Nuclear Mitochondrial DNA Segments Research Articles

Strong mitonuclear discordance in the phylogeny of Neodermata and evolutionary rates of Polyopisthocotylea

The genomic evolution of Polyopisthocotylea remains poorly understood in comparison to the remaining three classes of Neodermata: Monopisthocotylea, Cestoda, and Trematoda. Moreover, the evolutionary sequence of major events in the phylogeny of Neodermata remains unresolved. Herein we sequenced the mitogenome and transcriptome of the polyopisthocotylean Diplorchis sp., and conducted comparative evolutionary analyses using nuclear (nDNA) and mitochondrial (mtDNA) genomic datasets of Neodermata. We found strong mitonuclear discordance in the phylogeny of Neodermata. Polyopisthocotylea exhibited striking mitonuclear discordance in relative evolutionary rates: the fastest-evolving mtDNA in Neodermata and a comparatively slowly-evolving nDNA genome. This was largely attributable to its very long stem branch in mtDNA topologies, not exhibited by the nDNA data. We found indications that the fast evolution of mitochondrial genomes of Polyopisthocotylea may be driven both by relaxed purifying selection pressures and elevated levels of directional selection. We identified mitochondria-associated genes encoded in the nuclear genome: they exhibited unique evolutionary rates, but not correlated with the evolutionary rate of mtDNA, and there is no evidence for compensatory evolution (they evolved slower than the rest of the genome). Finally, there appears to exist an exceptionally large (≈6.3 kb) nuclear mitochondrial DNA segment (numt) in the nuclear genome of newly sequenced Diplorchis sp. A 3′-end segment of the 16S rRNA gene encoded by the numt was expressed, suggesting that this gene acquired novel, regulatory functions after the transposition to the nuclear genome. In conclusion, Polyopisthocotylea appears to be the lineage with the fastest-evolving mtDNA sequences among all of Bilateria, but most of the substitutions were accumulated deep in the evolutionary history of this lineage. As the nuclear genome does not exhibit a similar pattern, the circumstances underpinning this evolutionary phenomenon remain a mystery.

The Mighty NUMT: Mitochondrial DNA Flexing Its Code in the Nuclear Genome.

Nuclear-mitochondrial DNA segments (NUMTs) are mitochondrial DNA (mtDNA) fragments that have been inserted into the nuclear genome. Some NUMTs are common within the human population but most NUMTs are rare and specific to individuals. NUMTs range in size from 24 base pairs to encompassing nearly the entire mtDNA and are found throughout the nuclear genome. Emerging evidence suggests that the formation of NUMTs is an ongoing process in humans. NUMTs contaminate sequencing results of the mtDNA by introducing false positive variants, particularly heteroplasmic variants present at a low variant allele frequency (VAF). In our review, we discuss the prevalence of NUMTs in the human population, the potential mechanisms of de novo NUMT insertion via DNA repair mechanisms, and provide an overview of the existing approaches for minimizing NUMT contamination. Apart from filtering known NUMTs, both wet lab-based and computational methods can be used to minimize the contamination of NUMTs in analyses of human mtDNA. Current approaches include: (1) isolating mitochondria to enrich for mtDNA; (2) applying basic local alignment to identify NUMTs for subsequent filtering; (3) bioinformatic pipelines for NUMT detection; (4) k-mer-based NUMT detection; and (5) filtering candidate false positive variants by mtDNA copy number, VAF, or sequence quality score. Multiple approaches must be applied in order to effectively identify NUMTs in samples. Although next-generation sequencing is revolutionizing our understanding of heteroplasmic mtDNA, it also raises new challenges with the high prevalence and individual-specific NUMTs that need to be handled with care in studies of mitochondrial genetics.

Master graph: an essential integrated assembly model for the plant mitogenome based on a graph-based framework.

