Nucleotide Composition Bias Research Articles

Sequencing of the Complete Mitochondrial Genome of the Big Brown Mactra Clam, Mactra grandis (Venerida: Mactridae).

Mitochondrial genomes are playing an increasingly important role in molluscan taxonomy, germplasm, and evolution studies. The first complete mitochondrial genome of the commercial big brown mactra clam, Mactra grandis, was characterized using Illumina next-generation sequencing in this study. The 17,289 bp circular genome has a typical gene organization of 13 protein-coding genes (PCGs), 2 rRNAs, and 22 tRNAs, with an obvious (A + T)-bias of 64.54%. All PCGs exhibited a homogeneous bias in nucleotide composition with a (A + T)-bias, a positive GC skew, and a negative AT skew. Results of phylogenetic analysis showed that Mactra grandis was most closely related to Mactra cygnus. The functional gene arrangement of the two species was identical but different from other Mactra species. The congeneric relationships among Mactra species were demonstrated by genetic distance analysis. Additionally, the selective pressure analysis suggested that cox1 was highly efficient for discriminating closely related species in genus Mactra, while nad2 was the most appropriate marker for population genetic analysis.

Chargaff's second parity rule lies at the origin of additive genetic interactions in quantitative traits to make omnigenic selection possible.

Francis Crick's central dogma provides a residue-by-residue mechanistic explanation of the flow of genetic information in living systems. However, this principle may not be sufficient for explaining how random mutations cause continuous variation of quantitative highly polygenic complex traits. Chargaff's second parity rule (CSPR), also referred to as intrastrand DNA symmetry, defined as near-exact equalities G ≈ C and A ≈ T within a single DNA strand, is a statistical property of cellular genomes. The phenomenon of intrastrand DNA symmetry was discovered more than 50 years ago; at present, it remains unclear what its biological role is, what the mechanisms are that force cellular genomes to comply strictly with CSPR, and why genomes of certain noncellular organisms have broken intrastrand DNA symmetry. The present work is aimed at studying a possible link between intrastrand DNA symmetry and the origin of genetic interactions in quantitative traits. Computational analysis of single-nucleotide polymorphisms in human and mouse populations and of nucleotide composition biases at different codon positions in bacterial and human proteomes. The analysis of mutation spectra inferred from single-nucleotide polymorphisms observed in murine and human populations revealed near-exact equalities of numbers of reverse complementary mutations, indicating that random genetic variations obey CSPR. Furthermore, nucleotide compositions of coding sequences proved to be statistically interwoven via CSPR because pyrimidine bias at the 3rd codon position compensates purine bias at the 1st and 2nd positions. According to Fisher's infinitesimal model, we propose that accumulation of reverse complementary mutations results in a continuous phenotypic variation due to small additive effects of statistically interwoven genetic variations. Therefore, additive genetic interactions can be inferred as a statistical entanglement of nucleotide compositions of separate genetic loci. CSPR challenges the neutral theory of molecular evolution-because all random mutations participate in variation of a trait-and provides an alternative solution to Haldane's dilemma by making a gene function diffuse. We propose that CSPR is symmetry of Fisher's infinitesimal model and that genetic information can be transferred in an implicit contactless manner.

Different Patterns of Codon Usage and Amino Acid Composition across Primate Lentiviruses.

