Nuclear Mitochondrial Pseudogenes Research Articles

Genomic and transcriptomic perspectives on the origin and evolution of NUMTs in Orthoptera

Nuclear mitochondrial pseudogenes (NUMTs) result from the transfer of mitochondrial DNA (mtDNA) to the nuclear genome. NUMTs, as “frozen” snapshots of mitochondria, can provide insights into diversification patterns. In this study, we analyzed the origins and insertion frequency of NUMTs using genome assembly data from ten species in Orthoptera. We found divergences between NUMTs and contemporary mtDNA in Orthoptera ranging from 0 % to 23.78 %. The results showed that the number of NUMT insertions was significantly positively correlated with the content of transposable elements in the genome. We found that 39.09 %-68.65 % of the NUMTs flanking regions (2,000 bp) contained retrotransposons, and more NUMTs originated from mitochondrial rDNA regions. Based on the analysis of the mitochondrial transcriptome, we found a potential mechanism of NUMT integration: mitochondrial transcripts are reverse transcribed into double-stranded DNA and then integrated into the genome. The probability of this mechanism occurring accounts for 0.30 %-1.02 % of total mitochondrial nuclear transfer events. Finally, based on the phylogenetic tree constructed using NUMTs and contemporary mtDNA, we provide insights into ancient evolutionary events such as species-specific “autaponumts” and “synaponumts” shared among different species, as well as post-integration duplication events.

Development of short-target primers for species identification in biological studies of Carnivora.

Noninvasive genetic sampling greatly facilitates studies on the genetics, ecology, and conservation of threatened species. Species identification is often a prerequisite for noninvasive sampling-based biological studies. Due to the low quantity and quality of genomic DNA from noninvasive samples, high-performance short-target PCR primers are necessary for DNA barcoding applications. The order Carnivora is characterized by an elusive habit and threatened status. In this study, we developed three pairs of short-target primers for identifying Carnivora species. The COI279 primer pair was suitable for samples with better DNA quality. The COI157a and COI157b primer pairs performed well for noninvasive samples and reduced the interference of nuclear mitochondrial pseudogenes (numts). COI157a could effectively identify samples from Felidae, Canidae, Viverridae, and Hyaenidae, while COI157b could be applied to samples from Ursidae, Ailuridae, Mustelidae, Procyonidae, and Herpestidae. These short-target primers will facilitate noninvasive biological studies and efforts to conserve Carnivora species.

Numt Parser: Automated identification and removal of nuclear mitochondrial pseudogenes (numts) for accurate mitochondrial genome reconstruction in Panthera.

Nuclear mitochondrial pseudogenes (numts) may hinder the reconstruction of mtDNA genomes and affect the reliability of mtDNA datasets for phylogenetic and population genetic comparisons. Here, we present the program Numt Parser, which allows for the identification of DNA sequences that likely originate from numt pseudogene DNA. Sequencing reads are classified as originating from either numt or true cytoplasmic mitochondrial (cymt) DNA by direct comparison against cymt and numt reference sequences. Classified reads can then be parsed into cymt or numt datasets. We tested this program using whole genome shotgun-sequenced data from 2 ancient Cape lions (Panthera leo), because mtDNA is often the marker of choice for ancient DNA studies and the genus Panthera is known to have numt pseudogenes. Numt Parser decreased sequence disagreements that were likely due to numt pseudogene contamination and equalized read coverage across the mitogenome by removing reads that likely originated from numts. We compared the efficacy of Numt Parser to 2 other bioinformatic approaches that can be used to account for numt contamination. We found that Numt Parser outperformed approaches that rely only on read alignment or Basic Local Alignment Search Tool (BLAST) properties, and was effective at identifying sequences that likely originated from numts while having minimal impacts on the recovery of cymt reads. Numt Parser therefore improves the reconstruction of true mitogenomes, allowing for more accurate and robust biological inferences.

A DNA barcode library for katydids, cave crickets, and leaf-rolling crickets (Tettigoniidae, Rhaphidophoridae and Gryllacrididae) from Zhejiang Province, China.