Unlike the typical single circular structure of most animal mitochondrial genomes (mitogenome), the drastic structural variation of plant mitogenomes is a result of a mixture of molecules of various sizes and structures. Obtaining the full panoramic plant mitogenome is still considered a roadblock in evolutionary biology. In this study, we developed a graph-based sequence assembly toolkit (GSAT) to construct the pan-structural landscape of plant mitogenome with high-quality mitochondrial master graphs (MMGs) for model species including rice (Oryza sativa) and thale cress (Arabidopsis thaliana). The rice and thale cress MMGs have total lengths of 346562 and 358041bp, including 9 and 6 contigs and 12 and 8 links, respectively, and could be further divided into 6 and 3 minimum master circles and 4 and 2 minimum secondary circles separately. The nuclear mitochondrial DNA segments (NUMTs) in thale cress strongly affected the frequency evaluation of the homologous structures in the mitogenome, while the effects of NUMTs in rice were relatively weak. The mitochondrial plastid DNA segments (MTPTs) in both species had no effects on the assessment of the MMGs. All potential recombinant structures were evaluated, and the findings revealed that all, except for nuclear-homologous structures, MMG structures are present at a much higher frequency than non-MMG structures are. Investigations of potential circular and linear molecules further supported multiple dominant structures in the mitogenomes and could be completely summarized in the MMG. Our study provided an efficient and accurate model for assembling and applying graph-based plant mitogenomes to assess their pan-structural variations.

New methods for analysing circulating cell‐free mitochondrial DNA

Trauma, ischemia, inflammatory stress and environmental conditions can cause tissue injury and damage. This damage spurs the release of cellular components and debris into the extracellular environment, including damage associated molecular pattern (DAMP) molecules. These DAMPs are sensed by pattern recognition receptors that activate a cellular damage response to the pathological insult. Mitochondrial components have recently been recognised as potent DAMPs.1, 2 The mitochondrial genome shares features with bacterial genomes, due to their shared origin. Therefore, when mitochondrial DNA (mtDNA) is released into the extracellular space it is recognised as a foreign molecule and initiates an innate immune response.1, 2 Not only have DAMP molecules been of interest in studying the relationships between cellular damage and immune responses, but emerging evidence suggests that DAMPs may serve as ‘liquid biopsies’ to indicate tissue damage or disease status. For example, plasma levels of circulating cell-free mtDNA (ccf-mtDNA) can be isolated and quantified easily using quantitative real-time PCR (qPCR). The results of many studies suggest that there are higher levels of ccf-mtDNA after trauma, acute myocardial infarction, sepsis and in some inflammatory conditions.3 A recent systematic review examined published articles on utilising ccf-mtDNA as a predictor of mortality in critically ill patients.4 The authors noted that in their analysis there are inconsistencies in ccf-mtDNA isolation methods and quantification. For example, most often qPCR primers were designed against subunits of the NADH dehydrogenase (MT-ND, MT-ND1, MT-ND2 or MT-ND6).4 However, some studies use one primer set, others use multiple primer sets and some use primer sets recognising different genes in the mitochondrial genome. This may explain the discrepancies in the findings linking ccf-mtDNA to mortality in some studies but not in all. Therefore, there is a need for the development of technologies that improve upon these current methods to quantify ccf-mtDNA. Daly and colleagues recently developed a protocol that provides a new approach to analysing plasma ccf-mtDNA.5 In their approach, they utilised a target bait-capture kit consisting of biotinylated RNA probes complementary to the entire mitochondrial genome. First, the biotinylated probes bind to mitochondrial sequences and then are enriched, pooled, and sequenced on a standard Illumina instrument. Second, a Workflow Description Language-based computational pipeline aligns the reads to both the nuclear and mitochondrial genome and then custom programs call mitochondrial and nuclear coverage, insert fragment size and mtDNA variants. Nuclear genomes contain sequences of mitochondrial origin, called nuclear mitochondrial DNA segments (NUMTs). NUMT pseudogenes can also be enriched using these methods. Therefore, to exclude the possibility that reads are from the nuclear genome, this method excludes sequences that aligned to both nuclear and mitochondrial genomes. This approach may underestimate the mtDNA levels but increases classification accuracy. In fact, this protocol was shown to enrich for mean coverage of the mtDNA genome compared to whole genome sequencing. Therefore, this protocol allows for the simultaneous quantification of mtDNA levels across the entire mitochondrial genome, differentiation of fragment lengths, and the identification of mtDNA variants.5 The authors utilised their protocol to investigate ccf-mtDNA in comparison to clinical phenotypes of injury and severity scores in a cohort of 30 trauma patients.5 There were no significant differences in ccf-mtDNA abundance between patients with non-severe or severe injuries. The ccf-mtDNA fragment length was significantly shorter in patients with major trauma and in patients where acute lung injury occurred after admission compared to non-severe injury patients. There were also mtDNA variants that were discovered that were associated with various clinical scores of injuries and severity in this cohort. Therefore, the authors utilised samples from this trauma cohort to validate that their protocol can be implemented to examined mtDNA levels, fragment length, and variants.5 This technological advance has advantages over the standard qPCR methods, as the entire mitochondrial genome can be analysed in one protocol. However, additional experiments and validation studies should be performed to directly compare the sensitivity and accuracy of this protocol versus the standard qPCR methods. Nevertheless, these methods will give scientists an additional tool to examine ccf-mtDNA in relation to tissue damage or disease status. This work was funded by the Intramural Research Program of the National Institute on Aging, National Institutes of Health. The authors declare no conflict of interest.