A common feature of the mammalian Lentiviruses (family Retroviridae) is an RNA genome that contains an extremely high frequency of adenine (31.7-38.2%) while being extremely poor in cytosine (13.9-21.2%). Such a biased nucleotide composition has implications for codon usage, causing a striking difference between the frequency of synonymous codons in Lentiviruses and that in their hosts. To test whether primate Lentiviruses present differences in codon and amino acid composition, we assembled a dataset of genome sequences that includes SIV species infecting Old-World monkeys and African apes, HIV-2, and the four groups of HIV-1. Using principal component analysis, we found that HIV-1 shows a significant enrichment in adenine plus thymine in the third synonymous codon position and in adenine and guanine in the first and second nonsynonymous codon positions. Similarly, we observed an enrichment in adenine and in guanine in nonsynonymous first and second codon positions, which affects the amino acid composition of the proteins Gag, Pol, Vif, Vpr, Tat, Rev, Env, and Nef. This result suggests an effect of natural selection in shaping codon usage. Under the hypothesis that the use of synonyms in HIV-1 could reflect adaptation to that of genes expressed in specific cell types, we found a highly significant correlation between codon usage in HIV-1 and monocytes, which was remarkably higher than that with B and T lymphocytes. This finding is in line with the notion that monocytes represent an HIV-1 reservoir in infected patients, and it could help understand how this reservoir is established and maintained.

Extreme plastomes in holoparasitic Balanophoraceae are not the norm

BackgroundBalanophoraceae plastomes are known for their highly condensed and re-arranged nature alongside the most extreme nucleotide compositional bias known to date, culminating in two independent reconfigurations of their genetic code. Currently, a large portion of the Balanophoraceae diversity remains unexplored, hindering, among others, evolutionary pattern recognition. Here, we explored newly sequenced plastomes of Sarcophyte sanguinea and Thonningia sanguinea. The reconstructed plastomes were analyzed using various methods of comparative genomics based on a representative taxon sampling.ResultsSarcophyte, recovered sister to the other sampled Balanophoraceae s. str., has plastomes up to 50% larger than those currently published. Its gene set contains five genes lost in any other species, including matK. Five cis-spliced introns are maintained. In contrast, the Thonningia plastome is similarly reduced to published Balanophoraceae and retains only a single cis-spliced intron. Its protein-coding genes show a more biased codon usage compared to Sarcophyte, with an accumulation of in-frame TAG stop codons. Structural plastome comparison revealed multiple, previously unknown, structural rearrangements within Balanophoraceae.ConclusionsFor the “minimal plastomes” of Thonningia, we propose a genetic code change identical to sister genus Balanophora. Sarcophyte however differs drastically from our current understanding on Balanophoraceae plastomes. With a less-extreme nucleotide composition, there is no evidence for an altered genetic code. Using comparative genomics, we identified a hotspot for plastome reconfiguration in Balanophoraceae. Based on previously published and newly identified structural reconfigurations, we propose an updated model of evolutionary plastome trajectories for Balanophoraceae, illustrating a much greater plastome diversity than previously known.

Effect of Different Types of Sequence Data on Palaeognath Phylogeny.

Palaeognathae consists of five groups of extant species: flighted tinamous (1) and four flightless groups: kiwi (2), cassowaries and emu (3), rheas (4), and ostriches (5). Molecular studies supported the groupings of extinct moas with tinamous and elephant birds with kiwi as well as ostriches as the group that diverged first among the five groups. However, phylogenetic relationships among the five groups are still controversial. Previous studies showed extensive heterogeneity in estimated gene tree topologies from conserved nonexonic elements, introns, and ultraconserved elements. Using the noncoding loci together with protein-coding loci, this study investigated the factors that affected gene tree estimation error and the relationships among the five groups. Using closely related ostrich rather than distantly related chicken as the outgroup, concatenated and gene tree-based approaches supported rheas as the group that diverged first among groups (1)-(4). Whereas gene tree estimation error increased using loci with low sequence divergence and short length, topological bias in estimated trees occurred using loci with high sequence divergence and/or nucleotide composition bias and heterogeneity, which more occurred in trees estimated from coding loci than noncoding loci. Regarding the relationships of (1)-(4), the site patterns by parsimony criterion appeared less susceptible to the bias than tree construction assuming stationary time-homogeneous model and suggested the clustering of kiwi and cassowaries and emu the most likely with ∼40% support rather than the clustering of kiwi and rheas and that of kiwi and tinamous with 30% support each.