Barcode libraries are generally assembled with two main objectives in mind: specimen identification and species discovery/delimitation. In this study, the standard COI barcode region was sequenced from 681 specimens belonging to katydids (Tettigoniidae), cave crickets (Rhaphidophoridae), and leaf-rolling crickets (Gryllacrididae) from Zhejiang Province, China. Of these, four COI-5P sequences were excluded from subsequent analyses because they were likely NUMTs (nuclear mitochondrial pseudogenes). The final dataset consisted of 677 barcode sequences representing 90 putative species-level taxa. Automated cluster delineation using the Barcode of Life Data System (BOLD) revealed 118 BINs (Barcodes Index Numbers). Among these 90 species-level taxa, 68 corresponded with morphospecies, while the remaining 22 were identified based on reverse taxonomy using BIN assignment. Thirteen of these morphospecies were represented by a single barcode (so-called singletons), and each of 19 morphospecies were split into more than one BIN. The consensus delimitation scheme yielded 55 Molecular Operational Taxonomic Units (MOTUs). Only four morphospecies (I max > DNN) failed to be recovered as monophyletic clades (i.e., Elimaeaterminalis, Phyllomimusklapperichi, Sinochloraszechwanensis and Xizicushowardi), so it is speculated that these may be species complexes. Therefore, the diversity of katydids, cave crickets, and leaf-rolling crickets in Zhejiang Province is probably slightly higher than what current taxonomy would suggest.

Do pseudogenes pose a problem for metabarcoding marine animal communities?

Because DNA metabarcoding typically employs sequence diversity among mitochondrial amplicons to estimate species composition, nuclear mitochondrial pseudogenes (NUMTs) can inflate diversity. This study quantifies the incidence and attributes of NUMTs derived from the 658-bp barcode region of cytochrome c oxidase I (COI) in 156 marine animal genomes. NUMTs were examined to ascertain if they could be recognized by their possession of indels or stop codons. In total, 309 NUMTs ≥150 bp were detected, with an average of 1.98 per species (range=0-33) and a mean length of 391 ± 200 bp. Among this total, 75 (24.3%) lacked indels or stop codons. NUMTs appear to pose the greatest interpretational risk when short (<313 bp) amplicons are used, such as in environmental DNA studies, dietary analyses or processed fish identification. Employing the standard amplicon length (313 bp) for marine metabarcoding, NUMTs could potentially inflate the operational taxonomic unit (OTU) count by 21% above the true species count while also raising intraspecific variation at COI by 15%. However, when both amplicon length and position are considered, inflation in OTU counts and in barcode variation were just 9% and 10%, respectively, suggesting NUMTs will not seriously distort biodiversity assessments. There was a weak positive correlation between genome size and NUMT count but no variation among phyla or trophic groups. Until bioinformatic advances improve NUMT detection, the best defence involves targeting long amplicons and developing reference databases that include both mitochondrial sequences and their NUMT derivatives.

Ultra-deep whole genome bisulfite sequencing reveals a single methylation hotspot in human brain mitochondrial DNA

ABSTRACT While DNA methylation is established as a major regulator of gene expression in the nucleus, the existence of mitochondrial DNA (mtDNA) methylation remains controversial. Here, we characterized the mtDNA methylation landscape in the prefrontal cortex of neurological healthy individuals (n=26) and patients with Parkinson’s disease (n=27), using a combination of whole-genome bisulphite sequencing (WGBS) and bisulphite-independent methods. Accurate mtDNA mapping from WGBS data required alignment to an mtDNA reference only, to avoid misalignment to nuclear mitochondrial pseudogenes. Once correctly aligned, WGBS data provided ultra-deep mtDNA coverage (16,723 ± 7,711) and revealed overall very low levels of cytosine methylation. The highest methylation levels (5.49 ± 0.97%) were found on CpG position m.545, located in the heavy-strand promoter 1 region. The m.545 methylation was validated using a combination of methylation-sensitive DNA digestion and quantitative PCR analysis. We detected no association between mtDNA methylation profile and Parkinson’s disease. Interestingly, m.545 methylation correlated with the levels of mtDNA transcripts, suggesting a putative role in regulating mtDNA gene expression. In addition, we propose a robust framework for methylation analysis of mtDNA from WGBS data, which is less prone to false-positive findings due to misalignment of nuclear mitochondrial pseudogene sequences.