Complete mitochondrial genomes of three fairy shrimps from snowmelt pools in Japan

BackgroundFairy shrimps belong to order Anostraca, class Branchiopoda, subphylum Crustacea, and phylum Arthropoda. Three fairy shrimp species (Eubranchipus uchidai, E. asanumai, and E. hatanakai) that inhabit snowmelt pools are currently known in Japan. Whole mitochondrial genomes are useful genetic information for conducting phylogenetic analyses. Mitochondrial genome sequences for Branchiopoda members are gradually being collated.ResultsSix whole mitochondrial genomes from the three Eubranchipus species are presented here. Eubranchipus species share the anostracan pattern of gene arrangement in their mitochondrial genomes. The mitochondrial genomes of the Eubranchipus species have a higher GC content than those of other anostracans. Accelerated substitution rates in the lineage of Eubranchipus species were observed.ConclusionThis study is the first to obtain whole mitochondrial genomes for Far Eastern Eubranchipus species. We show that the nucleotide sequences of cytochrome oxidase subunit I and the 16S ribosomal RNA of E. asanumai presented in a previous study were nuclear mitochondrial DNA segments. Higher GC contents and accelerated substitution rates are specific characteristics of the mitochondrial genomes of Far Eastern Eubranchipus. The results will be useful for further investigations of the evolution of Anostraca as well as Branchiopoda.

Profile hidden Markov model sequence analysis can help remove putative pseudogenes from DNA barcoding and metabarcoding datasets

BackgroundPseudogenes are non-functional copies of protein coding genes that typically follow a different molecular evolutionary path as compared to functional genes. The inclusion of pseudogene sequences in DNA barcoding and metabarcoding analysis can lead to misleading results. None of the most widely used bioinformatic pipelines used to process marker gene (metabarcode) high throughput sequencing data specifically accounts for the presence of pseudogenes in protein-coding marker genes. The purpose of this study is to develop a method to screen for nuclear mitochondrial DNA segments (nuMTs) in large COI datasets. We do this by: (1) describing gene and nuMT characteristics from an artificial COI barcode dataset, (2) show the impact of two different pseudogene removal methods on perturbed community datasets with simulated nuMTs, and (3) incorporate a pseudogene filtering step in a bioinformatic pipeline that can be used to process Illumina paired-end COI metabarcode sequences. Open reading frame length and sequence bit scores from hidden Markov model (HMM) profile analysis were used to detect pseudogenes.ResultsOur simulations showed that it was more difficult to identify nuMTs from shorter amplicon sequences such as those typically used in metabarcoding compared with full length DNA barcodes that are used in the construction of barcode libraries. It was also more difficult to identify nuMTs in datasets where there is a high percentage of nuMTs. Existing bioinformatic pipelines used to process metabarcode sequences already remove some nuMTs, especially in the rare sequence removal step, but the addition of a pseudogene filtering step can remove up to 5% of sequences even when other filtering steps are in place.ConclusionsOpen reading frame length filtering alone or combined with hidden Markov model profile analysis can be used to effectively screen out apparent pseudogenes from large datasets. There is more to learn from COI nuMTs such as their frequency in DNA barcoding and metabarcoding studies, their taxonomic distribution, and evolution. Thus, we encourage the submission of verified COI nuMTs to public databases to facilitate future studies.