The complete mitochondrial genome of Peleteria iavana (Diptera, Tachinidae) in Guizhou, China, and its phylogenetic placement

The mitochondrial genome of the tachinid fly Peleteria iavana (Wiedemann, 1819), which belongs to the family Tachinidae, was obtained for the first time using high-throughput sequencing techniques. The complete mitochondrial genome is 15,697 bp in size and consists of 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNAs and a non-coding control region. The nucleotide composition biases A and T, the overall A + T percentage is up to 78.9% of the entire mitogenome. A phylogenetic analysis of 30 species within the family Tachinidae suggested that P. iavana is most closely related to (Janthinomyia sp.+Lydina aenea). The P. iavana mitochondrial genome will be a fundamental resource for understanding the molecular phylogenetic relationships of the species-rich subfamily Tachininae of Tachinidae.

Complete mitochondrial genome of Cylindromyia (Calocyptera) intermedia from Guiyang, China, and phylogeny of Phasiinae (Diptera: Tachinidae)

The mitochondrial genome of Cylindromyia (Calocyptera) intermedia (Meigen, 1824) was sequenced and assembled through high-throughput sequencing techniques and reference-based assembly methods. The mitochondrial genome is 15,114 bp in total, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 noncoding control region. The nucleotide composition biases toward A and T; the overall A + T content are up to 76.9% of the entire mitogenome. The result of phylogenetic analysis suggested a close relationship between C. intermedia and subfamily Dexiinae.

Mitochondrial Genomes Provide New Phylogenetic and Evolutionary Insights into Psilidae (Diptera: Brachycera)

Simple SummaryMembers of Psilidae are commonly known as rust flies. They constitute the largest family of Diopsoidea, with about 340 species being known worldwide. Several species of Psilidae show agricultural significance due to their severe damage of root crops. However, the systematic relationships and intrafamilial classification of Psilidae remained controversial. To provide further information on the phylogeny and evolution of Psilidae, mitogenomes of 6 psilid species are sequenced. Comparative analyses of the 6 newly obtained mitogenomes are conducted. Phylogenetic analyses based on the 6 psilid mitogenomes and public data are carried out, resulting in a monophyletic Psilidae and a non-monophyletic Diopsoidea. The sister relationship between Psilinae and Chylizinae is highly supported. This study provides several new insights into the phylogeny and evolution of Psilidae.Psilidae (Diptera: Brachycera) is a moderate-sized family currently placed in the superfamily Diopsoidea and contains some destructive agricultural and forestry pests. The systematic position and intrafamilial classification of rust flies are in need of further study, and the available molecular data of Psilidae are still limited. In this study, we present the mitochondrial genomes of 6 Psilidae species (Chamaepsila testudinaria Wang and Yang, Chyliza bambusae Wang and Yang, Chy. chikuni Wang, Loxocera lunata Wang and Yang, L. planivena Wang and Yang and L. sinica Wang and Yang). Comparative analyses show a conserved genome structure, in terms of gene composition and arrangement, and a highly Adenine plus Thymine biased nucleotide composition of the 6 psilid mitogenomes. Mitochondrial evolutionary rates vary among the 6 species, with species of Chylizinae exhibiting a slower average rate than species of Psilinae. The length, the nucleotide composition, and the copy number of repeat units of the control region are variable among the 6 species, which may offer useful information for phylogenetic and evolutionary studies of Psilidae. Phylogenetic analyses based on 4 mitogenomic datasets (AA, PCG, PCG12RNA, and PCGRNA) support the monophyly of Psilidae, and the sister relationship between Chylizinae and Psilinae, while Diopsoidea is suggested to be non-monophyletic. Our study enlightens the future application of mitogenomic data in the phylogenetic and evolutionary studies of Psilidae, based on denser taxon sampling.

Enabling genomic island prediction and comparison in multiple genomes to investigate bacterial evolution and outbreaks.

