Mitochondrial DNA Research Articles

Whole mitochondrial genome sequencing and phylogenetic analysis of Gangetic mystus (Mystus cavasius)

Mystus cavasius, known as Gangetic mystus, is a freshwater indigenous catfish. The present study represents the first-ever complete mitochondrial genome sequencing of M. cavasius. The whole mitochondrial genome size is 16,554 bp (GenBank accession number OR018997). The mitogenome organization consists of total 37 genes, with 13 protein-coding genes, two rRNA, 22 tRNAs, and a D-loop regulatory region. Among these, 28 genes are coded on the heavy strand, while eight tRNA genes and ND6 are encoded separately. Phylogenetic analysis reveals that M. cavasius clusters with other Mystus species and bagrid catfishes. This work contributes valuable insights into structural characterization and phylogenetic relationships.

Alternative oxidase of plants mitochondria is related with increased resistance of tomato mtDNA to the difenoconazole exposure

It is known that plant mitochondria and mitochondrial DNA (mtDNA) are more resistant to damage than animal mitochondria. We hypothesized that this phenomenon may be related to alternative respiratory pathways in plants mitochondria, in particular alternative oxidase (AOX). The results of a pot experiment demonstrated that the application of the fungicide difenoconazole at concentrations that were 3-, 5-, and 10-times higher than the recommended dosage resulted in a 106 %, 76 %, and 90 % increase in mitochondrial DNA damage in tomato shoots, respectively, in comparison to the shoots treated with difenoconazole at the dosage recommended by the manufacturer . Inhibition of shoot growth was observed in response to treatment with difenoconazole at a dose 10times higher than recommended. It is noteworthy that when tomatoes were treated with difenoconazole at this concentration, there was a tendency for the expression of inducible aox1a. In a field experiment, difenoconazole at a concentration of 5 times higher than recommended resulted in a 10 % increase in mtDNA damage in the fruits compared to the control. Similar results were obtained in an in vitro experiment. The addition of low doses of difenoconazole to intact tomato mitochondria did not cause mtDNA damage. The observed damages occured only when 200 μM difenoconazole was added. In contrast, incubation of 20 μM difenoconazole with SHAM, which inhibits AOX, resulted in a 115 % increase in mtDNA damage compared to the use of the same concentration without difenoconazole. This finding is consistent with the damaging effect induced by 200 μM difenoconazole. The increase in difenoconazole toxicity induced by SHAM and the elevation in aox1a gene expression resulting from the treatment with a 10 times higher than the recommended dose of difenoconazole may signify a pivotal function of AOX in the increased resistance of plant mtDNA to the pesticide exposure.

NLRX1 and STING alleviate renal ischemia-reperfusion injury by regulating LC3 lipidation during mitophagy

Mitophagy significantly influences renal ischemia/reperfusion (I/R) injury and recovery. NLRX1 is recognized for its regulatory role in governing mitochondrial damage, autophagy, and the expression of pro-inflammatory factors. Despite the acknowledged involvement of NLRX1 in these crucial cellular processes, its specific function in renal I/R injury remains unclear. We detected the expression of NLRX1, the cGAS-STING pathway, and autophagy-related proteins using Western Blot analysis. RT-qPCR was utilized to measure the expression of NLRX1 mRNA and cytokines, and changes in mitochondrial DNA (mtDNA) within the cytoplasm. Immunofluorescence was applied to observe alterations in DNA distribution within the cytoplasm. The EtBr drug, which depletes mtDNA, and the Mdivi-1 mitophagy inhibitor, were used to verify the promotion of mitophagy by NLRX1. The results demonstrated that NLRX1 was downregulated after H/R injury, and there was an increase in cytoplasmic DNA. NLRX1 overexpression not only reduced IL-1β and IL-6 levels, but also decreased mtDNA in the cytoplasm. Additionally, NLRX1 further increases mitochondrial LC3 lipidation after H/R injury, and this effect is inhibited by Mdivi-1 drugs. The activation of the cGAS-STING pathway after H/R injury is inhibited by EtBr drugs and NLRX1. Co-immunoprecipitation results showed that NLRX1 could bind to STING. Moreover, inhibiting STING reversed NLRX1-induced mitochondrial LC3 lipidation. Our study reveals that NLRX1 can bind to STING to promote mitophagy and inhibits inflammation caused by mtDNA/cGAS/STING signaling.

