Assessing the effects of primer specificity on eliminating numt coamplification in DNA barcoding: a case study from Orthoptera (Arthropoda: Insecta)

DNA barcoding is a diagnostic method of species identification based on sequencing a short mitochondrial DNA fragment of cytochrome oxidase I (COI), but its ability to correctly diagnose species is limited by the presence of nuclear mitochondrial pseudogenes (numts). Numts can be coamplified with the mitochondrial orthologue when using universal primers, which can lead to incorrect species identification and an overestimation of the number of species. Some researchers have proposed that using more specific primers may help eliminate numt coamplification, but the efficacy of this method has not been thoroughly tested. In this study, we investigate the taxonomic distribution of numts in 11 lineages within the insect order Orthoptera, by analysing cloned COI sequences and further test the effects of primer specificity on eliminating numt coamplification in four lineages. We find that numts are coamplified in all 11 taxa using universal (barcoding) primers, which suggests that numts may be widespread in other taxonomic groups as well. Increased primer specificity is only effective at reducing numt coamplification in some species tested, and only eliminates it in one species tested. Furthermore, we find that a number of numts do not have stop codons or indels, making it difficult to distinguish them from mitochondrial orthologues, thus putting the efficacy of barcoding quality control measures under question. Our findings suggest that numt coamplification is a serious problem for DNA barcoding and more quality control measures should be implemented to identify and eliminate numts prior to using mitochondrial barcodes for species diagnoses.     

Defeating numts: semi-pure mitochondrial DNA from eggs and simple purification methods for field-collected wildlife tissues.

Mitochondrial DNA (mtDNA) continues to play a pivotal role in phylogeographic, phylogenetic, and population genetic studies. PCR amplification with mitochondrial primers often yields ambiguous sequences, in part because of the co-amplification of nuclear copies of mitochondrial genes (numts) and true mitochondrial heteroplasmy arising from mutations, hybridization with paternal leakage, gene duplications, and recombination. Failing to detect numts or to distinguish the origin of such homologous sequences results in the incorrect interpretation of data. However, few studies obtain purified mtDNA to confirm the mitochondrial origin of the first reference sequences for a species. Here, we demonstrate the importance and ease of obtaining semi-pure mtDNA from wildlife tissues, preserved under various typical field conditions, and investigate the success of 3 commercial extraction kits, cesium-chloride gradient mtDNA purification, long-template PCR amplification, cloning, and more species-specific degenerate primers. Using more detailed avian examples, we illustrate that unfertilized or undeveloped eggs provide the purest sources of mtDNA; that kits provide an alternative to cesium-chloride gradient methods; and that long-template PCR, cloning, and degenerate primers cannot be used to produce reliable mitochondrial reference sequences, but can be powerful tools when used in conjunction with purified mtDNA stocks to distinguish numts from true heteroplasmy.     

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

Accurate species-level identifications underpin many aspects of basic and applied biology;however,identifications can be hampered by a lack of discriminating morphological characters,taxonomic expertise or time.Molecular approaches,such as DNA"barcoding"of the cytochrome c oxidase(COI)gene,are argued to overcome these issues.However,nuclear encoding of mitochondrial genes(numts)and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications.One insect group for which these molecular and morphological problems are significant are the dacine fruit flies(Diptera:Tephritidae:Dacini).We addressed these issues associated with COI barcoding in the dacines by first assessing several"universal"COI primers against public mitochondrial genome and numt sequences for dacine taxa.We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region.Our new primers were tested alongside universal primers on a selection of dacine species,including both fresh preserved and decades-old dry specimens.Additionally,Bactrocera tiyoni mitochondrial and nuclear genomes were compared to identify putative numts.Four numt clades were identified,three of which were amplified using existing universal primers.In contrast,our new primers preferentially amplified the"true"mitochondrial COI barcode in all dacine species tested.The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable.Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.     