Outbreaks of virulent and/or drug-resistant bacteria have a significant impact on human health and major economic consequences. Genomic islands (GIs; defined as clusters of genes of probable horizontal origin) are of high interest because they disproportionately encode virulence factors, some antimicrobial-resistance (AMR) genes, and other adaptations of medical or environmental interest. While microbial genome sequencing has become rapid and inexpensive, current computational methods for GI analysis are not amenable for rapid, accurate, user-friendly and scalable comparative analysis of sets of related genomes. To help fill this gap, we have developed IslandCompare, an open-source computational pipeline for GI prediction and comparison across several to hundreds of bacterial genomes. A dynamic and interactive visualization strategy displays a bacterial core-genome phylogeny, with bacterial genomes linearly displayed at the phylogenetic tree leaves. Genomes are overlaid with GI predictions and AMR determinants from the Comprehensive Antibiotic Resistance Database (CARD), and regions of similarity between the genomes are also displayed. GI predictions are performed using Sigi-HMM and IslandPath-DIMOB, the two most precise GI prediction tools based on nucleotide composition biases, as well as a novel blast-based consistency step to improve cross-genome prediction consistency. GIs across genomes sharing sequence similarity are grouped into clusters, further aiding comparative analysis and visualization of acquisition and loss of mobile GIs in specific sub-clades. IslandCompare is an open-source software that is containerized for local use, plus available via a user-friendly, web-based interface to allow direct use by bioinformaticians, biologists and clinicians (at https://islandcompare.ca).

Comparative Genome Analysis of 'Candidatus Phytoplasma luffae' Reveals the Influential Roles of Potential Mobile Units in Phytoplasma Evolution.

Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of ‘Candidatus Phytoplasma luffae’ strain NCHU2019 that is associated with witches’ broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has a remarkably repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ∼25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for subfunctionalization or neofunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

The complete mitochondrial genome of Metidiocerus impressifrons (Kirschbaum, 1868) (Hemiptera: Cicadellidae: Idiocerinae)

We determined the complete mitochondrial genome (mitogenome) of the leafhopper Metidiocerus impressifrons by next-generation sequencing. The mitogenome sequence was 16,426 bp in length and consists of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region. Moreover, the nucleotide composition biases toward A and T, which together made up 78.2% of the entirety. The complete mitochondrial genomes of Metidiocerus impressifrons and other 27 species were used for phylogenetic analysis using the Bayesian method. The above results would facilitate our understanding of the evolution of Idiocerinae mitochondrial genome.

Dinucleotide tag-based parallel reporter gene assay method enables efficient identification of regulatory mutations.

The causal single nucleotide polymorphisms (SNPs) leading to increased cancer predisposition mainly function as gene regulatory elements, the evaluation of which largely relies on the parallel reporter gene assay system. However, the common DNA barcodes used in parallel reporter gene assay systems typically because nucleotide composition bias, and many barcodes must be allocated for each sequence to reduce the bias effect. Here, a versatile dinucleotide-tag reporter system (DiR) that enables parallel analysis of regulatory elements with minimized bias based on next-generation sequencing is described. The DiR system is more robust than the classical luciferase assay method, particularly for the investigation of moderate-level regulatory elements. The authors applied the DiR-seq assay in the functional evaluation of SNPs with prostate cancer risk and nominated two and six regulatory SNPs in PC-3 and LNCaP cells, respectively. The DiR system has great potential to advance the functional study of SNPs associated with polygenic disease risks.

Nucleotide composition bias of rDNA sequences as a source of phylogenetic artifacts in Basidiomycota—a case of a new lineage of a uredinicolous Ramularia-like anamorph with affinities to Ustilaginomycotina

Nucleotide composition bias of rDNA sequences as a source of phylogenetic artifacts in Basidiomycota—a case of a new lineage of a uredinicolous Ramularia-like anamorph with affinities to Ustilaginomycotina

The mitochondrial genome of Calohilara tibetensis Ding, He, Lin, and Yang, 2020 (Diptera: Empididae)

The dance fly Calohilara tibetensis Ding, He, Lin and Yang, 2020 belongs to the subfamily Empidinae of Empididae. The mitochondrial genome of C. tibetensis was sequenced as the new representative of the subfamily Empidinae. The nearly complete mitogenome was 15,354 bp, consisting of 13 protein-coding genes, two rRNAs, and 22 tRNAs. All genes have the similar locations and strands with that of other published species of Empididae. The nucleotide composition biases toward A and T is 77.4% of the entirety. All PCGs start with ATN codons except COI and NAD1, and end with TAA or incomplete stop codon. Bayesian inference (BI) analysis strongly supported the monophyly of both Empididae and Dolichopodidae and the monophyly of subfamily Empidinae. It suggested that Calohilara is the sister group of Hilara.

