first sequences from species within the Hawaiian Cixiidae. The sequence data here will provide a foundation for continued studies of speciation patterns and dynamics of evolutionary radiation across Hawaiian planthoppers.

Gene arrangement, phylogeny and divergence time estimation of mitogenomes in Thrips.

The metazoan mitogenomes usually display conserved gene arrangement while thrips are known for their extensive gene rearrangement, and duplication of the control region. We sequenced complete mitogenomes of eight species of thrips to determine the gene arrangement, phylogeny and divergence time estimation. All contain 37 genes and one control region, (CR) except four species with two CRs. Duplicated tRNAs were detected in Mycterothrips nilgiriensis and Thrips florum. nad4-nad4L were not found adjacent to each other in Phibalothrips peringueyi and Plicothrips apicalis. Both Bayesian and likelihood phylogenetic analyses of thrips mitogenomes supported the monophyly of two suborders (Terebrantia and Tubulifera) and the two largest families (Phlaeothripidae and Thripidae). Out of seven earlier proposed ancestral gene blocks, six are conserved in Panchaetothripinae, three in Thripinae and two in Phlaeothripidae. Additionally, eight Thrips Gene Blocks were identified, of which, three conserved in Tubulifera, four in Terebrantia, and one only in Aeolothripidae. Forty-two gene boundaries (15 from previous study + 27 new) were identified. The molecular divergence time is estimated for the order Thysanoptera and suggested that these insects may have been diversified from hemipterans in the late Permian period. The most recent ancestors belong to family Thripidae and Phlaeothripidae, which were diversified in upper Cretaceous period and showed higher rates of rearrangement from the ancestral gene order. The current study is the first largest effort to provide the new insights into the mitogenomic features, gene arrangement, phylogeny and divergence time estimation of thrips belonging to the order Thysanoptera.

Complete mitochondrial genome of the Mexican marine littoral hygrophilous Spinactaletes boneti (Collembola: Actaletidae) and its phylogenetic placement

Spinactaletes boneti mitochondrial genome was assembled and annotated. It has 14,766 bp in length, all 37 genes are present and the gene order is the same as the Pancrustacean ancestral gene order. Phylogenetic analyses based on maximum likelihood placed the species as a sister group of the remaining Entomobryomorpha, not closely related to the Isotomoidea superfamily, contradicting the actual systematics of the group.

The mitochondrial genome of Chelonus formosanus (Hymenoptera: Braconidae) with novel gene orders and phylogenetic implications.

Chelonus formosanus Sonan is an important egg‐larval parasitoid of noctuid moths and a potential candidate for understanding interactions between host and parasitoid mediated by polydnavirues (PDVs). We sequenced and annotated the mitochondrial genome of C. formosanus, which is 15,466 bp in length and possesses 38 mitochondrial genes. However, unlike most animal mitochondrial genomes, it contains one extra trnF gene. There are five transfer RNA (tRNA) rearrangement events compared with the ancestral gene order, which is a novel rearrangement type in Hymenoptera for all published mitogenomes so far. Phylogenetic trees supported C. formosanus from the subfamily Cheloninae was closely related to the subfamily Cardiochilinae and Microgastrinae.

Small parsimony for natural genomes in the DCJ-indel model.

The Small Parsimony Problem (SPP) aims at finding the gene orders at internal nodes of a given phylogenetic tree such that the overall genome rearrangement distance along the tree branches is minimized. This problem is intractable in most genome rearrangement models, especially when gene duplication and loss are considered. In this work, we describe an Integer Linear Program algorithm to solve the SPP for natural genomes, i.e. genomes that contain conserved, unique, and duplicated markers. The evolutionary model that we consider is the DCJ-indel model that includes the Double-Cut and Join rearrangement operation and the insertion and deletion of genome segments. We evaluate our algorithm on simulated data and show that it is able to reconstruct very efficiently and accurately ancestral gene orders in a very comprehensive evolutionary model.

The Mitochondrial Genomes of 18 New Pleurosticti (Coleoptera: Scarabaeidae) Exhibit a Novel trnQ-NCR-trnI-trnM Gene Rearrangement and Clarify Phylogenetic Relationships of Subfamilies within Scarabaeidae.