Platinum-Quality Mitogenome Haplotypes from United States Populations.

A total of 1327 platinum-quality mitochondrial DNA haplotypes from United States (U.S.) populations were generated using a robust, semi-automated next-generation sequencing (NGS) workflow with rigorous quality control (QC). The laboratory workflow involved long-range PCR to minimize the co-amplification of nuclear mitochondrial DNA segments (NUMTs), PCR-free library preparation to reduce amplification bias, and high-coverage Illumina MiSeq sequencing to produce an average per-sample read depth of 1000 × for low-frequency (5%) variant detection. Point heteroplasmies below 10% frequency were confirmed through replicate amplification, and length heteroplasmy was quantitatively assessed using a custom read count analysis tool. Data analysis involved a redundant, dual-analyst review to minimize errors in haplotype reporting with additional QC checks performed by EMPOP. Applying these methods, eight sample sets were processed from five U.S. metapopulations (African American, Caucasian, Hispanic, Asian American, and Native American) corresponding to self-reported identity at the time of sample collection. Population analyses (e.g., haplotype frequencies, random match probabilities, and genetic distance estimates) were performed to evaluate the eight datasets, with over 95% of haplotypes unique per dataset. The platinum-quality mitogenome haplotypes presented in this study will enable forensic statistical calculations and thereby support the usage of mitogenome sequencing in forensic laboratories.

Distinguishing mitochondrial DNA and NUMT sequences amplified with the precision ID mtDNA whole genome panel

Nuclear mitochondrial DNA segments (NUMTs) are generated via transfer of portions of the mitochondrial genome into the nuclear genome. Given their common origin, there is the possibility that both the mitochondrial and NUMT segments may co-amplify using the same set of primers. Thus, analysis of the variation of the mitochondrial genome must take into account this co-amplification of mitochondrial and NUMT sequences. The study herein builds on data from the study by Strobl et al. (Strobl et al., 2019), in which multiple point heteroplasmies were called with an “N” to prevent labeling NUMT sequences mimicking mitochondrial heteroplasmy and being interpreted as true mitochondrial in origin sequence variants. Each of these point heteroplasmies was studied in greater detail, both molecularly and bioinformatically, to determine whether NUMT or true mitochondrial DNA variation was present. The bioinformatic and molecular tools available to help distinguish between NUMT and mitochondrial DNA and the effect of NUMT sequences on interpretation were discussed.

Nuclear-mitochondrial DNA segments resemble paternally inherited mitochondrial DNA in humans

Several strands of evidence question the dogma that human mitochondrial DNA (mtDNA) is inherited exclusively down the maternal line, most recently in three families where several individuals harbored a ‘heteroplasmic haplotype’ consistent with biparental transmission. Here we report a similar genetic signature in 7 of 11,035 trios, with allelic fractions of 5–25%, implying biparental inheritance of mtDNA in 0.06% of offspring. However, analysing the nuclear whole genome sequence, we observe likely large rare or unique nuclear-mitochondrial DNA segments (mega-NUMTs) transmitted from the father in all 7 families. Independently detecting mega-NUMTs in 0.13% of fathers, we see autosomal transmission of the haplotype. Finally, we show the haplotype allele fraction can be explained by complex concatenated mtDNA-derived sequences rearranged within the nuclear genome. We conclude that rare cryptic mega-NUMTs can resemble paternally mtDNA heteroplasmy, but find no evidence of paternal transmission of mtDNA in humans.

Falcon genomics in the context of conservation, speciation, and human culture.