Rates of DNA Duplication and Mitochondrial DNA Insertion in the Human Genome

The hundreds of mitochondrial pseudogenes in the human nuclear genome sequence (numts) constitute an excellent system for studying and dating DNA duplications and insertions. These pseudogenes are associated with many complete mitochondrial genome sequences and through those with a good fossil record. By comparing individual numts with primate and other mammalian mitochondrial genome sequences, we estimate that these numts arose continuously over the last 58 million years. Our pairwise comparisons between numts suggest that most human numts arose from different mitochondrial insertion events and not by DNA duplication within the nuclear genome. The nuclear genome appears to accumulate mtDNA insertions at a rate high enough to predict within-population polymorphism for the presence/absence of many recent mtDNA insertions. Pairwise analysis of numts and their flanking DNA produces an estimate for the DNA duplication rate in humans of 2.2 × 10^–9 per numt per year. Thus, a nucleotide site is about as likely to be involved in a duplication event as it is to change by point substitution. This estimate of the rate of DNA duplication of noncoding DNA is based on sequences that are not in duplication hotspots, and is close to the rate reported for functional genes in other species.     

Factors Affecting the Relative Abundance of Nuclear Copies of Mitochondrial DNA (Numts) in Hominoids

Although nuclear copies of mitochondrial DNA (numts) can originate from any portion of the mitochondrial genome, evidence from humans suggests that more variable parts of the mitochondrial genome, such as the mitochondrial control region (MCR), are under-represented in the nucleus. This apparent deficit might arise from the erosion of sequence identity in numts originating from rapidly evolving mitochondrial sequences. However, the extent to which mitochondrial sequence properties impacts the number of numts detected in genomic surveys has not been evaluated. In order to address this question, we: (1) conducted exhaustive BLAST searches of MCR numts in three hominoid genomes; (2) assessed numt prevalence across the four MCR sub-domains (HV1, CCD, HV2, and MCR_F); (3) estimated their insertion rates in great apes (Hominoidea); and (4) examined the relationship between mitochondrial DNA variability and numt prevalence in sequences originating from MCR and coding regions of the mitochondrial genome. Results indicate a marked deficit of numts from HV2 and MCR_F MCR sub-domains in all three species. These MCR sub-domains exhibited the highest proportion of variable sites and the lowest number of detected numts per mitochondrial site. Variation in MCR insertion rate between lineages was also observed with a pronounced burst in recent integrations within chimpanzees and orangutans. A deficit of numts from HV2/MCR_F was observed regardless of age, whereas HV1 is under-represented only in older numts (>25 million years). Finally, more variable mitochondrial genes also exhibit a lower identity with nuclear copies and because of this, appear to be under-represented in human numt databases.     

Heteroplasmy and Ancient Translocation of Mitochondrial DNA to the Nucleus in the Chinese Horseshoe Bat (Rhinolophus sinicus) Complex

The utility and reliability of mitochondrial DNA sequences in phylogenetic and phylogeographic studies may be compromised by widespread and undetected nuclear mitochondrial copies (numts) as well as heteroplasmy within individuals. Both numts and heteroplasmy are likely to be common across diverse taxa yet few studies have characterised their frequencies and variation at the intra-specific level. Here we report the presence of both numts and heteroplasmy in the mitochondrial control region of the Chinese horseshoe bat Rhinolophus sinicus. In total we generated 123 sequences from 18 bats, which contained two different numt clades (i.e. Numt-1 and Numt-2) and one mtDNA clade. The sequence divergence between Numt-1 and Numt-2 was 16.8% and each numt type was found in all four R. sinicus taxa, suggesting either two ancient translocations of mitochondrial DNA into the nucleus from the same source taxon, or a single translocation from different source taxa that occurred before the split of R. sinicus into different lineages. Within the mtDNA clade, phylogenetic relationships among the four taxa of R. sinicus were similar to those seen in previous results. Based on PCR comparisons, heteroplasmy was inferred between almost all individuals of R. sinicus with respect to sequence variation. Consistent with introgression of mtDNA between Central sinicus and septentrionalis, individuals from these two taxa exhibited similar signatures of repeated sequences in the control region. Our study highlights the importance of testing for the presence of numts and heteroplasmy when applying mtDNA markers to phylogenetic studies.     