Gene Flow Patterns of the Aedes aegypti (Diptera: Culicidae) Mosquito in Colombia: a Continental Comparison Suggests Multiple Invasion Routes and Gene Exchange

In Colombia Aedes aegypti is present in 80% of the country up to 2,300 m; however, little is known of its genetic relations within a country context and, hence, within a broader context, for example, America. The aforementioned, herein, analyzed the gene flow within a context of the Americas, its directionality and genetic diversity of the mitochondrial lineages in the A. aegypti populations for Colombia. This called for the use of the sequences for A. aegypti available of the mitochondrial ND4 gene in the GenBank for Colombia and the American continent. No presence was found of nuclear mitochondrial pseudogenes (NUMTs) for Colombia. It is estimated that in Colombia the gene flow of the A. aegypti populations is occurring from the southeast and northeast toward the center of the country. In comparison with the mitochondrial sequences for America, the vector’s haplotypes in Colombia suggest connections between the populations of mosquitoes from the south with those from the north of the continent. The gene flow model at continental scale suggests bidirectional connections between the populations from the north of the continent with those from the south, while at South American scale it proposes the gene flow in all the directions with respect to the Colombian. The Colombian A. aegypti vector monitoring and control strategies must pay special attention to the vector’s points of entry into Colombia related with Peru, Venezuela, Brazil, Mexico, and North America to avoid the entry of populations with characteristics like resistance to insecticides or vector competition.

Detection of heteroplasmy and nuclear mitochondrial pseudogenes in the Japanese spiny lobster Panulirus japonicus

Partial mtDNA cytochrome oxidase subunit I (COI) fragments and near entire stretch of 12S rDNA (12S) and control region (Dloop) of the Japanese spiny lobster (Panulirus japonicus) (n = 3) were amplified by PCR and used for direct nucleotide sequencing and for clone library-based nucleotide sequence analysis. Nucleotide sequences of a total of 75 clones in COI, 77 in 12S and 92 in Dloop were determined. Haplotypes of the clones matched with those obtained by direct sequencing were determined to be genuine mtDNA sequence of the individual. Phylogenetic analysis revealed several distinct groups of haplotypes in all three regions. Genuine mtDNA sequences were observed to form a group with their closely related variables, and most of these variables may be due to amplification error but a few to be heteroplasmy. Haplotypes determined as nuclear mitochondrial pseudogenes (NUMTs) formed distinct groups. Nucleotide sequence divergence (K2P distance) between genuine haplotypes and NUMTs were substantial (7.169–23.880% for COI, 1.336–23.434% for 12S, and 7.897–71.862% for Dloop). These values were comparable to or smaller than those between species of the genus Panulirus, indicating that integration of mtDNA into the nuclear genome is a continuous and dynamic process throughout pre- and post-speciation events. Double peaks in electropherograms obtained by direct nucleotide sequencing were attributed to common nucleotides shared by multiple NUMTs. Information on the heteroplasmy and NUMTs would be very important for addressing their impact on direct nucleotide sequencing and for quality control of nucleotide sequences obtained.

Validated removal of nuclear pseudogenes and sequencing artefacts from mitochondrial metabarcode data.

Metabarcoding of Metazoa using mitochondrial genes may be confounded by both the accumulation of PCR and sequencing artefacts and the co-amplification of nuclear mitochondrial pseudogenes (NUMTs). The application of read abundance thresholds and denoising methods is efficient in reducing noise accompanying authentic mitochondrial amplicon sequence variants (ASVs). However, these procedures do not fully account for the complex nature of concomitant sequences and the highly variable DNA contribution of specimens in a metabarcoding sample. We propose, as a complement to denoising, the metabarcoding Multidimensional Abundance Threshold Evaluation (metaMATE) framework, a novel approach that allows comprehensive examination of multiple dimensions of abundance filtering and the evaluation of the prevalence of unwanted concomitant sequences in denoised metabarcoding datasets. metaMATE requires a denoised set of ASVs as input, and designates a subset of ASVs as being either authentic (mitochondrial DNA haplotypes) or nonauthentic ASVs (NUMTs and erroneous sequences) by comparison to external reference data and by analysing nucleotide substitution patterns. metaMATE (i) facilitates the application of read abundance filtering strategies, which are structured with regard to sequence library and phylogeny and applied for a range of increasing abundance threshold values, and (ii) evaluates their performance by quantifying the prevalence of nonauthentic ASVs and the collateral effects on the removal of authentic ASVs. The output from metaMATE facilitates decision-making about required filtering stringency and can be used to improve the reliability of intraspecific genetic information derived from metabarcode data. The framework is implemented in the metaMATE software (available at https://github.com/tjcreedy/metamate).