Ketogenic diet in adult patients with mitochondrial myopathy

BackgroundThis study aimed to explore the feasibility, safety and efficacy of a Modified Atkins Diet (MAD) in patients with mitochondrial myopathy (MM). MethodsPatients with genetically proven mitochondrial disorder and exercise intolerance or muscle weakness followed a twelve week MAD. Feasibility was measured by diet duration and ketone levels. Safety was assessed by monitoring adverse events (AE). Efficacy was assessed by a maximal incremental test and a muscle performance test. ResultsEight out of twenty patients completed the twelve week intervention. Reasons to discontinue were the occurrence of AE: rhabdomyolysis (n = 3), vomiting (n = 1), fatigue (n = 6), constipation (n = 1), in combination with a lack of improvement and adherence difficulties. On an individual level, various positive effects were reported including improvements in VO2peak (n = 6), anaerobic threshold (n = 9), muscle fatigue resistance (n = 5), muscle strength (n = 7), fatigue (n = 6), glucose tolerance (n = 7), migraine (n = 3), sleep (n = 3), and gastrointestinal complaints (n = 2). Lipid profile improved and thirteen patients lost weight. All patients with mitochondrial DNA (mtDNA) deletions, experienced muscle related AE. The five patients with the m.3243A>G mutation achieved the longest diet duration. Discussion/conclusionMAD feasibility, safety and efficacy is variable in MD patients. MAD appears to be unsuitable for MD patients with mtDNA deletions. All patients should be monitored closely for adverse events when initiating the diet. Further research should focus on predictive factors to consider the diet, effectiveness of less stringent carbohydrate restricted diets.

Comprehensive phenotypic assessment of nonsense mutations in mitochondrial ND5 in mice.

Mitochondrial dysfunction induced by mitochondrial DNA (mtDNA) mutations has been implicated in various human diseases. A comprehensive analysis of mitochondrial genetic disorders requires suitable animal models for human disease studies. While gene knockout via premature stop codons is a powerful method for investigating the unique functions of target genes, achieving knockout of mtDNA has been rare. Here, we report the genotypes and phenotypes of heteroplasmic MT-ND5 gene-knockout mice. These mutant mice presented damaged mitochondrial cristae in the cerebral cortex, hippocampal atrophy, and asymmetry, leading to learning and memory abnormalities. Moreover, mutant mice are susceptible to obesity and thermogenetic disorders. We propose that these mtDNA gene-knockdown mice could serve as valuable animal models for studying the MT-ND5 gene and developing therapies for human mitochondrial disorders in the future.

Mitogenome diversity and evolution of Bos indicus cattle in India.

Mitochondrial DNA has been widely utilized as a valuable tool for studying the evolutionary and demographic history both within and between different livestock speciesover the past three decades. Evaluation of the evolutionary history, population structure and genetic diversity is imperative for their productivity, ecosystem services, and breeding and conservation strategies for effective management. The present study included complete mitogenome of 78 cattle, out of which 33 samples belonged to 6 Bos indicus breeds of India. Mitogenome diversity of zebu cattle within population (π- nucleotide, haplotype diversity) was estimated using DnaSP v6 software and between populations (FST ratio, AMOVA analysis) was estimated using Arlequin 3.5.2.2. Ladakhi breed showed maximum (π = 0.00645) while Gir (π = 0.00042) and Tharparkar (π = 0.00053) showed minimum diversity. The diversity between the breeds of Indian cattle was 16.34 %. There were 13 and 14 haplotypes in each of I1 and I2 halogroups respectively suggesting that the divergence of Bos indicus haplotypes likely occurred within the Indian subcontinent. The Bos indicus and Bos taurus cattle lineages separated approximately 0.75 million years ago. The divergence pattern observed in zebu cattle highlighted the probability of a distinct ancestor and supported notion of independent divergence of Bos indicus.

Mitochondrial nucleoids

Eve Kakudji and Samantha Lewis discuss the structure and function of mitochondrial nucleoids - large nucleoprotein complexes containing mitochondrial DNA and the regulatory factors necessary for its packaging, replication, transcription, and repair.

