Codon usage bias: causative factors,quantification methods and genome‐wide patterns: with emphasis on insect genomes

Codon usage bias refers to the phenomenon where specific codons are used more often than other synonymous codons during translation of genes, the extent of which varies within and among species. Molecular evolutionary investigations suggest that codon bias is manifested as a result of balance between mutational and translational selection of such genes and that this phenomenon is widespread across species and may contribute to genome evolution in a significant manner. With the advent of whole‐genome sequencing of numerous species, both prokaryotes and eukaryotes, genome‐wide patterns of codon bias are emerging in different organisms. Various factors such as expression level, GC content, recombination rates, RNA stability, codon position, gene length and others (including environmental stress and population size) can influence codon usage bias within and among species. Moreover, there has been a continuous quest towards developing new concepts and tools to measure the extent of codon usage bias of genes. In this review, we outline the fundamental concepts of evolution of the genetic code, discuss various factors that may influence biased usage of synonymous codons and then outline different principles and methods of measurement of codon usage bias. Finally, we discuss selected studies performed using whole‐genome sequences of different insect species to show how codon bias patterns vary within and among genomes. We conclude with generalized remarks on specific emerging aspects of codon bias studies and highlight the recent explosion of genome‐sequencing efforts on arthropods (such as twelve Drosophila species, species of ants, honeybee, Nasonia and Anopheles mosquitoes as well as the recent launch of a genome‐sequencing project involving 5000 insects and other arthropods) that may help us to understand better the evolution of codon bias and its biological significance.     

Evolutionary Patterns of Codon Usage in the Chloroplast Gene rbcL

In this study we reconstruct the evolution of codon usage bias in the chloroplast gene rbcL using a phylogeny of 92 green-plant taxa. We employ a measure of codon usage bias that accounts for chloroplast genomic nucleotide content, as an attempt to limit plausible explanations for patterns of codon bias evolution to selection- or drift-based processes. This measure uses maximum likelihood-ratio tests to compare the performance of two models, one in which a single codon is overrepresented and one in which two codons are overrepresented. The measure allowed us to analyze both the extent of bias in each lineage and the evolution of codon choice across the phylogeny. Despite predictions based primarily on the low G+C content of the chloroplast and the high functional importance of rbcL, we found large differences in the extent of bias, suggesting differential molecular selection that is clade specific. The seed plants and simple leafy liverworts each independently derived a low level of bias in rbcL, perhaps indicating relaxed selectional constraint on molecular changes in the gene. Overrepresentation of a single codon was typically plesiomorphic, and transitions to overrepresentation of two codons occurred commonly across the phylogeny, possibly indicating biochemical selection. The total codon bias in each taxon, when regressed against the total bias of each amino acid, suggested that twofold amino acids play a strong role in inflating the level of codon usage bias in rbcL, despite the fact that twofolds compose a minority of residues in this gene. Those amino acids that contributed most to the total codon usage bias of each taxon are known through amino acid knockout and replacement to be of high functional importance. This suggests that codon usage bias may be constrained by particular amino acids and, thus, may serve as a good predictor of what residues are most important for protein fitness. Present address (Joshua T. Herbeck): JBP Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA 02543, USA     

Causal analysis of CpG suppression in the Mycoplasma genome

Some bacterial genomes are known to have low CpG dinucleotide frequencies. While their causes are not clearly understood, the frequency of CpG is suppressed significantly in the genome of Mycoplasma genitalium, but not in that of Mycoplasma pneumoniae. We compared orthologous gene pairs of the two closely related species to analyze CpG substitution patterns between these two genomes. We also divided genome sequences into three regions: protein-coding, noncoding, and RNA-coding, and obtained the CpG frequencies for each region for each organism. It was found that the observed/expected ratio of CpG dinucleotides is low in both the protein-coding and noncoding regions; while that ratio is in the normal range in the RNA-coding region. Our results indicate that CpG suppression of the Mycoplasma genome is not caused by (1) biased usage amino acid; (2) biased usage of synonymous codon; or (3) methylation effects by the CpG methyltransferase in the genomes of their hosts. Instead, we consider it likely that a certain global pressure, such as genome-wide pressure for the advantages of DNA stability or replication, has the effect of decreasing CpG over the entire genome, which, in turn, resulted in the biased codon usage.     