Coil: an R package for cytochrome c oxidase I (COI) DNA barcode data cleaning, translation, and error evaluation.

Biological conclusions based on DNA barcoding and metabarcoding analyses can be strongly influenced by the methods utilized for data generation and curation, leading to varying levels of success in the separation of biological variation from experimental error. The 5' region of cytochrome c oxidase subunit I (COI-5P) is the most common barcode gene for animals, with conserved structure and function that allows for biologically informed error identification. Here, we present coil ( https://CRAN.R-project.org/package=coil ), an R package for the pre-processing and frameshift error assessment of COI-5P animal barcode and metabarcode sequence data. The package contains functions for placement of barcodes into a common reading frame, accurate translation of sequences to amino acids, and highlighting insertion and deletion errors. The analysis of 10 000 barcode sequences of varying quality demonstrated how coil can place barcode sequences in reading frame and distinguish sequences containing indel errors from error-free sequences with greater than 97.5% accuracy. Package limitations were tested through the analysis of COI-5P sequences from the plant and fungal kingdoms as well as the analysis of potential contaminants: nuclear mitochondrial pseudogenes and Wolbachia COI-5P sequences. Results demonstrated that coil is a strong technical error identification method but is not reliable for detecting all biological contaminants.

Identifying a “pseudogene” for the mitochondrial DNA COI region of the corixid aquatic insect, Hesperocorixa distanti (Heteroptera, Corixidae)

DNA barcoding has been actively used as a method for species identification, and it will become an increasingly important method in the future. However, DNA barcoding can occasionally encounter a major problem, namely “nuclear mitochondrial DNA pseudogenes (NUMTs)”. In this paper, we report the discovery of a pseudogene in the mitochondrial COI region from aquatic heteropterans. PCR with Folmer’s universal primer set produced two different lengths of DNA sequences: a standard 658 bp sequence and a slightly shorter 652 bp sequence. The presence of an “indel” and “stop codon” in the shorter sequence made them separable. We recommend using the “HCOoutout” primer as a downward primer for amplification of the COI region of aquatic heteropterans, especially corixid water bugs.

Conserved numts mask a highly divergent mitochondrial-COI gene in a species complex of Australian stingless bees Tetragonula (Hymenoptera: Apidae)

Tetragonula carbonaria, Tetragonula davenporti, Tetragonula hockingsi and Tetragonula mellipes comprise a species complex of Australian stingless bee species known as the ‘Carbonaria’ group. The species are difficult to distinguish morphologically and the major species-defining characters relate to comb architecture and nest entrance ornamentation. The taxonomy of the group is further complicated by likely nuclear mitochondrial pseudogenes (numts) and inter-specific hybrids. Here we demonstrate the existence of COI numts and isolate and characterize the ‘true’ mt-COI gene in T. carbonaria and T. hockingsi. Numts were isolated from enriched-nuclear DNA extraction followed by PCR amplification and Sanger sequencing, and were recognized by the presence of deletions and/or premature stop codons in the translated sequences. The mt-COI sequences were obtained from NGS sequencing using purified mtDNA. In T. carbonaria, two numts (numt1 and numt2) were identified and a third (numt3) was identified in T. hockingsi. Numt2 and numt3 are similar (1.2% sequence divergence), indicating a recent common origin. The genetic distance between the mt-COI of the two Tetragonula species was higher than might be expected for closely related species, 16.5%, corroborating previous studies in which T. carbonaria and T. hockingsi were regarded as separate species. The three numts are more similar to the COI of other stingless bee species, including Australian Austroplebia australis and South American Melipona bicolor (81.7–83.9%) than to the mt-COI of their own species (70–71.4%). This is because the mt-COI of T. carbonaria and T. hockingsi differ greatly from other Meliponinae. Our findings explain some formerly puzzling aspects of Carbonaria biogeography, and misinterpreted amplifications.