First Complete Genome Sequence of Nigrospora osmanthi: A Fundamental Resource for Studying the Plant Pathogenic Fungus

Nigrospora osmanthi strain Z1 is a potential bioherbicide for the biological control of water lettuce, one of the most common invasive weeds. Here, the first high-quality genome assembly of N. osmanthi was obtained by a combination of PacBio and Illumina sequencing technologies. Twelve contigs representing the nuclear genome and one mitochondrial DNA were generated, with a total length of 45.94 Mb, an N50 equal to 6.99 Mb, and a GC content of 57.91%; 9 of the 12 nuclear contigs represented chromosomes from telomere to telomere characterized by telomeric-like repeats. A total of 11,051 genes were predicted using a combination of gene prediction tools, including 336 noncoding RNAs, 1,298 genes encoding secretory proteins, 1,735 genes encoding carbohydrate-active enzymes, 1,955 fungal virulence factors, and 50 putative secondary metabolite gene clusters. This complete genome sequence can serve as a valuable resource for future comparative genomic studies of the fungus Nigrospora for effective application in the biocontrol of invasive species and the discovery of fungal secondary metabolites. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The evolutionarily diverged single-stranded DNA-binding proteins SSB1/SSB2 differentially affect the replication, recombination, and mutation of organellar genomes in Arabidopsis thaliana

Single-stranded DNA-binding proteins (SSBs) play essential roles in the replication, recombination, and repair processes of organellar DNA molecules. In Arabidopsis thaliana, SSBs are encoded by a small family of two genes (SSB1 and SSB2). However, the functional divergence of these two SSB copies in plants remains largely unknown, and detailed studies regarding their roles in the replication and recombination of organellar genomes are still incomplete. In this study, phylogenetic, gene structure, and protein motif analyses all suggested that SSB1 and SSB2 probably diverged during the early evolution of seed plants. Based on accurate long-read sequencing results, ssb1, and ssb2 mutants had decreased copy numbers for both mitochondrial DNA (mtDNA) and plastid DNA (ptDNA), accompanied by a slight increase in structural rearrangements mediated by intermediate-sized repeats in mt genome and small-scale variants in both genomes. Our findings provide an important foundation for further investigating the effects of DNA dosage in the regulation of mutation frequencies in plant organellar genomes.

Mitochondrial Dysfunction in Environmental Toxicology: Mechanisms, Impacts, and Health Implications.

Mitochondria, pivotal to cellular metabolism, serve as the primary sources of biological energy and are key regulators of intracellular calcium ion storage, crucial for maintaining cellular calcium homeostasis. Dysfunction in these organelles impairs ATP synthesis, diminishing cellular functionality. Emerging evidence implicates mitochondrial dysfunction in the etiology and progression of diverse diseases. Environmental factors that induce mitochondrial dysregulation raise significant public health concerns, necessitating a nuanced comprehension and classification of mitochondrial-related hazards. This review systematically adopts a toxicological perspective to illuminate the biological functions of mitochondria, offering a comprehensive exploration of how toxicants instigate mitochondrial dysfunction. It delves into the disruption of energy metabolism, the initiation of mitochondrial fragility and autophagy, and the induction of mutations in mitochondrial DNA by mutagens. The overarching objective is to enhance our understanding of the repercussions of mitochondrial damage on human health.

Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks

Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause discernible transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that mitochondrial editors, including mitoTALEN and recently developed base editor DdCBE, can also enhance crosstalk between mtDNA and the nuclear genome. Moreover, we provide a practical solution by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings imply genome instability of mitochondria during induced DNA breaks and explain the origins of mitochondrial-nuclear DNA segments.

Fasciola hepatica and Fasciola hybrid form co-existence in yak from Tibet of China: application of rDNA internal transcribed spacer.

Fasciolosis is a parasitic disease affecting humans and livestock, caused by digenean trematodes of the genus Fasciola, primarily F. hepatica and F. gigantica. This study investigates the coexistence of these species and their hybrids in yaks from Tibet, China. We analyzed the nuclear rDNA internal transcribed spacer (ITS) regions, including ITS1 and ITS2, through Sanger sequencing and Next-Generation Sequencing (NGS) to assess single-nucleotide polymorphisms (SNPs). Our results reveal that one specimen (NM008B) is identical to pure F. hepatica, while another (NM008A) contains genetic information from both F. hepatica and F. gigantica, indicating potential hybridization or introgression. The morphological analysis reveals that the collected adult F. hepatica specimens exhibit distinct characteristics, while the hybrid specimens display "intermediate" features of F. hepatica and F. gigantica. This study is the first to document the coexistence of F. hepatica and hybrid Fasciola forms in a single yak. The findings underscore the complexities of hybridization dynamics and the necessity for advanced molecular techniques in accurately identifying Fasciola species. Future research should focus on mitochondrial DNA and other nuclear gene analysis to further elucidate the nature of these hybrids and their ecological implications.

Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis.

Mitochondrial DNA (mtDNA) mutations are frequent in cancer, yet their precise role in cancer progression remains debated. To functionally evaluate the impact of mtDNA variants on tumor growth and metastasis, we developed an enhanced cytoplasmic hybrid (cybrid) generation protocol and established isogenic human melanoma cybrid lines with wild-type mtDNA or pathogenic mtDNA mutations with partial or complete loss of mitochondrial oxidative function. Cybrids with homoplasmic levels of pathogenic mtDNA reliably established tumors despite dysfunctional oxidative phosphorylation. However, these mtDNA variants disrupted spontaneous metastasis from primary tumors and reduced the abundance of circulating tumor cells. Migration and invasion of tumor cells were reduced, indicating that entry into circulation is a bottleneck for metastasis amid mtDNA dysfunction. Pathogenic mtDNA did not inhibit organ colonization following intravenous injection. In heteroplasmic cybrid tumors, single-cell analyses revealed selection against pathogenic mtDNA during melanoma growth. Collectively, these findings experimentally demonstrate that functional mtDNA is favored during melanoma growth and supports metastatic entry into the blood.

Cutaneous myiasis by Calliphoridae dipterans in dogs from Chad

Cutaneous myiasis caused by various Calliphoridae dipteran species is prevalent worldwide and is of particular veterinary and public health concern. Recently, in a scientific exploration of the Guinea Worm Eradication Program to Chad, Africa, we observed that dogs with mutilated ears, based on local awareness, were caused by cutaneous myiasis. In this study, we analyzed epidemiological, morphological, and molecular data on cutaneous myiasis in dogs from Chad. From September to October 2022, dogs (n = 1,562) from 56 villages situated along the Chari River were physically inspected for cutaneous myiasis. All larvae were collected and identified morphologically and by molecular analysis of the partial cytochrome oxidase c subunit I (COI) mitochondrial gene. The prevalence of myiasis infestation along with 95% confidence intervals (95% CI) was determined using the modified Wilson method. Myiasis was detected in dogs from 21 villages (37.5%; 95% CI 26 - 50%), predominating in the southernmost region. Of 1,562 dogs, 66 (4.22%; 95% CI 3.34 - 5.34%) were infested by calliphorid larvae, with a mean infestation of 2.28 larvae per animal (range = 1 to 24). Specimens were morphologically identified as Cordylobia anthropophaga (n = 94), Chrysomya bezziana (n = 54), and Chrysomya sp. (n = 3), which were detected in 57, eight and one dog, respectively. No co-infestations were observed. The molecular analyses confirmed the morphological identification and revealed the presence of 17 haplotypes for C. anthropophaga, 2 for C. bezziana, and one for Chrysomya sp. Our study emphasizes the veterinary importance of myiasis in dogs in Africa and proposes measures to assure their health and well-being.

Increase in blood derived mitochondrial DNA copy number in strabismus patients

BackgroundAbnormalities in mitochondrial energy homeostasis can lead to various disorders, including ocular motility aberrations. Previous studies have suggested the involvement of mitochondrial aberrations in strabismus etiology. We compared the blood-derived mitochondrial DNA (mtDNA) copy number from comitant strabismus patients with that from age-matched controls, and also compared expression of mitochondrial biogenesis genes in a separate set of extraocular muscle samples from strabismic and control subjects. MethodsBlood samples from 93 strabismic (39 esotropic, 54 exotropic) and 93 control subjectswere analyzed for mtDNA copy number through quantitative polymerase chain reaction. We also examined the expression of 6 genes involved in mitochondrial biogenesis in cDNA obtained from extraocular muscles of a separate group of 26 strabismus patients and 4 healthy controls. ResultsThe mtDNA content was significantly higher in strabismus patients as compared to the control group, both overall (fold change, 1.39; Z = −2.43 [P = 0.01]) and in strabismus subgroups (esotropia: fold change,1.42; Z = 2.59 [P = 0.0096]; exotropia: fold change, 1.41, Z = 3.35 [P = 0.00078]). No significant difference was observed in the expression of the examined biogenesis genes between strabismus and control groups. ConclusionsOur results suggest an association between mtDNA copy number and strabismus; however, further studies are required to elucidate the significance of altered mtDNA in strabismus and its possible significance with regard to the etiology of strabismus.