Simple SummaryThe family Scarabaeidae is one of the largest families in the insect order Coleoptera and is comprised of two quasi-systematics groups, Pleurosticti and Laparosticti. Pleurosticti is an economically important scarab group comprising about 20,000 species, the majority of which are phytophagous. Despite the innumerable studies based on ecological, molecular, and morphological characteristics, their taxonomy is still unclear and subject to many scientific hypotheses. The mitochondrial (mt) genome can provide tangible information to resolve the phylogenetic relationships within the family Scarabaeidae. However, the available mt genomes of Scarabaeidae in GenBank are underrepresented. Thus, we sequenced and analyzed 18 new phytophagous Scarabaeidae mitochondrial genomes from two subfamilies, Cetoniinae and Dynastinae, to conduct phylogenetic analyses within Scarabaeidae. This study contributes to increasing our knowledge about phytophagous Scarabaeidae.The availability of next-generation sequencing (NGS) in recent years has facilitated a revolution in the availability of mitochondrial (mt) genome sequences. The mt genome is a powerful tool for comparative studies and resolving the phylogenetic relationships among insect lineages. The mt genomes of phytophagous scarabs of the subfamilies Cetoniinae and Dynastinae were under-represented in GenBank. Previous research found that the subfamily Rutelinae was recovered as a paraphyletic group because the few representatives of the subfamily Dynastinae clustered into Rutelinae, but the subfamily position of Dynastinae was still unclear. In the present study, we sequenced 18 mt genomes from Dynastinae and Cetoniinae using next-generation sequencing (NGS) to re-assess the phylogenetic relationships within Scarabaeidae. All sequenced mt genomes contained 37 sets of genes (13 protein-coding genes, 22 tRNAs, and two ribosomal RNAs), with one long control region, but the gene order was not the same between Cetoniinae and Dynastinae species. All mt genomes of Dynastinae species showed the same gene rearrangement of trnQ-NCR-trnI-trnM, whereas all mt genomes of Cetoniinae species showed the ancestral insect gene order of trnI-trnQ-trnM. Phylogenetic analyses (IQ-tree and MrBayes) were conducted using 13 protein-coding genes based on nucleotide and amino acid datasets. In the ML and BI trees, we recovered the monophyly of Rutelinae, Cetoniinae, Dynastinae, and Sericinae, and the non-monophyly of Melolonthinae. Cetoniinae was shown to be a sister clade to (Dynastinae + Rutelinae).

BOPAL 2.0 and a study of tRNA and rRNA gene evolution in Clostridium.

We present BOPAL 2.0, an improved version of the BOPAL algorithm for the evolutionary history inference of tRNA and rRNA genes in bacterial genomes. Our approach can infer complete evolutionary scenarios and ancestral gene orders on a phylogeny and considers a wide range of events such as duplications, deletions, substitutions, inversions and transpositions. It is based on the fact that tRNA and rRNA genes are often organized in operons/clusters in bacteria, and this information is used to help identify orthologous genes for each genome comparison. BOPAL 2.0 introduces new features, such as a triple-wise alignment step, context-aware singleton matching and a second pass of the algorithm. Evaluation on simulated datasets shows that BOPAL 2.0 outperforms the original BOPAL in terms of the accuracy of inferred events and ancestral genomes. We also present a study of the tRNA/rRNA gene evolution in the Clostridium genus, in which the organization of these genes is very divergent. Our results indicate that tRNA and rRNA genes in Clostridium have evolved through numerous duplications, losses, transpositions and substitutions, but very few inversions were inferred.

Ancestral Flowering Plant Chromosomes and Gene Orders Based on Generalized Adjacencies and Chromosomal Gene Co-Occurrences.

Recurrent whole genome duplication and the ensuing loss of redundant genes-fractionation-complicate efforts to reconstruct the gene orders and chromosomes of the ancestors associated with the nodes of a phylogeny. Loss of genes disrupts the gene adjacencies key to current techniques. With our RACCROCHE pipeline, instead of starting with the inference of short ancestral segments, we suggest delaying the choice of gene adjacencies while we accumulate many more syntenically validated generalized (gapped) adjacencies. We obtain longer ancestral contigs using maximum weight matching (MWM). Similarly, we do not construct chromosomes by successively piecing together contigs into larger segments, but rather compile counts of pairwise contig co-occurrences on the set of extant genomes and use these to cluster the contigs. Chromosome-level contig assemblies for a monoploid genome emerge naturally at each node of the phylogeny and the contigs then can be ordered along the chromosome. Sampling alternative MWM solutions, visualizing heat maps, and applying gap statistics allow us to estimate the number of chromosomes in the reconstruction. We introduce several measures of quality: length of contigs, continuity of contig structure on successive ancestors, coverage of the extant genome by the reconstruction, and rearrangement relations among the inferred chromosomes. The reconstructed ancestors are visualized by painting the ancestral projections on the descendant genomes. We submit genomes drawn from a broad range of monocot orders to our pipeline, confirming the tetraploidization event "tau" in the stem lineage between the alismatids and the lilioids. We show additional applications to the Solanaceae and to four Brassica genomes, producing evidence about the monoploid ancestor in each case.