Elicitin genes in <Emphasis Type="Italic">Phytophthora infestans</Emphasis> are clustered and interspersed with various transposon-like elements

Sequencing and annotation of a contiguous stretch of genomic DNA (112.3 kb) from the oomycete plant pathogen Phytophthora infestans revealed the order, spacing and genomic context of four members of the elicitin (inf) gene family. Analysis of the GC content at the third codon position (GC3) of six genes encoded in the region, and a set of randomly selected coding regions as well as random genomic regions, showed that a high GC3 value is a general feature of Phytophthora genes that can be exploited to optimize gene prediction programs for Phytophthora species. At least one-third of the annotated 112.3-kb P. infestans sequence consisted of transposons or transposon-like elements. The most prominent were four Tc3/gypsy and Tc1/copia type retrotransposons and three DNA transposons that belong to the Tc1/mariner, Pogo and PiggyBac groups, respectively. Comparative analysis of other available genomic sequences suggests that transposable elements are highly heterogeneous and ubiquitous in the P. infestans genome.Electronic Supplementary Material Supplementary material is available for this article at     

沙枣叶绿体全基因组序列及其使用密码子偏性分析

该研究以2株野生沙枣(Elaeagnus angustifolia Linn.)嫩枝经温室水培后的嫩叶为材料,采用CTAB法分别提取总DNA,并利用第二代测序技术进行总DNA从头测序,组装后得到2株沙枣叶绿体基因组全序列,并详细分析了其蛋白质编码基因密码子使用的偏好性及其原因,为沙枣叶绿体基因工程和分子系统进化等研究奠定基础。结果显示:(1)组装得到沙枣叶绿体基因组序列全长150 546 bp,由长度为81 113 bp的长单拷贝(LSC)区域和25 494 bp的短单拷贝(SSC)区域,以及1对分隔开它们的长18 445 bp的反向重复序列(IRS)组成;注释共得到132个基因,包括86个蛋白编码基因、38个tRNA基因和8个rRNA基因。(2)沙枣叶绿体基因组蛋白编码基因密码子的第三位碱基GC含量(GC_3)为28.47%,明显低于整个叶绿体基因组GC含量(37%),也低于第一位(GC_1)和第二位(GC_2)碱基的GC含量,说明密码子对AT碱基结尾有偏好性;其中, UCU、CCU、UGU、GCU、CUU、GAU、UCA和UAA为最优密码子。(3)同义密码子相对使用频率(RSCU)分析发现,影响密码子使用模式的因素并不单一,密码子的偏好性受到突变、选择及其他因素的共同影响,并且自然选择表达引起的序列差异比突变对密码子偏好性的影响要显著;中性绘图分析、有效密码子数(ENC-plot)分析和奇偶偏好性(PR2-plot)分析表明,沙枣叶绿体基因组使用密码子的偏性受选择的影响更大。(4)通过最大似然法、最大简约法和贝叶斯方法对胡颓子科6个物种和1个枣的叶绿体基因序列构建系统发育树,与它们使用密码子偏性聚类的结果一致,表明叶绿体基因组使用密码子偏性与物种的亲缘关系相关。     

新型水果空心泡叶绿体基因组特征及其系统发育分析北大核心CSCD

为探究空心泡(Rubus rosaefolius)叶绿体基因组特征,本研究以空心泡为试验材料,采用Illumina NovaSeq平台进行高通量测序,获得空心泡完整的叶绿体基因组序列,并进行空心泡叶绿体基因序列特征和系统发育分析。结果表明:空心泡的完整叶绿体基因组总长度为155650 bp,具有典型的四分体结构,包括2个反向重复序列(各25748 bp)、1个大拷贝区(85443 bp)、1个小拷贝区(18711 bp)。空心泡叶绿体全基因组共鉴定出131个基因,包括86个蛋白质编码基因、37个tRNA基因和8个rRNA基因,全基因组的GC含量为36.9%。空心泡叶绿体基因组包含47个散在重复序列、72个简单重复序列(simple sequence repeating,SSR)位点,密码子偏好性为亮氨酸密码子,偏好使用A/U结尾的密码子。系统发育分析表明,空心泡与小叶悬钩子(Rubus taiwanicola)亲缘关系最近,其次是能高悬钩子(Rubus rubroangustifolius)和腺萼悬钩子(Rubus glandulosopunctatus)。空心泡的叶绿体基因组特征及其系统发育分析,为空心泡的遗传多样性研究和叶绿体开发利用提供理论依据。     