Analysis of nuclear copies of mitochondrial sequences in honeybee (Apis mellifera) genome

At least 0.08% of the Apis mellifera nuclear genome contains sequences that originated from mitochondria. These nuclear copies of mitochondrial sequences (numts) are scattered all over the honeybee chromosomes and have originated by multiple independent insertions of mitochondrial DNA (mtDNA) as evident by phylogenetic analysis. Apart from original insertions, moderate duplications of numts also contributed to the present pattern and distribution of mitochondrial sequences in honeybee chromosomes. Assimilation of mitochondrial genes in the nuclear genome is mediated by extensive fragmentations of the original inserts. Replication slippage seems to be a major mechanism by which small sequences are inserted or deleted from mtDNA destined to nucleus. Most of the honeybee numts (84%) are located in the nongenic regions. The majority (94%) of the numts that are located in predicted nuclear genes have originated from mitochondrial genes coding for cytochrome oxidase and NADH dehydrogenase subunits. On the other hand, the mitochondrial rRNA or tRNA gene sequences are predominantly (88%) located in nongenic regions of the genome. Evidences also support for exertion of purifying selection on numts located in specific genes. Comparative analysis of numts of European, African, and Africanized honeybees suggests that numt evolution in A. mellifera is probably not demarked by speciation time frame but may be a continuous and dynamic process.     

Rampant Nuclear Insertion of mtDNA across Diverse Lineages within Orthoptera (Insecta)

Nuclear mitochondrial pseudogenes (numts) are non-functional fragments of mtDNA inserted into the nuclear genome. Numts are prevalent across eukaryotes and a positive correlation is known to exist between the number of numts and the genome size. Most numt surveys have relied on model organisms with fully sequenced nuclear genomes, but such analyses have limited utilities for making a generalization about the patterns of numt accumulation for any given clade. Among insects, the order Orthoptera is known to have the largest nuclear genome and it is also reported to include several species with a large number of numts. In this study, we use Orthoptera as a case study to document the diversity and abundance of numts by generating numts of three mitochondrial loci across 28 orthopteran families, representing the phylogenetic diversity of the order. We discover that numts are rampant in all lineages, but there is no discernable and consistent pattern of numt accumulation among different lineages. Likewise, we do not find any evidence that a certain mitochondrial gene is more prone to nuclear insertion than others. We also find that numt insertion must have occurred continuously and frequently throughout the diversification of Orthoptera. Although most numts are the result of recent nuclear insertion, we find evidence of very ancient numt insertion shared by highly divergent families dating back to the Jurassic period. Finally, we discuss several factors contributing to the extreme prevalence of numts in Orthoptera and highlight the importance of exploring the utility of numts in evolutionary studies.     

A cryptic, intergeneric cytochrome c oxidase I pseudogene in tyrant flycatchers (family: Tyrannidae)

Nuclear mitochondrial pseudogenes, or "numts", are nonfunctional copies of mitochondrial genes that have been translocated to the nuclear genome. Numts have been used to study differences in mutation rates between the nuclear and mitochondrial genomes, but have also been implicated as troublesome for phylogenetic studies and DNA-based species identification (i.e., DNA barcoding). In this study, a suspected numt discovered during a study of mitochondrial cytochrome c oxidase I (COI) diversity in North American birds was targeted and sequenced from tyrant flycatchers (family: Tyrannidae). In total, the numt was found in five taxa representing two genera. Substitution rates were compared between COI and numt sequences. None of the numt sequences harboured stop codons nor frameshift mutations, but phylogenetic analysis revealed they had accumulated more amino acid substitutions than the mitochondrial COI sequences. Mitochondrial COI appeared to be preferentially amplified in most cases, but methods for numt detection are discussed for cases like this where sequences lack obvious features for identification. Because of its persistence across a broad taxonomic lineage, this numt could form a valuable model system for studying evolution in numts. The full size of the numt and its location within the nuclear genome are yet to be determined.     

Mitochondrial DNA insertions in the nuclear horse genome

The insertion of mitochondrial DNA in the nuclear genome generates numts, nuclear sequences of mitochondrial origin. In the horse reference genome, we identified 82 numts and showed that the entire horse mitochondrial DNA is represented as numts without gross bias. Numts were inserted in the horse nuclear genome at random sites and were probably generated during the repair of DNA double-strand breaks. We then analysed 12 numt loci in 20 unrelated horses and found that null alleles, lacking the mitochondrial DNA insertion, were present at six of these loci. At some loci, the null allele is prevalent in the sample analysed, suggesting that, in the horse population, the number of numt loci may be higher than 82 present in the reference genome. Contrary to humans, the insertion polymorphism of numts is extremely frequent in the horse population, supporting the hypothesis that the genome of this species is in a rapidly evolving state.     