Genome Comparisons of the Fission Yeasts Reveal Ancient Collinear Loci Maintained by Natural Selection.

Fission yeasts have a unique life history and exhibit distinct evolutionary patterns from other yeasts. Besides, the species demonstrate stable genome structures despite the relatively fast evolution of their genomic sequences. To reveal what could be the reason for that, comparative genomic analyses were carried out. Our results provided evidence that the structural and sequence evolution of the fission yeasts were correlated. Moreover, we revealed ancestral locally collinear blocks (aLCBs), which could have been inherited from their last common ancestor. These aLCBs proved to be the most conserved regions of the genomes as the aLCBs contain almost eight genes/blocks on average in the same orientation and order across the species. Gene order of the aLCBs is mainly fission-yeast-specific but supports the idea of filamentous ancestors. Nevertheless, the sequences and gene structures within the aLCBs are as mutable as any sequences in other parts of the genomes. Although genes of certain Gene Ontology (GO) categories tend to cluster at the aLCBs, those GO enrichments are not related to biological functions or high co-expression rates, they are, rather, determined by the density of essential genes and Rec12 cleavage sites. These data and our simulations indicated that aLCBs might not only be remnants of ancestral gene order but are also maintained by natural selection.

Massive gene rearrangement in mitogenomes of phytoseiid mites

Mitochondrial (mt) gene sequences have been widely used to infer phylogeny in animals. The relative order of mt genes in the mitogenome can also be a useful marker for evolution, but the propensity of mt gene rearrangements vary tremendously among taxa. Ticks and mites in Acari exemplify this trend as some families retain the ancestral arthropod gene order, while others show highly divergent gene orders. Mites in Phytoseiidae, many of which are effective biological control agents, show some of the most divergent gene orders. However, the diversity of mitogenome order within this family is little known. We thus sequenced three mt genomes of phytoseiid mites from two of the most speciose genera: Amblyseius swirskii (Athias-Henriot), Amblyseius tsugawai (Ehara) and Neoseiulus womersleyi (Schicha). We find differences in mt GC skew and nucleotide composition, especially between N. womersleyi and the two Amblyseius species. Each species within Phytoseiidae (including three previously available sequences) present a unique gene order. Phytoseiid mitogenomes show some of the highest numbers of breakpoints when compared to the ancestral arthropod order (up to 33), as well as high numbers of breakpoints within the family (14-30). This suggests a history of massive, ongoing mitogenome rearrangements in the family. Phylogenetic analyses of mt sequences confirm that the degree of gene rearrangements follows phylogenetic relatedness. We discuss possible causes for the high degree of mt gene rearrangement within phytoseiid mites as well as selection in the mt and nuclear genome tied to the independent evolution of many diverse feeding strategies in the family. Finally, we suggest N. womersleyi should be used instead of the synonym Amblyseius pseudolongispinosus.

Validation of Automated Chromosome Recovery in the Reconstruction of Ancestral Gene Order

The RACCROCHE pipeline reconstructs ancestral gene orders and chromosomal contents of the ancestral genomes at all internal vertices of a phylogenetic tree. The strategy is to accumulate a very large number of generalized adjacencies, phylogenetically justified for each ancestor, to produce long ancestral contigs through maximum weight matching. It constructs chromosomes by counting the frequencies of ancestral contig co-occurrences on the extant genomes, clustering these for each ancestor and ordering them. The main objective of this paper is to closely simulate the evolutionary process giving rise to the gene content and order of a set of extant genomes (six distantly related monocots), and to assess to what extent an updated version of RACCROCHE can recover the artificial ancestral genome at the root of the phylogenetic tree relating to the simulated genomes.