Codon Usage and Selection on Proteins

Selection pressures on proteins are usually measured by comparing homologous nucleotide sequences (Zuckerkandl and Pauling 1965). Recently we introduced a novel method, termed volatility, to estimate selection pressures on proteins on the basis of their synonymous codon usage (Plotkin and Dushoff 2003; Plotkin et al. 2004). Here we provide a theoretical foundation for this approach. Under the Fisher-Wright model, we derive the expected frequencies of synonymous codons as a function of the strength of selection on amino acids, the mutation rate, and the effective population size. We analyze the conditions under which we can expect to draw inferences from biased codon usage, and we estimate the time scales required to establish and maintain such a signal. We find that synonymous codon usage can reliably distinguish between negative selection and neutrality only for organisms, such as some microbes, that experience large effective population sizes or periods of elevated mutation rates. The power of volatility to detect positive selection is also modest—requiring approximately 100 selected sites—but it depends less strongly on population size. We show that phenomena such as transient hyper-mutators can improve the power of volatility to detect selection, even when the neutral site heterozygosity is low. We also discuss several confounding factors, neglected by the Fisher-Wright model, that may limit the applicability of volatility in practice. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Lauren Meyers]     

Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species

A detailed comparison was made of codon usage of chloroplast genes with their host (nuclear) genes in the four angiosperm speciesOryza sativa, Zea mays, Triticum aestivum andArabidopsis thaliana. The average GC content of the entire genes, and at the three codon positions individually, was higher in nuclear than in chloroplast genes, suggesting different genomic organization and mutation pressures in nuclear and chloroplast genes. The results of Nc-plots and neutrality plots suggested that nucleotide compositional constraint had a large contribution to codon usage bias of nuclear genes inO. sativa, Z. mays, andT. aestivum, whereas natural selection was likely to be playing a large role in codon usage bias in chloroplast genomes. Correspondence analysis and chi-test showed that regardless of the genomic environment (species) of the host, the codon usage pattern of chloroplast genes differed from nuclear genes of their host species by their AU-richness. All the chloroplast genomes have predominantly A- and/or U-ending codons, whereas nuclear genomes have G-, C- or U-ending codons as their optimal codons. These findings suggest that the chloroplast genome might display particular characteristics of codon usage that are different from its host nuclear genome. However, one feature common to both chloroplast and nuclear genomes in this study was that pyrimidines were found more frequently than purines at the synonymous codon position of optimal codons.     

The Genomic Signature of Human Rhinoviruses A,B and C

Human rhinoviruses are single stranded positive sense RNA viruses that are presented in more than 50% of acute upper respiratory tract infections. Despite extensive studies on the genetic diversity of the virus, little is known about the forces driving it. In order to explain this diversity, many research groups have focused on protein sequence requirements for viable, functional and transmissible virus but have missed out an important aspect of viral evolution such as the genomic ontology of the virus. This study presents for the first time the genomic signature of 111 fully sequenced HRV strains from all three groups HRV-A, HRV-B and HRV-C. We observed an HRV genome tendency to eliminate CpG and UpA dinucleotides, coupling with over-representation of UpG and CpA. We propose a specific mechanism which describes how rapid changes in the HRV genomic sequence can take place under the strict control of conservation of the polypeptide backbone. Moreover, the distribution of the observed under- and over-represented dinucleotides along the HRV genome is presented. Distance matrice tables based on CpG and UpA odds ratios were constructed and viewed as heatmaps and distance trees. None of the suppressions can be attributed to codon usage or in RNA secondary structure requirements. Since viral recognition is dependent on RNA motifs rich in CpG and UpA, it is possible that the overall described genome evolution mechanism acts in order to protect the virus from host recognition.     

Factors influencing synonymous codon and amino acid usage biases in Mimivirus

Synonymous codon and amino acid usage biases have been investigated in 903 Mimivirus protein-coding genes in order to understand the architecture and evolution of Mimivirus genome. As expected for an AT-rich genome, third codon positions of the synonymous codons of Mimivirus carry mostly A or T bases. It was found that codon usage bias in Mimivirus genes is dictated both by mutational pressure and translational selection. Evidences show that four factors such as mean molecular weight (MMW), hydropathy, aromaticity and cysteine content are mostly responsible for the variation of amino acid usage in Mimivirus proteins. Based on our observation, we suggest that genes involved in translation, DNA repair, protein folding, etc., have been laterally transferred to Mimivirus a long ago from living organism and with time these genes acquire the codon usage pattern of other Mimivirus genes under selection pressure.     