Mitochondrial COII Introgression into the Nuclear Genome of Gorilla gorilla

Numts are nonfunctional mitochondrial sequences that have translocated into nuclear DNA, where they evolve independently from the original mitochondrial DNA (mtDNA) sequence. Numts can be unintentionally amplified in addition to authentic mtDNA, complicating both the analysis and interpretation of mtDNA-based studies. Amplification of numts creates particular issues for studies on the noncoding, hypervariable 1 mtDNA region of gorillas. We provide data on putative numt sequences of the coding mitochondrial gene cytochrome oxidase subunit II (COII). Via polymerase chain reaction (PCR) and cloning, we obtained COII sequences for gorilla, orangutan, and human high-quality DNA and also from a gorilla fecal DNA sample. Both gorilla and orangutan samples yielded putative numt sequences. Phylogenetically more anciently transferred numts were amplified with a greater incidence from the gorilla fecal DNA sample than from the high-quality gorilla sample. Data on phylogenetically more recently transferred numts are equivocal. We further demonstrate the need for additional investigations into the use of mtDNA markers for noninvasively collected samples from gorillas and other primates.     

Extensive mitochondrial DNA transfer in a rapidly evolving rodent has been mediated by independent insertion events and by duplications

Mitochondrial DNA translocations to the nucleus (numt pseudogenes) are pervasive among eukaryotes, but copy number within the nuclear genome varies widely among taxa. As an increasing number of genomes are sequenced in their entirety, the origins, transfer mechanisms and insertion sites of numts are slowly being characterized. We investigated mitochondrial transfers within a genetically diverse rodent lineage and here report 15 numts totaling 21.8 kb that are harbored within the nuclear genome of the vole Microtus rossiaemeridionalis. The 15 numts total 21.8 kb and range from 0.39 to over 3.0 kb in length. Phylogenetic analyses revealed that these numts resulted from three independent insertions to the nucleus, two of which were followed by subsequent nuclear duplication events. The dates of the two translocations that led to subsequent duplications were estimated at 1.97 and 1.19 MYA, which coincide with the origin and radiation of the genus Microtus. Numt sequence data from five Microtus species were used to estimate an average rate of nucleotide substitution as 2.6x10(-8) subs/site/yr. This substitution rate is higher than in many other mammals, but is concordant with the elevated rate of mtDNA substitution in this lineage. Our data suggest that numt translocation in Microtus is more extensive than in either Mus or in Rattus, consistent with the elevated rate of speciation, karyotypic rearrangement, and mitochondrial DNA evolution in Microtus.     

A comparative analysis of numt evolution in human and chimpanzee

Mitochondrial DNA sequences are frequently transferred into the nuclear genome, giving rise to numts (nuclear DNA sequences of mitochondrial origin). So far, the evolutionary history of numts has largely been studied by using single genomes. Here, we present the first attempt to study numt evolution in a comparative manner by using a pairwise genomic alignment. The total number of numts was estimated to be 452 in human and 469 in chimpanzee. numts that were found in both genomes at identical loci were deemed to be orthologous; 391 numts (>80%) were classified as such. The preponderance of orthologous numts is due to the very short divergence time between the 2 hominoids. The rest of numts were deemed to be nonorthologous. Nonorthologous numts were subdivided into 1) ancestral numts that have lost an ortholog in one species through deletion (12 in human and 11 in chimpanzee), 2) new numts acquired by the insertion of a mitochondrial sequence after the divergence of the 2 species (34 in human and 46 in chimpanzee), and 3) paralogous numts created by the tandem duplication of a preexisting numt (2 in human). This approach also enabled us to reconstruct the numt repertoire in the common ancestor of humans and chimpanzees (409 numts). Our comparative approach is also useful in identifying the exact boundaries of numts.     

Mitochondrial pseudogenes: evolution's misplaced witnesses

Nuclear copies of mitochondrial DNA (mtDNA) have contaminated PCR-based mitochondrial studies of over 64 different animal species. Since the last review of these nuclear mitochondrial pseudogenes (Numts) in animals, Numts have been found in 53 of the species studied. The recent evidence suggests that Numts are not equally abundant in all species, for example they are more common in plants than in animals, and also more numerous in humans than in Drosophila. Methods for avoiding Numts have now been tested, and several recent studies demonstrate the potential utility of Numt DNA sequences in evolutionary studies. As relics of ancient mtDNA, these pseudogenes can be used to infer ancestral states or root mitochondrial phylogenies. Where they are numerous and selectively unconstrained, Numts are ideal for the study of spontaneous mutation in nuclear genomes.     