Here, we review the diversity, evolutionary history, and genomics of falcons in the context of their conservation and interactions with humans, and provide a perspective on how new genomic approaches may be applied to expand our knowledge of these topics. For millennia, humans and falcons (genus Falco) have developed unique relationships through falconry, religious rituals, conservation efforts, and human lifestyle transitions. From an evolutionary perspective, falcons remain an enigma. Having experienced several recent radiations, they have reached an unparalleled and almost global distribution, with an intrageneric species richness that is roughly an order of magnitude higher than typical within their family (Falconidae) and across other birds (Phylum: Aves). This diversity has evolved in the context of unusual genomic architecture that includes unique chromosomal rearrangements, relatively low chromosome counts, extremely low microdeletion rates, and high levels of nuclear mitochondrial DNA segments (NUMTs). These genomic peculiarities combine with high levels of ecological and organismal diversity and a legacy of human interactions to make falcons obvious candidates for evolutionary studies, providing unique research opportunities in common topics, including chromosomal evolution, the mechanics of speciation, local adaptation, domestication, and urban adaptation.

Mitotypes Based on Structural Variation of Mitochondrial Genomes Imply Relationships With Morphological Phenotypes and Cytoplasmic Male Sterility in Peppers.

Plant mitochondrial genomes characteristically contain extensive structural variation that can be used to define and classify cytoplasm types. We developed markers based on structural variation in the mitochondrial genomes of fertile and cytoplasmic male sterility (CMS) pepper lines and applied them to a panel of Capsicum accessions. We designed a total of 20 sequence characterized amplified region (SCAR) markers based on DNA rearrangement junctions or cytoplasm-specific segments that did not show high similarity to any nuclear mitochondrial DNA segments. We used those markers to classify the mitotypes of 96 C. annuum accessions into 15 groups. Precise genotyping of other Capsicum species (C. frutescens, C. chinense, and C. baccatum) was hampered because of various stoichiometric levels of marker amplicons. We developed a multiplex PCR system based on four of the markers that efficiently classified the C. annuum accessions into five mitotype groups. Close relationships between specific mitotypes and morphological phenotypes implied that diversification or domestication of C. annuum germplasm might have been accompanied by structural rearrangements of mitochondrial DNA or the selection of germplasms with specific mitotypes. Meanwhile, CMS lines shared the same amplification profile of markers with another mitotype. Further analysis using mitochondrial DNA (mtDNA) markers based on single-nucleotide polymorphisms (SNPs) or insertions and deletions (InDels) and CMS-specific open reading frames (orfs) provided new information about the origin of the CMS-specific mitotype and evaluation of candidates for CMS-associated genes, respectively.

Complete mitogenome of Anopheles sinensis and mitochondrial insertion segments in the nuclear genomes of 19 mosquito species

Anopheles sinensis is a major malarial vector in China and Southeast Asia. The mitochondria is involved in many important biological functions. Nuclear mitochondrial DNA segments (NUMTs) are common in eukaryotic organisms, but their characteristics are poorly understood. We sequenced and analyzed the complete mitochondrial (mt) genome of An. sinensis. The mt genome is 15,418 bp long and contains 13 protein-coding genes (PCGs), two rRNAs, 22 tRNAs and a large non-coding region. Its gene arrangement is similar to previously published mosquito mt genomes. We identified and analyzed the NUMTs of 19 mosquito species with both nuclear genomes and mt genome sequences. The number, total length and density of NUMTs are significantly correlated with genome size. About half of NUMTs are short (< 200 bp), but larger genomes can house longer NUMTs. NUMTs may help explain genome size expansion in mosquitoes. The expansion due to mitochondrial insertion segments is variable in different insect groups. PCGs are transferred to nuclear genomes at a higher frequency in mosquitoes, but NUMT origination is more different than in mammals. Larger-sized nuclear genomes have longer mt genome sequences in both mosquitoes and mammals. The study provides a foundation for the functional research of mitochondrial genes in An. sinensis and helps us understand the characteristics and origin of NUMTs and the potential contribution to genome expansion.

Bioinformatic removal of NUMT-associated variants in mitotiling next-generation sequencing data from whole blood samples.