The mitochondrial genomes of five spring and groundwater amphipods of the family Crangonyctidae (Crustacea: Amphipoda) from eastern North America

We sequenced the mitochondrial genomes of one spring-dwelling (Crangonyx forbesi) and four groundwater amphipods (Bactrurus brachycaudus, Stygobromus allegheniensis, S. pizzinii, and S. t. potomacus) from eastern North America using a shotgun sequencing approach on an Illumina HiSeq 4000 (Illumina, San Diego, CA). All five mitochondrial genomes encoded 13 protein-coding genes, 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs) representative of subphylum Crustacea. Although the four groundwater species exhibited gene orders nearly identical to the ancestral pancrustacean gene order, the spring-dwelling species, C. forbesi, possessed a transposition of the trnH–nad4–nad4l loci downstream after nad6–cytb–trnS2. Moreover, a long nad5 locus, longer rrnL, and rrnS loci, and unconventional start codons distinguished C. forbesi from the four groundwater amphipods. Overall, our five amphipod mitogenomes add to the increasing publicly available mitogenome resources for amphipods that are not only valuable for studying the evolutionary relationships of this diverse group of crustaceans but for exploring the evolution of mitochondrial genomes in general.

Comparative mitochondrial genome analysis of Eudocima salaminia (Cramer, 1777) (Lepidoptera: Noctuoidea), novel gene rearrangement and phylogenetic relationship within the superfamily Noctuoidea.

The species Eudocima salaminia (Cramer, 1777) commonly known as the fruit-piercing moth belongs to family Erebidae. Its distribution varies from India and across South-east Asia, pacific islands and parts of Australia. The insect is a devastating pest of citrus, longans and lychees. In the present study, complete mitochondrial genome of Eudocima salaminia was sequenced and analyzed using Illumina sequencer. The phylogenetic tree was reconstructed based on nucleotide sequences of 13 PCGs using Maximum likelihood method-General Reversible mitochondrial (mtREV) model. The mitogenome has 15,597 base pairs (bp) in length, comprising of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and A + T-rich region. All protein-coding genes (PCGs) initiate with canonical start codon ATN. The gene order (trnQ-trnI-trnM) of tRNA shows a different rearrangement compared to ancestral insect gene order (trnI-trnQ-trnM). Almost all tRNAs have a typical cloverleaf secondary structure except for trnS1 (AGN) which lacks the dihydrouridine arm. At the beginning of the control region, we observed a conserved polyT", motif "ATTTA" and microsatellite (TA)n element. There are 21 intergenic regions and five overlapping regions ranging from 1 to 73bp and 1 to 8bp, respectively. The phylogenetic relationships based on nucleotide sequences of 13 PCGs using Maximum likelihood method showed the family level relationships as (Notodontidae + (Euteliidae + Noctuidae + (Erebidae + Nolidae))). The present study represents the similarity to phylogenetic analysis of Noctuoidea mitogenome. Moreover, the family Erebidae is the sister to the families of (Euteliidae + Noctuidae + Nolidae).

Complete mitogenome of endemic plum-headed parakeet Psittacula cyanocephala - characterization and phylogenetic analysis.

Psittacula cyanocephala is an endemic parakeet from the Indian sub-continent that is widespread in the illegal bird trade. Previous studies on Psittacula parakeets have highlighted taxonomic ambiguities, warranting studies to resolve the issues. Since the mitochondrial genome provides useful information concerning the species evolution and phylogenetics, we sequenced the complete mitogenome of P. cyanocephala using NGS, validated 38.86% of the mitogenome using Sanger Sequencing and compared it with other available whole mitogenomes of Psittacula. The complete mitogenome of the species was 16814 bp in length with 54.08% AT composition. P. cyanocephala mitogenome comprises of 13 protein-coding genes, 2 rRNAs and 22 tRNAs. P. cyanocephala mitogenome organization was consistent with other Psittacula mitogenomes. Comparative codon usage analysis indicated the role of natural selection on Psittacula mitogenomes. Strong purifying selection pressure was observed maximum on nad1 and nad4l genes. The mitochondrial control region of all Psittacula species displayed the ancestral avian CR gene order. Phylogenetic analyses revealed the Psittacula genus as paraphyletic nature, containing at least 4 groups of species within the same genus, suggesting its taxonomic reconsideration. Our results provide useful information for developing forensic tests to control the illegal trade of the species and scientific basis for phylogenetic revision of the genus Psittacula.