Uncovering historical signature of mitochondrial DNA hidden in the nuclear genome: the biogeography of Schistocerca revisited

Inadvertent coamplification of nuclear mitochondrial pseudogenes (numts) is a serious problem in mitochondrial systematics, but numts can also be a valuable source of information because they represent ancient forms of mtDNA. We present a conceptual framework of numt accumulation, which states that in a given species there can be two types of numts, synaponumts and autaponumts, resulting from integration occurring respectively before and after a speciation event. In a given clade, a species that diverged early can only have its own autaponumts as well as synaponumts that were already present in the genome of the last common ancestor. A species that diverged more recently may, however, have many different synaponumts integrated at each different divergence as well as its own autaponumts. Therefore it is possible to decipher the evolutionary history of a species based on the phylogenetic distribution of numts in a simultaneous analysis of numts and extant mtDNA. In this study, we test this idea empirically in the context of addressing a controversial question regarding the biogeography of the grasshopper genus Schistocerca Stål (Orthoptera: Acrididae), based on numts of the cytochrome c oxidase subunit I (COI) gene. We find that our empirical data can be explained adequately by our conceptual framework, and that the phylogenetic distribution of COI numts reveals intricate evolutionary histories about past speciation events that are otherwise difficult to detect using conventional markers. Our study strongly favours the Old World origin of the desert locust, Schistocerca gregaria and the New World Schistocerca species are descendants from an ancestral gregaria‐like species that colonized the New World via westward transatlantic flight. However, the phylogenetic distribution of S. gregaria numts raises a distinct possibility that there might have been multiple founding events from Africa to America to give rise to the present‐day diversity of the genus. This is a case study for a creative use of numts as molecular fossils, and we demonstrate that numts provide an interesting and powerful phylogenetic signal, much more than what extant mtDNA or nuclear gene sequences might be able to provide.     

Ribosomal DNA: molecular evolution and phylogenetic inference.

Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organisms, and this approach has provided a wealth of information about phylogenetic relationships. Studies of rDNA sequences have been used to infer phylogenetic history across a very broad spectrum, from studies among the basal lineages of life to relationships among closely related species and populations. The reasons for the systematic versatility of rDNA include the numerous rates of evolution among different regions of rDNA (both among and within genes), the presence of many copies of most rDNA sequences per genome, and the pattern of concerted evolution that occurs among repeated copies. These features facilitate the analysis of rDNA by direct RNA sequencing, DNA sequencing (either by cloning or amplification), and restriction enzyme methodologies. Constraints imposed by secondary structure of rRNA and concerted evolution need to be considered in phylogenetic analyses, but these constraints do not appear to impede seriously the usefulness of rDNA. An analysis of aligned sequences of the four nuclear and two mitochondrial rRNA genes identified regions of these genes that are likely to be useful to address phylogenetic problems over a wide range of levels of divergence. In general, the small subunit nuclear sequences appear to be best for elucidating Precambrian divergences, the large subunit nuclear sequences for Paleozoic and Mesozoic divergences, and the organellar sequences of both subunits for Cenozoic divergences. Primer sequences were designed for use in amplifying the entire nuclear rDNA array in 15 sections by use of the polymerase chain reaction; these "universal" primers complement previously described primers for the mitochondrial rRNA genes. Pairs of primers can be selected in conjunction with the analysis of divergence of the rRNA genes to address systematic problems throughout the hierarchy of life.     

Comparative analysis of mitochondrial fragments transferred to the nucleus in vertebrate

Mitochondfial DNA sequences transferred to the nucleus give rise to the so-called nuclear mitochondrial DNA (numt). In the GenBank database, 244 numts have been found in six orders of birds (Anseriformes, Columbiformes, Falconiformes, Charadriiformes, Galliformes and Passeriformes). Sequences alignment (NCBI-BLASTN) was carried out with mitochondrial and corresponding nuclear genome sequences in nine vertebrate species. The sequences with high homology were considered as numts. The number of numts ranged from 15 in chicken to 159 in chimpanzee. The sequences of numts in macaque, chimpanzee, and human spanned 100% of the entire mammalian mitochondrial genome. The reconstructed frequency of the mitochondrial gene transferred to the nucleus demonstrated that the rRNA genes had high frequencies than other mitochondrial genes. Using the RepeatMasker program, the transposable elements were detected in the flanking regions of each numt. The results showed that less than 5% of the flanking sequences were made up of repetitive elements in chicken. The GC content of 5′- and 3′-flanking regions of numts in nine species was less than 44%. The analysis of the flanking sequences provided a valuable understanding for future study on mechanism of mitochondrial gene transfer to the nucleus and the site of numt integration.     