Nuclear mitochondrial DNA segments (NUMTs) have arisen because of the transposition of segments of the mitochondrial DNA genome (mitogenome) into the nuclear genome. When using a "mitotiling" strategy, NUMTs may be more readily amplified when targeting the entire mitogenome compared to the control region, as hundreds of primers are required for complete sequencing coverage. In samples with a high percentage of nuclear DNA copies per cell, such as whole blood, NUMT coenrichment may be exacerbated. The present study examined bioinformatic approaches for removing NUMTs and NUMT-associated variants (NAVs) from next-generation sequence data generated using two mitotiling kits (Precision ID and QIAseq). Across 16 samples with low mtDNA copy number, NUMT coenrichment produced 890 NAVs with >5% variant frequency. The use of the consensus sequence to eliminate NUMT reads proved to be effective for QIAseq data, and resulted in >85% NAV removal in Precision ID data. This method was bolstered by NAV filtering in Precision ID analysis. Alternative high stringency mapping to the revised Cambridge Reference Sequence (rCRS) and the human genome reference GRCh38 for the QIAseq data caused a reduction in mitogenome coverage without complete NUMT removal. These bioinformatic solutions facilitate mitotiling sequence data analysis for low-level variant detection.

Complete mitogenomes from Kurdistani sheep: abundant centromeric nuclear copies representing diverse ancestors

The geographical centre of domestication and species diversity for sheep (Ovis aries) lies around the Kurdistan region of Northern Iraq, within the ‘Fertile Crescent’. From whole genome sequence reads, we assembled the mitochondrial genomes (mtDNA or mitogenome) of five animals of the two main Kurdistani sheep breeds Hamdani and Karadi and found they fitted into known sheep haplogroups (or matrilineages), with some SNPs. Haplotyping 31 animals showed presence of the main Asian (hpgA) and European (hpgB) haplogroups, as well as the rarer Anatolian haplogroup hpgC. From the sequence reads, near-complete genomes of mitochondria from wild sheep species (or subspecies), and even many sequences similar to goat (Capra) mitochondria, could be extracted. Analysis suggested that these polymorphic reads were nuclear mitochondrial DNA segments (numts). In situ hybridization with seven regions of mitochondria chosen from across the whole genome showed strong hybridization to the centromeric regions of all autosomal sheep chromosomes, but not the Y. Centromeres of the three submetacentric pairs and the X chromosomes showed fewer copies of numts, with varying abundance of different mitochondrial regions. Some mitochondrial-nuclear transfer presumably occurred before species divergence within the genus, and there has been further introgression of sheep mitochondrial sequences more recently. This high abundance of nuclear mitochondrial sequences is not reflected in the whole nuclear genome assemblies, and the accumulation near major satellite sequences at centromeres was unexpected. Mitochondrial variants including SNPs, numts and heteroplasmy must be rigorously validated to interpret correctly mitochondrial phylogenies and SNPs.

A rapid and efficient method for enriching mitochondrial DNA from plants

Current mitochondrial purification techniques are tedious and protracted due to their emphasis on recovering physiologically active mitochondria. However, for studies that are exclusively interested in isolating mitochondrial DNA (mtDNA) for applications such as PCR and sequencing, respiring mitochondria − and the complex procedures that stem from the need to retain their function − are unnecessary. Still, global DNA extraction methods have proven insufficient for mitochondrial DNA isolation because nuclear mitochondrial DNA segments (NUMTs) pose unique challenges to accurate mtDNA quantification and characterization. We present a rapid and simple extraction technique that maximizes recovery of mitochondrial DNA from plant cells, while minimizing the presence of nuclear DNA. Through real-time PCR, we show that this method provides a significant increase in the enrichment of mitochondrial DNA compared to that of nuclear DNA in both Arabidopsis thaliana and Brassica rapa. This method has important implications for future mitochondrial DNA analyses as it possesses few procedural limitations and minimizes the analytical problems typically associated with mtDNA purification by other techniques.

Hominin Evolution Was Caused by Introgression from Gorilla

The discovery of Paranthropus deyiremeda in 3.3-3.5 million year old fossil sites in Afar, together with 30% of the gorilla genome showing lineage sorting between humans and chimpanzees, and a NUMT ("nuclear mitochondrial DNA segment") on chromosome 5 that is shared by both gorillas, humans and chimpanzees, and shown to have diverged at the time of the Pan-Homo split rather than the Gorilla/Pan-Homo split, provides conclusive evidence that introgression from the gorilla lineage caused the Pan-Homo split, and the speciation of both the Australopithecus lineage and the Paranthropus lineage.