Prediction of two novel overlapping ORFs in the genome of SARS-CoV-2

Six candidate overlapping genes have been detected in SARS-CoV-2, yet current methods struggle to detect overlapping genes that recently originated. However, such genes might encode proteins beneficial to the virus, and provide a model system to understand gene birth. To complement existing detection methods, I first demonstrated that selection pressure to avoid stop codons in alternative reading frames is a driving force in the origin and retention of overlapping genes. I then built a detection method, CodScr, based on this selection pressure. Finally, I combined CodScr with methods that detect other properties of overlapping genes, such as a biased nucleotide and amino acid composition. I detected two novel ORFs (ORF-Sh and ORF-Mh), overlapping the spike and membrane genes respectively, which are under selection pressure and may be beneficial to SARS-CoV-2. ORF-Sh and ORF-Mh are present, as ORF uninterrupted by stop codons, in 100% and 95% of the SARS-CoV-2 genomes, respectively.

The mitochondrial genome of Lichtwardtia dentalis Zhang, Masunaga et Yang, 2009 (Diptera: Dolichopodidae)

The long-legged fly Lichtwardtia dentalis Zhang, Masunaga et Yang, 2009 belongs to the subfamily Dolichopodinae of Dolichopodidae. The newly sequenced mitogenome of L. dentalis is a new representative of the subfamily. The nearly complete mitogenome is 15,124 bp in length, consisting of 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), and 22 transfer RNA genes (tRNAs). All genes have similar locations and encoding directions with that of other published mitogenomes of Dolichopodidae. The nucleotide composition biases toward A and T with the overall A + T % is 73.9%. All protein-coding genes initiate with standard start codon ATN except COI and NAD1, and TAA/TAG are conventionally used as stop codons. All tRNAs, ranging from 62 to 71 bp, have a clover-leaf structure. Based on the result of the phylogenetic analysis, Dolichopodidae and Empididae were monophyletic, and the relationships among subfamilies of Dolichopodidae were Diaphorinae + (Peloropeodinae + (Xanthochlorinae + (Medeterinae + Dolichopodinae))). The monophyly of the subfamily Dolichopodinae and the sister relationship between Dolichopus and Lichtwardtia were also strongly supported.

Mitochondrial Genomic Landscape: A Portrait of the Mitochondrial Genome 40 Years after the First Complete Sequence.

Notwithstanding the initial claims of general conservation, mitochondrial genomes are a largely heterogeneous set of organellar chromosomes which displays a bewildering diversity in terms of structure, architecture, gene content, and functionality. The mitochondrial genome is typically described as a single chromosome, yet many examples of multipartite genomes have been found (for example, among sponges and diplonemeans); the mitochondrial genome is typically depicted as circular, yet many linear genomes are known (for example, among jellyfish, alveolates, and apicomplexans); the chromosome is normally said to be “small”, yet there is a huge variation between the smallest and the largest known genomes (found, for example, in ctenophores and vascular plants, respectively); even the gene content is highly unconserved, ranging from the 13 oxidative phosphorylation-related enzymatic subunits encoded by animal mitochondria to the wider set of mitochondrial genes found in jakobids. In the present paper, we compile and describe a large database of 27,873 mitochondrial genomes currently available in GenBank, encompassing the whole eukaryotic domain. We discuss the major features of mitochondrial molecular diversity, with special reference to nucleotide composition and compositional biases; moreover, the database is made publicly available for future analyses on the MoZoo Lab GitHub page.