伪狂犬病病毒基因编码区碱基组成与密码子使用偏差

由于伪狂犬病病毒(PRV)中G C含量高达74％,至今尚没有一个毒株完成全基因组测序。对已知的68个PRV基因编码区序列碱基组成及密码子使用现象进行了统计分析,结果发现PRV基因中存在非常强的密码子使用偏差。所有68个PRV基因编码区密码子第三位总的G C含量为96．24％,其中UL48基因高达99．52％。PRV基因偏向于使用富含GC的密码子,特别是以C或G结尾的密码子。此外,还发现PRV中G C含量变化较大的UL48、UL40、UL14和IE180等基因附近正好与已知的PRV基因组复制起始区相对应。根据基因功能将PRV基因分为6类进行分析发现,基因功能相同或相近的基因其密码子使用模式相似,其中调节基因的同义密码子相对使用度(RSCU)与其他基因有显著差异,在调节基因中以C结尾的密码子的RSCU值远大于其他同义密码子。最后,对PRV基因氨基酸组成差异进行多元分析,发现不同功能的PRV基因在对应分析图上分布不同,表明PRV基因密码子使用模式可能与基因功能相关。     

Development of a BAC library for yellow-poplar (Liriodendron tulipifera) and the identification of genes associated with flower development and lignin biosynthesis

Liriodendron tulipifera L., a member of the Magnoliaceae, occupies an important phylogenetic position as a basal angiosperm that has retained numerous putatively ancestral morphological characters, and thus has often been used in studies of the evolution of flowering plants and of specific gene families. However, genomic resources for these early branching angiosperm lineages are very limited. In this study, we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from L. tulipifera. Flow cytometry estimates that this nuclear genome is approximately 1,802 Mbp per haploid genome (±16 SD). The BAC library contains 73,728 clones, a 4.8-fold genome coverage, with an average insert size of 117 kb, a chloroplast DNA content of 0.2%, and little to no bacterial sequences nor empty vector content clones. As a test of the utility of this BAC library, we screened the library with six single/low-copy genic probes. We obtained at least two positive clones for each gene and confirmed the clones by DNA sequencing. A total of 182 paired end sequences were obtained from 96 of the BAC clones. Using BLAST searches, we found that 25% of the BAC end sequences were similar to DNA sequences in GenBank. Of these, 68% shared sequence with transposable elements and 25% with genes from other taxa. This result closely reflected the content of random sequences obtained from a small insert genomic library for L. tulipifera, indicating that the BAC library construction process was not biased. The first genomic DNA sequences for Liriodendron genes are also reported. All the Liriodendron genomic sequences described in this paper have been deposited in the GenBank data library. The end sequences from shotgun genomic clones and BAC clones are under accession DU169330–DU169684. Partial sequences of Gigantea, Frigida, LEAFY, cinnamyl alcohol dehydrogenase, 4-coumarate:CoA ligase, and phenylalanine ammonia-lyase genes are under accession DQ223429–DQ223434. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

滇黄精叶绿体全基因组序列及其密码子使用偏性分析

为探究滇黄精(Polygonatum kingianum)叶绿体全基因组特征和密码子使用偏性,利用第二代测序技术对滇黄精嫩叶进行测序,再经组装与注释后得到其叶绿体基因组全序列,通过MISA、EMBOSS和CodonW等软件对滇黄精叶绿体全基因组的SSR位点、系统发育及密码子偏好性进行分析。结果表明,滇黄精完整叶绿体基因组长度为155 852 bp,基因组平均GC含量为37.7%,其大、小单拷贝区(LSC)长度分别为84 633和185 25 bp,反向重复区长度为26 347 bp,注释了132个基因,包括86个蛋白编码基因、38个tRNA基因和8个核糖rRNA基因。叶绿体基因组中共有69个SSR位点,绝大多数属于单碱基重复的A/T类型。系统发育分析表明滇黄精与格脉黄精(P. tessellatum)亲缘关系近,可能与分布地域有关。密码子偏好性分析表明,滇黄精叶绿体基因组密码子使用模式受到自然选择影响大于突变因素,最终确定9个最优密码子。因此, 滇黄精叶绿体基因组遗传结构和系统发育位置及其密码子偏倚的分析,为叶绿体基因工程研究提供理论依据。     

The tendency of lentiviral open reading frames to become A-rich: constraints imposed by viral genome organization and cellular tRNA availability