High mitochondrial DNA levels accelerate lung adenocarcinoma progression.

Lung adenocarcinoma is a common aggressive cancer and a leading cause of mortality worldwide. Here, we report an important in vivo role for mitochondrial DNA (mtDNA) copy number during lung adenocarcinoma progression in the mouse. We found that lung tumors induced by KRASG12D expression have increased mtDNA levels and enhanced mitochondrial respiration. To experimentally assess a possible causative role in tumor progression, we induced lung cancer in transgenic mice with a general increase in mtDNA copy number and found that they developed a larger tumor burden, whereas mtDNA depletion in tumor cells reduced tumor growth. Immune cell populations in the lung and cytokine levels in plasma were not affected by increased mtDNA levels. Analyses of large cancer databases indicate that mtDNA copy number is also important in human lung cancer. Our study thus reports experimental evidence for a tumor-intrinsic causative role for mtDNA in lung cancer progression, which could be exploited for development of future cancer therapies.

Cryopreservation of yellowtail kingfish (Seriola lalandi) sperm: Effects on metabolic enzymes and sperm quality

This study aimed to determine the effects of cryopreservation on sperm functionality and assess post-thaw sperm quality in yellowtail kingfish (Seriola lalandi). Milt designated for cryopreservation was frozen in a solution containing Cortland® medium +1.3 M Me2SO + 0.2 M glucose +2 % BSA in a 1:3 ratio (milt:cryoprotectant). Fresh milt was used as the control. The samples were frozen in 0.5 mL straws using a programmable freezing system. After thawing, the samples were evaluated for motility using Computer-Assisted Sperm Analysis (CASA) with a phase-contrast optical microscope. Additionally, flow cytometry and a multimodal reader were employed to assess various sperm parameters, including plasma membrane integrity (SYBR-14/PI), mitochondrial membrane potential (∆ΨMMit: JC-1), DNA fragmentation (TUNEL), ATP content (CellTiter-Glo®), intracellular calcium levels (Fluo-4 AM), lipid peroxidation (LPO), intracellular superoxide (DHE/SYTOX) and glutathione (GSH/GSSG) production, activities of glutathione peroxidase (GPx), catalase (CAT) and 8-oxoguanine glycosylase (OGG1), and detection of 8-hydroxydeoxyguanosine (8-OHdG). In cryopreserved milt, significant differences in motility (64.14 ± 2.2 %), plasma membrane integrity (61.5 ± 2.7 %), mitochondrial membrane potential (55.4 ± 2.4 %), DNA fragmentation (6.7 ± 1.1 %), ATP content (7.5 ± 1.5 nmol/109 spermatozoa), and intracellular calcium levels (59.8 ± 2.5 nM/109 spermatozoa) were observed compared to fresh milt. Differences were also noted in LPO, O2−i, GPx, GSH/GSSG, CAT, 8-OHdG and OGG1 between the cryopreserved and control groups. Additionally, correlations between sperm quality parameters, oxidative stress markers, and enzyme activity in cryopreserved spermatozoa were determined. These results suggest that cryopreservation induces significant alterations derived from oxidative damage in the functionality of yellowtail kingfish spermatozoa, and thus further research is necessary to explore the need for using antioxidants in the cryopreservation process of sperm from this species.

Mitochondrial Bioenergetics Deficiency in cisd-1 Mutants is Linked to AMPK-Mediated Lipid Metabolism