The mitochondrial genome of Telenomus remus (Hymenoptera: Platygastridae)

Telenomus remus Nixon, 1937 is an important parasitoid of lepidopterans. We sequenced the mitochondrial genome of T. remus, 15,500 bp in size, and possessed all 37 typical mitochondrial genes. A few tRNAs show gene arrangements compared with the ancestral gene order, mainly involving in the four tRNA clusters (E-C-Y-Q-I-A, D-K, N-F-S1-R, and M-V). The nucleotide sequences of 13 protein-coding genes of this sequence and another seven species from Platygastridae were used for phylogenetic analysis by MrBayes, with two species from Cynipoidea as an outgroup. The topology demonstrated that T. remus was most closely related to Telenomus sp.

Mitochondrial gene order of the freshwater bryozoan Cristatella mucedo retains ancestral lophotrochozoan features

Bryozoans are aquatic colonial suspension-feeders abundant in many marine and freshwater benthic communities. At the same time, the phylum is under studied on both morphological and molecular levels, and its position on the metazoan tree of life is still disputed. Bryozoa include the exclusively marine Stenolaemata, predominantly marine Gymnolaemata and exclusively freshwater Phylactolaemata. Here we report the mitochondrial genome of the phylactolaemate bryozoan Cristatella mucedo. This species has the largest (21,008 bp) of all currently known bryozoan mitogenomes, containing a typical metazoan gene compendium as well as a number of non-coding regions, three of which are longer than 1500 bp. The trnS1/trnG/nad3 region is presumably duplicated in this species. Comparative analysis of the gene order in C. mucedo and another phylactolaemate bryozoan, Pectinatella magnifica, confirmed their close relationships, and revealed a stronger similarity to mitogenomes of phoronids and other lophotrochozoan species than to marine bryozoans, indicating the ancestral nature of their gene arrangement. We suggest that the ancestral gene order underwent substantial changes in different bryozoan cladesshowing mosaic distribution of conservative gene blocks regardless of their phylogenetic position. Altogether, our results support the early divergence of Phylactolaemata from the rest of Bryozoa.

Comparative Mitogenomic Analysis of Two Cuckoo Bees (Apoidea: Anthophila: Megachilidae) with Phylogenetic Implications.

Simple SummaryMegachilidae plays an important role in natural and agricultural ecosystems. There is uncertainty in the phylogenetic relationship among tribes of Megachilidae. Due to the lack of basic analysis of the mitogenomic structure of the cuckoo bees (cleptoparasitic bees) of the Megachilidae, the risk of insect cleptoparasitism in the Megachilidae is not fully understood. To further provide a new perspective on the phylogenetic relationship of Megachilidae and enrich the basic theory of cleptoparasitic controls, two mitogenomes of cuckoo bees (Coelioxys fenestrata and Euaspis polynesia) were sequenced and analyzed. Different mitogenomic structures and base compositions were found between two cuckoo bees based on comparative analyses of general characteristics of the mitochondrial, noncoding region and gene rearrangement pattern. In addition, the phylogenetic results strongly supported that the tribe-level relationship of Megachilidae was Osmiini + (Anthidiini + Megachilini). Our findings clarified the phylogenetic position among tribes from the mitogenome level so as to provide a further basis to study the evolution of Megachilidae.Bees (Hymenoptera, Apoidea and Anthophila) are distributed worldwide and considered the primary pollinators of angiosperm. Megachilidae is one of the largest families of Anthophila. In this study, two complete mitogenomes of cuckoo bees in Megachilidae, namely Coelioxys fenestrata and Euaspis polynesia, were amplified and sequenced, with a length of 17,004 bp (C. fenestrata) and 17,682 bp (E. polynesia). The obtained results show that 37 mitogenomic genes and one putative control region were conserved within Hymenoptera. Truncated stop codon T was found in the cox3 gene of E. polynesia. The secondary structure of small (rrnS) and large (rrnL) rRNA subunits contained three domains (28 helices) and five domains (44 helices) conserved within Hymenoptera, respectively. Compared with ancestral gene order, gene rearrangement events included local inversion and gene shuffling. In order to reveal the phylogenetic position of cuckoo bees, we performed phylogenetic analysis. The results supported that all families of Anthophila were monophyletic, the tribe-level relationship of Megachilidae was Osmiini + (Anthidiini + Megachilini) and Coelioxys fenestrata was clustered to the Megachile genus, which was more closely related to Megachile sculpturalis and Megachile strupigera than Euaspis polynesia.