Human immunodeficiency virus type 1 (HIV-1) and other lentiviridae demonstrate a strong preference for the A-nucleotide, which can account for up to 40% of the viral RNA genome. The biological mechanism responsible for this nucleotide bias is currently unknown. The increased A-content of these viral genomes corresponds to the typical use of synonymous codons by all members of the lentiviral family (HIV, SIV, BIV, FIV, CAEV, EIAV, visna) and the human spuma retrovirus, but not by other retroviruses like the human T-cell leukemia viruses HTLV-I and HTLV-II. In this article, we analyzed A-bias for all codon groups in all open reading frames of several lentiviruses. The extent of lentiviral codon bias could be related to host cellular translation. By calculating codon bias indices (CBIs), we were able to demonstrate an inverse correlation between the extent of codon bias and the rate of translation of individual reading frames in these viruses. Specifically, the shift toward A-rich codons is more pronounced in pol than in gag lentiviral genes. Since it is known that Gag synthesis exceeds Pol synthesis by a factor of 20 due to infrequent ribosomal frame-shifting during translation of the gap-pol mRNA molecule, we propose that the aminoacyl-tRNA availability in the host cell restricts the lentiviral preference for A-rich codons. In addition, less A-nucleotides were found in regions of the viral genome encoding multiple functions; e.g., overlapping reading frames (tat-rev-env) or in genes that overlap regulatory sequences (nef-LTR region). Finally, the characteristics of lentiviral codon usage are presented as a phylogenetic tree without the need for prior sequence alignment.Correspondence to: B. Berkhout     

Analysis of synonymous codon usage bias in 09H1N1

A novel subtype of influenza A virus 09H1N1 has rapidly spread across the world. Evolutionary analyses of this virus have revealed that 09H1N1 is a triple reassortant of segments from swine, avian and human influenza viruses. In this study, we investigated factors shaping the codon usage bias of 09H1N1 and carried out cluster analysis of 60 strains of influenza A virus from different subtypes based on their codon usage bias. We discovered that more preferentially used codons of 09H1N1 are A-ended or U-ended, and the intra-genomic codon usage bias of 09H1N1 is quite low. Base composition constraint, dinucleotide biases and translational selection are the main factors influencing the codon usage bias of 09H1N1. At the genome level, we find that the codon usage bias of 09H1N1 is similar to H1N1 (A/swine/Kansas/77778/2007H1N1), H9N2 from Asia, H1N2 from Asia and North America and H3N2 from North America. Our results provide insight for understanding the processes governing evolution, regulation of gene expression, and revealing the evolution of 09H1N1.     

Comparative organization of wheat homoeologous group 3S and 7L using wheat-rice synteny and identification of potential markers for genes controlling xanthophyll content in wheat

EST and genomic DNA sequencing efforts for rice and wheat have provided the basis for interpreting genome organization and evolution. In this study we have used EST and genomic sequencing information and a bioinformatic approach in a two-step strategy to align portions of the wheat and rice genomes. In the first step, wheat ESTs were used to identify rice orthologs and it was shown that wheat 3S and rice 1 contain syntenic units with intrachromosomal rearrangements. Further analysis using anchored rice contiguous sequences and TBLASTX alignments in a second alignment step showed interruptions by orthologous genes that map elsewhere in the wheat genome. This indicates that gene content and order is not as conserved as large chromosomal blocks as previously predicted. Similarly, chromosome 7L contains syntenic units with rice 6 and 8 but is interrupted by combinations of intrachromosomal and interchromosomal rearrangements involving syntenic units and single gene orthologs from other rice chromosome groups. We have used the rice sequence annotations to identify genes that can be used to develop markers linked to biosynthetic pathways on 3BS controlling xanthophyll production in wheat and thus involved in determining flour colour.Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Patterns of evolution and host gene mimicry in influenza and other RNA viruses