Analysis of Nuclear Mitochondrial DNA Segments of Nine Plant Species: Size, Distribution, and Insertion Loci

Nuclear mitochondrial DNA segment (Numt) insertion describes a well-known phenomenon of mitochondrial DNA transfer into a eukaryotic nuclear genome. However, it has not been well understood, especially in plants. Numt insertion patterns vary from species to species in different kingdoms. In this study, the patterns were surveyed in nine plant species, and we found some tip-offs. First, when the mitochondrial genome size is relatively large, the portion of the longer Numt is also larger than the short one. Second, the whole genome duplication event increases the ratio of the shorter Numt portion in the size distribution. Third, Numt insertions are enriched in exon regions. This analysis may be helpful for understanding plant evolution.

Full-length Numt analysis provides evidence for hybridization between the Asian colobine genera Trachypithecus and Semnopithecus.

The phylogenetic position of the genus Semnopithecusis unresolved because of topological incongruence when inferred using different molecular markers. Although some studies proposed hybridization between the genera Semnopithecus and Trachypithecus to explain the discordance, no conclusive evidence for hybridization has been identified. To address this issue, we used DNA walking and long-range PCR to describe a nuclear mitochondrial DNA (Numt) segment present in Trachypithecus pileatus which extends over more than 15 kb, and represents approximately 92% of the entire mitochondrial genome. We assessed the presence of this Numt in 16 other colobine species, including four species of the genus Trachypithecus, six species of the genus Semnopithecus, and representative species of six other genera belonging to the subfamily Colobinae. We failed to detect a Numt sequence in any of the other colobine species except for T. shortridgei, which is closely related to T. pileatus. The sister relationship of this Numt within the genus Semnopithecus suggests that it was derived from the mt genome of the genus Semnopithecus and invaded the nuclear genome of T. pileatus by unidirectional introgression hybridization. These results offer the most conclusive evidence for the existence of hybridization between Semnopithecus and Trachypithecus.

Characterisation of full-length mitochondrial copies and partial nuclear copies (numts) of the cytochrome b and cytochrome c oxidase subunit I genes of Toxoplasma gondii, Neospora caninum, Hammondia heydorni and Hammondia triffittae (Apicomplexa: Sarcocystidae)