Investigation of frequent somatic mutations of MTND5 gene in gastric cancer cell lines and tissues

The present study investigated the single nucleotide variants (SNVs) in mitochondrial DNA (mtDNA) of 13 paired gastric cancer tissue samples and seven gastric cancer cell lines using direct sequencing analysis of the MTND5 region. Results showed that nuclear mitochondrial pseudogenes (NUMTs) and mitochondrial copy number affected the detection of the SNV frequency in gastric cancer tissue and cell line samples using high-throughput sequencing technique. The heteroplasmic point mutation C12474T and G12835A happened in AGS and BGC823 cell lines, respectively. A total of seven SNVs were found in three paired gastric cancer tissue samples, including five heteroplasmic point mutations (A12406G, C12705T, T12882C, G12501A, and A12584G) and two homoplasmic point mutations (G12561A and C13590T). Gastric cancer tissue sample 16 exhibited the highest SNVs frequency with four SNVs (np 12406, np 12705, np 12882, and np 12501), whereas no SNVs or SNPs were detected in the tissue sample 4. SNP 12705 turned out to be an SNV in gastric cancer tissue sample 16. SNV 12338 detected by exome sequencing approach appeared to be an SNP in this study.

Next-Generation Sequencing of the Complete Mitochondrial Genome of the Endangered Species Black Lion Tamarin Leontopithecus chrysopygus (Primates) and Mitogenomic Phylogeny Focusing on the Callitrichidae Family.

We describe the complete mitochondrial genome sequence of the Black Lion Tamarin, an endangered primate species endemic to the Atlantic Rainforest of Brazil. We assembled the Leontopithecus chrysopygus mitogenome, through analysis of 523M base pairs (bp) of short reads produced by next-generation sequencing (NGS) on the Illumina Platform, and investigated the presence of nuclear mitochondrial pseudogenes and heteroplasmic sites. Additionally, we conducted phylogenetic analyses using all complete mitogenomes available for primates until June 2017. The single circular mitogenome of BLT showed organization and arrangement that are typical for other vertebrate species, with a total of 16618 bp, containing 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 non-coding region (D-loop region). Our full phylogenetic tree is based on the most comprehensive mitogenomic dataset for Callitrichidae species to date, adding new data for the Leontopithecus genus, and discussing previous studies performed on primates. Moreover, the mitochondrial genome reported here consists of a robust mitogenome with 3000X coverage, which certainly will be useful for further phylogenetic and evolutionary analyses of Callitrichidae and higher taxa.

Massive parallel sequencing of mitochondrial DNA genomes from mother-child pairs using the ion torrent personal genome machine (PGM)

Mitochondrial genome analysis is a potent tool in forensic practice and in the understanding of human phylogeny in the maternal lineage. With the development of molecular biology and bioinformatics techniques, high-throughput sequencing has enabled mtDNA analysis during whole genome sequencing, which provides more comprehensive information and raises the power of discrimination. In this study, peripheral blood samples were taken from 194 mother-offspring pairs and sequenced by Ion Torrent Personal Genome Machine and obtained high-coverage mitochondrial sequencing data, demonstrating the mutation levels at each position in the mitochondrial DNA (mtDNA) between maternally related pairs. A total of 14,332 variants were observed at 891 nucleotide positions from 388 individuals, and the result shows that all maternally related pairs shared the same detailed homoplasmic SNPs and haplotypes. With appropriate criteria for avoiding false positives due to sequencing errors and contamination by nuclear mitochondrial pseudogenes, we identified 33 heteroplasmies at a frequency of ≥10% at 32 sites in 30 pairs. The maternally related pairs had the same heteroplasmic sites but with different allele frequencies. The dataset is available through EMPOP under accession number EMP00684 and will serve as an mtDNA reference database in forensic casework in Eastern China.

Population genetic structure of Calanoides natalis (Copepoda, Calanoida) in the eastern Atlantic Ocean and Benguela upwelling system

The population genetic structure of Calanoides natalis (ex Calanoides carinatus; Copepoda, Calanoida), an ecologically important component of African upwelling systems, was studied in order to (i) search for potential cryptic species, (ii) describe spatial patterns in the distribution of genetic variance and (iii) identify potential barriers to gene flow. Samples were obtained in the eastern Atlantic Ocean from the Iberian Peninsula to Namibia. Analysis of mitochondrial (cytochrome c oxidase subunit I; COI) and nuclear (citrate synthase; CS) marker genes revealed a genetically cohesive population of C. natalis with a prevalent shift in allele frequencies. The discovery of a deep split solely present in the mitochondrial dataset does not point to cryptic speciation, but rather suggests the occurrence of nuclear mitochondrial pseudogenes or incomplete reproductive isolation upon secondary contact. Genetic differentiation between the northern and southern hemisphere was significant, which may point to a potential, but permeable barrier close to the equator. No vertical genetic structuring was detected in the northern Benguela implying that horizontal differentiation was more pronounced than vertical structuring. Retention mechanisms and the oxygen minimum zone did not have a strong impact on genetic differentiation of C. natalis in the Benguela region.