It is well known that the dinucleotide CpG is under-represented in the genomic DNA of many vertebrates. This is commonly thought to be due to the methylation of cytosine residues in this dinucleotide and the corresponding high rate of deamination of 5-methycytosine, which lowers the frequency of this dinucleotide in DNA. Surprisingly, many single-stranded RNA viruses that replicate in these vertebrate hosts also have a very low presence of CpG dinucleotides in their genomes. Viruses are obligate intracellular parasites and the evolution of a virus is inexorably linked to the nature and fate of its host. One therefore expects that virus and host genomes should have common features. In this work, we compare evolutionary patterns in the genomes of ssRNA viruses and their hosts. In particular, we have analyzed dinucleotide patterns and found that the same patterns are pervasively over- or under-represented in many RNA viruses and their hosts suggesting that many RNA viruses evolve by mimicking some of the features of their host's genes (DNA) and likely also their corresponding mRNAs. When a virus crosses a species barrier into a different host, the pressure to replicate, survive and adapt, leaves a footprint in dinucleotide frequencies. For instance, since human genes seem to be under higher pressure to eliminate CpG dinucleotide motifs than avian genes, this pressure might be reflected in the genomes of human viruses (DNA and RNA viruses) when compared to those of the same viruses replicating in avian hosts. To test this idea we have analyzed the evolution of the influenza virus since 1918. We find that the influenza A virus, which originated from an avian reservoir and has been replicating in humans over many generations, evolves in a direction strongly selected to reduce the frequency of CpG dinucleotides in its genome. Consistent with this observation, we find that the influenza B virus, which has spent much more time in the human population, has adapted to its human host and exhibits an extremely low CpG dinucleotide content. We believe that these observations directly show that the evolution of RNA viral genomes can be shaped by pressures observed in the host genome. As a possible explanation, we suggest that the strong selection pressures acting on these RNA viruses are most likely related to the innate immune response and to nucleotide motifs in the host DNA and RNAs.     

普通野生稻线粒体蛋白质编码基因密码子使用偏好性的分析

以普通野生稻(Oryza rufipogon Griff.)线粒体基因组为对象,分析其蛋白质编码基因的密码子使用特征及与亚洲栽培稻(O. sativa L.)的差异,探讨其密码子偏性形成的影响因素和进化过程。结果显示:普通野生稻线粒体基因组编码序列第1、第2和第3位碱基的GC含量依次为49.18%、42.67%和40.86%;有效密码子数(Nc)分布于45.32～61.00之间,其密码子偏性较弱; Nc值仅与GC_3呈显著相关,密码子第3位的碱基组成对密码子偏性影响较大;第1向量轴上显示9.91%的差异,其与GC_3s、Nc、密码子偏好指数(CBI)和最优密码子使用频率(Fop)的相关性均达到显著水平;而GC_3和GC₁₂的相关性未达到显著水平。因此,普通野生稻线粒体基因组密码子的使用偏性主要受自然选择压力影响而形成。本研究确定了21个普通野生稻线粒体基因组的最优密码子,大多以A或T结尾,与叶绿体密码子具有趋同进化,但是与核基因组具有不同的偏好性。同义密码子相对使用度(RSCU)、PR2偏倚分析和中性绘图分析显示,普通野生稻线粒体基因功能和其密码子使用密切相关,且线粒体密码子使用在普通野生稻、粳稻(O. sativa L. subsp. japonica Kato)和籼稻(O. sativa L. subsp.indica Kato)内具有同质性。     

The Complete Mitochondrial Genome of the Entomopathogenic Nematode Steinernema carpocapsae: Insights into Nematode Mitochondrial DNA Evolution and Phylogeny

We determined the complete sequence of the mitochondrial DNA of the entomopathogenic nematode Steinernema carpocapsae and analyzed its structure and composition as well as the secondary structures predicted for its tRNAs and rRNAs. Almost the complete genome has been amplified in one fragment with long PCR and sequenced using a shotgun strategy. The 13,925-bp genome contains genes for 2 rRNAs, 22 tRNAs, and 12 proteins and lacks an ORF encoding ATPase subunit 8. Four initiation codons were inferred, TTT, TTA, ATA, and ATT, most of the genes ended with TAA or TAG, and only two had a T as an incomplete stop codon. All predicted tRNAs showed the nonconventional secondary structure typical of Secernentea. Although we were able to fold the sequences of trnN, trnD, and trnC into more conventional cloverleaf structures after adding adjacent nucleotides, northern blot experiments showed that the nonstandard tRNAs are actually expressed. Phylogenetic and comparative analyses showed that the mitochondrial genome of S. carpocapsae is more closely related to the genomes of A. suum and C. elegans than to that of Strongyloides stercoralis. This finding does not support the phylogeny based on nuclear small subunit ribosomal DNA sequences previously published. This discrepancy may result from differential reproductive strategies and/or differential selective pressure acting on nuclear and mitochondrial genes. The distinctive characteristics observed among mitochondrial genomes of Secernentea may have arisen to counteract the deleterious effects of Muller’s ratchet, which is probably enhanced by the reproductive strategies and selective pressures referred to above. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]