Genomic DNA was extracted from three oocyst isolates of Hammondia triffittae from foxes and two oocyst isolates of Hammondia heydorni from dogs, as well as from cell culture-derived tachyzoites of Toxoplasma gondii (RH strain) and Neospora caninum (NC-Liverpool strain), and examined by PCR with primers targeting the cytochrome b (cytb) and the cytochrome c oxidase subunit I (cox1) genes in order to characterise both genes and, if possible, the remainder of the mitochondrial genome of these species. Several primers were designed and used in various combinations to amplify regions within and between both genes and to determine gene order. When certain forward primers targeting cytb were used in combination with certain reverse primers targeting cox1, two overlapping sequences were obtained for each species and isolate studied, which showed that a full-length copy of cytb was followed 36-37bp downstream by a full-length copy of cox1, and these sequences are believed to represent the true mitochondrial genes and the gene order in the mitochondrial genome of the four species examined. The cytb of T. gondii, N. caninum, H. heydorni and H. triffittae comprised a total of 1,080bp (359 amino acids) and used ATG and TAA as start and stop codon, respectively. The cox1 of these species also used TAA as stop codon, whereas the most likely start codon was ATG, resulting in a gene comprising 1,491bp (496 amino acids). Pair-wise sequence comparisons based on either cytb or cox1 clearly separated T. gondii from N. caninum and both of these species from the two Hammondia species, whereas the latter two species were 100% identical at cytb and shared 99.3% identity at cox1. Phylogenetic analyses using the maximum-likelihood method confirmed these findings and placed T. gondii in a clade separate from the three other species and all four Toxoplasmatinae in a sister clade to Eimeria spp. PCR with other primers and/or primer pairs than those used to obtain the full-length mitochondrial genes yielded several types of about 1-1.5kb long sequences, which comprised stretches of the primer-targeted genes at both ends and an intervening non-coding sequence of various length and composition. Thus, portions of cytb could be found both upstream and downstream from portions of cox1 and portions of the same gene could be found adjacent to each other (cytb→cox1; cox1→cytb; cytb→cytb; cox1→cox1). Sequence comparisons revealed that some of these gene fragments were truncated genes, whereas others included the putative start or stop codon of the full-length mitochondrial genes. From the nature of the gene fragments and/or their flanking sequences, they are assumed to be located on the chromosomes of the nuclear genome and to represent nuclear mitochondrial DNA segments (numts) or pseudogenes. In the four species examined, there were no nucleotide differences between the full-length mitochondrial copies of cytb and cox1 and their various incomplete nuclear counterparts. With a few exceptions, identical numt types and closely similar flanking sequences were obtained for all four species, which would indicate that the original transfer of these mitochondrial genes to the nuclear genome and/or the majority of any subsequent rearrangements of these gene fragments within the nuclear genome happened before the four species diverged. Yet, there were species-specific differences in the nucleotide composition of the nuclear gene fragments, identical to the differences in the mitochondrial genes, which would indicate that the incomplete nuclear copies of cytb and cox1 have been continuously updated during evolution to conform to their mitochondrial parent genes. The PCR-based findings of numts were further supported by Basic Local Alignment Search Tool (BLAST) searches against genome sequences of T. gondii and N. caninum using the concatenated mitochondrial cytb/cox1 sequences as queries. These searches revealed the presence of numerous numts of eighth distinct types in both species, with each one having a fixed starting and end point with respect to the nucleotide positions in the full-length mitochondrial genes. Four numt types were completely homologous between both species, whereas four other types differed with respect to their end point and/or the absence/presence of a 96-bp deletion. Each starting and end point was associated with a unique 100-200-bp long flanking sequence, which further revealed the presence of numts. For both species, the numt types and their various arrangements with respect to each other were identical or similar to those obtained by PCR in all four species examined. None of the identified numts covered a full-length gene, but together, the various numts covered the entire mitochondrial cytb and cox1 genes in an overlapping manner. In addition, they were fairly closely spaced on the chromosomes, and these features may explain why the nuclear copies were preferentially amplified to the exclusion of the true mitochondrial genes with most primers and primer pairs used in the present study. The possibility of a similar high prevalence of numts occurring in the nuclear genome of dinoflagellates is discussed.

Numt, a recent transfer and tandem amplification of mitochondrial DNA to the nuclear genome of the domestic cat

The mitochondrial DNA of plant and animal cells is a transcriptionally active genome that traces its origins to a symbiotic infection of eucaryotic cells by bacterial progenitors. As prescribed by the Serial Endosymbiosis Theory, symbiotic organelles have gradually transferred their genes to the eucaryotic genome, producing a functional interaction of nuclear and mitochondrial genes in organelle function. We report here a recent remarkable transposition of 7.9 kb of a typically 17.0-kb mitochondrial genome to a specific nuclear chromosomal position in the domestic cat. The intergrated segment has subsequently become amplified 38-76 times and now occurs as a tandem repeat macrosatellite with multiple-length alleles resolved by pulse-field gel electrophoresis (PFGE) segregating in cat populations. Sequence determination of the nuclear mitochondrial DNA segment, Numt, revealed a d(CA)-rich 8-bp motif [ACACACGT] repeated imperfectly five times at the deletion junction that is a likely target for recombination. The extent and pattern of sequence divergence of Numt genes from the cytoplasmic mtDNA homologues plus the occurrence of Numt in other species of the family Felidae allowed an estimate for the origins of Numt at 1.8-2.0 million years ago in an ancestor of four modern species in the genus Felis. Numt genes do not function in cats; rather, the locus combines properties of nuclear minisatellites and pseudogenes. These observations provide an empirical glimpse of historic genomic events that may parallel the accommodation of organelles in eucaryotes.