Reliability of mitochondrial DNA in an acanthocephalan: the problem of pseudogenes

The utility of mitochondrial DNA as a molecular marker for evolutionary studies is well recognized. However, several problems can arise when using mitochondrial DNA, one of which is the presence of nuclear mitochondrial pseudogenes, or Numts. Pseudogenes of cytochrome oxidase I were preferentially amplified from Acanthocephalus lucii (Acanthocephala) using a universal PCR approach. To verify the presence and abundance of pseudogenes, length heterogeneity analysis of the PCR fragments was performed. PCR products obtained with universal primers often contained fragments of different sizes. Cloned sequences from universal PCR products nearly always contained sequence abnormalities such as indels and/or stop codons. Based on these sequences, new primers were developed to specifically target mitochondrial DNA. Sequences obtained with these specific primers lacked abnormalities. Phylogenetic analysis produced a single most parsimonious tree in which pseudogenes obtained with universal primers grouped together as did putative mitochondrial DNA sequences obtained with specific primers. The pattern of codon bias observed in the pseudogenes suggests a single nuclear integration event from the mitochondria. This is the first reported occurrence of pseudogenes in an acanthocephalan, and it demonstrates the potential dangers associated with the use of universal primers.     

Using secondary structure to identify ribosomal numts: cautionary examples from the human genome.

The identification of inadvertently sequenced mitochondrial pseudogenes (numts) is critical to any study employing mitochondrial DNA sequence data. Failure to discriminate numts correctly can confound phylogenetic reconstruction and studies of molecular evolution. This is especially problematic for ribosomal mtDNA genes. Unlike protein-coding loci, whose pseudogenes tend to accumulate diagnostic frameshift or premature stop mutations, functional ribosomal genes are not constrained to maintain a reading frame and can accumulate insertion-deletion events of varying length, particularly in nonpairing regions. Several authors have advocated using structural features of the transcribed rRNA molecule to differentiate functional mitochondrial rRNA genes from their nuclear paralogs. We explored this approach using the mitochondrial 12S rRNA gene and three known 12S numts from the human genome in the context of anthropoid phylogeny and the inferred secondary structure of primate 12S rRNA. Contrary to expectation, each of the three human numts exhibits striking concordance with secondary structure models, with little, if any, indication of their pseudogene status, and would likely escape detection based on structural criteria alone. Furthermore, we show that the unwitting inclusion of a particularly ancient (18-25 Myr old) and surprisingly cryptic human numt in a phylogenetic analysis would yield a well-supported but dramatically incorrect conclusion regarding anthropoid relationships. Though we endorse the use of secondary structure models for inferring positional homology wholeheartedly, we caution against reliance on structural criteria for the discrimination of rRNA numts, given the potential fallibility of this approach.     

Primate numts and reticulate evolution of capped and golden leaf monkeys (Primates: Colobinae)

A recent phylogenetic study of langurs and leaf monkeys of South Asia suggested a reticulate evolution of capped and golden leaf monkeys through ancient hybridization between Semnopithecus and Trachypithecus. To test this hybridization scenario, I analysed nuclear copies of the mitochondrial cytochrome b gene (numts) from capped, golden and Phayre’s leaf monkeys. These numts were aligned with mitochondrial cytochrome b sequences of various species belonging to the genera Semnopithecus and Trachypithecus. In the phylogenetic tree derived from this alignment, the numts fell into three distinct clades (A, B and C) suggesting three independent integration events. Clade A was basal to Semnopithecus, and clades B and C were basal to Trachypithecus. Among the numts in clades A and C were sequences derived from species not represented in their respective sister mitochondrial groups. This unusual placement of certain numts is taken as additional support for the hybridization scenario. Based on the molecular dating of these integration events, hybridization is estimated to have occurred around 7.1 to 3.4 million years ago. Capped and golden leaf monkeys might have to be assigned to a new genus to reconcile their unique evolutionary history. Additionally, northeast India appears to be a ‘hot spot’ for lineages that might have evolved through reticulate evolution.