BackgroundCISD-1 is a mitochondrial iron-sulfate [2Fe-2S] protein known to be associated with various human diseases, including cancer and diabetes. Previously, we demonstrated that CISD-1 deficiency in worms lowers glucose and ATP levels. In this study, we further explored how worms compensate for lower ATP levels by analyzing changes in cytoplasmic and mitochondrial iron content, AMPK activities, and total lipid profiles. Materials and MethodsExpression levels of CISD-1 and CISD-1::GFP fusion proteins in wild-type worms (N2), cisd-1-deletion mutants (tm4993 and syb923) and GFP insertion transgenic worms (PHX953 and SJL40) were examined by western blot. Fluorescence microscopy analyzed CISD-1::GFP pattern in PHX953 embryos and adults, and lipid droplet sizes in N2, cisd-1, aak-2 and aak-2;cisd-1 worms. Total and mitochondrial iron content, electron transport complex profiles, and AMPK activity were investigated in tm4993 and syb923 mutants. mRNA levels of mitochondrial β-oxidation genes, acs-2, cpt-5, and ech-1, were quantified by RT-qPCR in various genetic worm strains. Lipidomic analyses were performed in N2 and cisd-1(tm4993) worms. ResultsDefects in cisd-1 lead to an imbalance in iron transport and cause proton leak, resulting in lower ATP production by interrupting the mitochondrial electron transport chain. We identified a signaling pathway that links ATP deficiency-induced AMPK (AMP activated protein kinase) activation to the expression of genes that facilitate lipolysis via β-oxidation. ConclusionOur data provide a functional coordination between CISD-1 and AMPK constitutes a mitochondrial bioenergetics quality control mechanism that provides compensatory energy resources.

A rapid and parallel Late Pleistocene/Holocene morphological radiation in a predaceous planktonic water flea: the case of Bythotrephes (Cladocera: Cercopagididae)

Abstract The predatory cladoceran Bythotrephes is one of North America’s most successful and impactive invasive species in freshwater plankton communities. The taxonomic status of the genus Bythotrephes Leydig, 1860 (Crustacea: Cladocera: Cercopagididae) has remained unclear and a subject of intensive debate for over 150 years. We applied an integrative taxonomy approach with multi-gene analysis (mitochondrial COI and 12S genes, and nuclear 18S and 28S genes) on 80 individuals (representing at least four morphospecies) from various regions spanning the genus distribution (North America, Europe, and Asia) to resolve the taxonomic status of species within Bythotrephes. The results of our study strongly support the hypothesis of a single species—Bythotrephes longimanus Leydig, 1860, and ecological morphs should be accepted as junior synonyms. Our work also elucidates the very recent radiation of Bythotrephes, which may have begun rapidly and parallel during the Late Pleistocene, or even after the last glaciation. Finally, we provide a comprehensive biogeographic reconstruction of Bythotrephes dispersal within the Holarctic realm. Europe likely served as a dispersal centre for Bythotrephes from where they spread relatively recently (possibly, less than 10 kyr) and we distinguish five possible dispersal events in its evolutionary history.

A footworm in the door: revising Onchocerca phylogeny with previously unknown cryptic species in wild North American ungulates

Onchocerca is an important genus of vector-borne filarial nematodes that infect both humans and animals worldwide. Many Onchocerca spp., most of medical and veterinary health relevance, are the focus of a variety of diagnostic and molecular research. However, despite the importance of these parasites, there is growing evidence of previously unexplored genetic diversity of these nematodes, particularly among wild ungulate hosts in North America. These understudied parasites prevent us from comprehending the evolutionary history of the genus Onchocerca, monitoring potential One Health threats, and improving our filarioid diagnostic capabilities. In order to fill these knowledge gaps, we identified five uncharacterized Onchocerca lineages and compared them with other well-known filarioid species using single and concatenated gene regions (i.e., nd5, cox1, 12S, 18S, 28S, hsp70, MyoHC, rbp1). Phylogenetic analyses revealed that the novel Onchocerca lineages of wild North American ungulates segregate into two clades. One clade comprised Onchocerca lineages II, IV, and V and other species found mainly in domestic animals and humans, and the second comprised Onchocerca lineages I and III and other species from a variety of hosts including cervids, bovids, and equids. The formation of two clearly separate clades supports the idea of at least two independent expansion events of ancestral Onchocerca spp. into the North American continent via the Bering land bridge. Cophylogenetic analysis shows evidence of ancestral Onchocerca spp. of Bovidae host-switching to wild Cervidae and giving rise to the novel Onchocerca spp. Lastly, pairwise analysis confirms informative molecular markers of diagnostic relevance in both mitochondrial and nuclear gene regions of filarioid nematodes. The overall information provides greater context to the genus Onchocerca and emphasizes the need to discover, characterize, and monitor neglected parasites, especially those of wildlife origin.