GRINS: Genetic elements that recode assembly-line polyketide synthases and accelerate their diversification

Assembly-line polyketide synthases (PKSs) are large and complex enzymatic machineries with a multimodular architecture, typically encoded in bacterial genomes by biosynthetic gene clusters. Their modularity has led to an astounding diversity of biosynthesized molecules, many with medical relevance. Thus, understanding the mechanisms that drive PKS evolution is fundamental for both functional prediction of natural PKSs as well as for the engineering of novel PKSs. Here, we describe a repetitive genetic element in assembly-line PKS genes which appears to play a role in accelerating the diversification of closely related biosynthetic clusters. We named this element GRINS: genetic repeats of intense nucleotide skews. GRINS appear to recode PKS protein regions with a biased nucleotide composition and to promote gene conversion. GRINS are present in a large number of assembly-line PKS gene clusters and are particularly widespread in the actinobacterial genus Streptomyces While the molecular mechanisms associated with GRINS appearance, dissemination, and maintenance are unknown, the presence of GRINS in a broad range of bacterial phyla and gene families indicates that these genetic elements could play a fundamental role in protein evolution.

The complete mitochondrial genome of capillariid nematodes (Eucoleus annulatus): A novel gene arrangement and phylogenetic implications

Capillariid nematode is a group of endoparasites of vertebrates with a complex taxonomy, causing significant economic losses to poultry industry. The taxonomic status of the genus Eucoleus remained controversial for several decades. Mitochondrial (mt) DNA provides useful genetic markers for accurate identification of species, but complete mt genome sequences have been lacking for any Capillariid nematodes. In the present study, we decoded the complete mt genome of E. annulatus and examined its phylogenetic relationship with selected members of the class Enoplea nematodes. The circular mt genome of E. annulatus was 14,118 bp, encoded 37 genes with a single non-coding region and showed substantial gene rearrangements (especially tRNA genes) compared to other nematodes studied to date. The complete mt genome of E. annulatus showed a clear A + T bias in nucleotide composition. The number of A (5404) was approximately equal to T (5405) and the GC-skew was negative on average (−0.073). Phylogenetic analyses based on 18S rDNA placed Eucoleus spp. well apart from each other and supported the proposal that Eucoleus and Capillaria are two distinct genera. Similarly, Bayesian inference (BI) and Maximum likelihood (ML) phylogenies based on mtDNA sequences revealed that the family Capillariidae is more closely related to the family Trichuridae than to the family Trichinellidae. This is the first report of the complete mt genome of capillariid nematodes, and it will provide additional genetic markers for studying the molecular epidemiology, population genetics and systematics of capillariid nematodes and should have implications for the molecular diagnosis, prevention, and control of capillariosis in animals.

Plastomes in the holoparasitic family Balanophoraceae: Extremely high AT content, severe gene content reduction, and two independent genetic code changes

The transition to a heterotrophic lifestyle in angiosperms is characterized by convergent evolutionary changes. Plastid genome remodeling includes dramatic functional and physical reductions with the highest degrees observed in fully heterotrophic plants. Genes related to photosynthesis are generally absent or pseudogenized, while a few genes related to other metabolic processes that take place within the plastid are almost invariably maintained. The family Balanophoraceae consists of root holoparasites that present reduced plastid genomes with an extraordinarily elevated AT content and the single genetic code change ever documented in land plant plastomes (the stop codon TAG now codes for tryptophan). Here, we studied the plastomes of Lophophytum leandri and Ombrophytum subterraneum (Balanophoraceae) that showed the remarkable absence of the gene trnE, a highly biased nucleotide composition, and an independent genetic code change (the standard stop codon TGA codes for tryptophan). This is the second genetic code change identified in land plant plastomes. Analysis of the transcriptome of Lophophytum indicated that the entire C5 pathway typical of plants is conserved despite the lack of trnE in its plastome. A hypothetical model of plastome evolution in the Balanophoraceae is presented.