Mitochondrial DNA insertions in the nuclear Capra hircus genome.

Nuclear mitochondrial pseudogenes (numts), originating from mtDNA insertions into the nuclear genome, have been detected in many species. However, the distribution of numts in the newly published nuclear genome of domestic goat (Capra hircus) has not yet been explored. We used the entire goat mtDNA sequence and nuclear genome, to identify 118 numts using BLAST. Of these, 79 were able to map sequences to the genome. Further analysis showed that the size of the numts ranged from 318 to 9608 bp, and the homologous identity between numts and their respective corresponding mtDNA fragments varied between 65 and 99%. The identified Yunnan black goat numts covered nearly all the mitochondrial genes including mtDNA control region, and were distributed over all chromosomes with the exception of chromosomes 18, 21, and 25. The Y chromosome was excluded from our analysis, as sequence data are currently not available. Among the discovered 79 numts that we were able to map to the genome, 26 relatively complete mitochondrial genes were detected. Our results constitute valuable information for subsequent studies related to mitochondrial genes and goat evolution.

DNA barcoding of crickets, katydids and grasshoppers (Orthoptera) from Central Europe with focus on Austria, Germany and Switzerland.

We present a DNA barcoding study on the insect order Orthoptera that was generated in collaboration between four barcoding projects in three countries, viz. Barcoding Fauna Bavarica (Germany), German Barcode of Life, Austrian Barcode of Life and Swiss Barcode of Life. Our data set includes 748 COI sequences from 127 of the 162 taxa (78.4%) recorded in the three countries involved. Ninety-three of these 122 species (76.2%, including all Ensifera) can be reliably identified using DNA barcodes. The remaining 26 caeliferan species (families Acrididae and Tetrigidae) form ten clusters that share barcodes among up to five species, in three cases even across different genera, and in six cases even sharing individual barcodes. We discuss incomplete lineage sorting and hybridization as most likely causes of this phenomenon, as the species concerned are phylogenetically young and hybridization has been previously observed. We also highlight the problem of nuclear mitochondrial pseudogenes (numts), a known problem in the barcoding of orthopteran species, and the possibility of Wolbachia infections. Finally, we discuss the possible taxonomic implications of our barcoding results and point out future research directions.

A comparison of mitochondrial DNA isolation methods in frozen post-mortem human brain tissue--applications for studies of mitochondrial genetics in brain disorders.

Given that many brain disorders are characterized by mitochondrial dysfunction, there is a growing interest in investigating genetic and epigenetic variation in mitochondrial DNA (mtDNA). One major caveat for such studies is the presence of nuclear-mitochondrial pseudogenes (NUMTs), which are regions of the mitochondrial genome that have been inserted into the nuclear genome over evolution and, if not accounted for, can confound genetic studies of mtDNA. Here we provide the first systematic comparison of methods for isolating mtDNA from frozen post-mortem human brain tissue. Our data show that a commercial method from Miltenyi Biotec, which magnetically isolates mitochondria using antibodies raised against the mitochondrial import receptor subunit TOM22, gives significant mtDNA enrichment and should be considered the method of choice for mtDNA studies in frozen brain tissue.

Population structure of the Indonesian giant tiger shrimp Penaeus monodon: a window into evolutionary similarities between paralogous mitochondrial DNA sequences and their genomes.

Here we used both microsatellites and mtCR (mitochondrial DNA control region) sequences as genetic markers to examine the genetic diversity and population structure of Penaeus monodon shrimp from six Indonesian regions. The microsatellite data showed that shrimp from the Indian and the Pacific Ocean were genetically distinct from each other. It has been reported previously that P. monodon mtCR sequences from the Indo-Pacific group into two major paralogous clades of unclear origin. Here we show that the population structure inferred from mtCR sequences matches the microsatellite-based population structure for one of these clades. This is consistent with the notion that this mtCR clade shares evolutionary history with nuclear DNA and may thus represent nuclear mitochondrial pseudogenes (Numts).