mtDNA Sequences Research Articles

Mitochondrial Genome Instability in W303-SK1 Yeast Cytoplasmic Hybrids

Unlike most animals, some fungi, including baker’s yeast, inherit mitochondrial DNA (mtDNA) from both parents. When haploid yeast cells fuse, they form a heteroplasmic zygote, whose offspring retain one or the other variant of mtDNA. Meanwhile, some mutant mtDNA (rho−), with large deletions in the nucleotide sequence, can displace wild-type (rho+) mtDNA. Consequently, offspring of zygotes with such rho− mtDNA predominantly carry the mutant variant. This phenomenon is called suppressivity. In this study, we investigated how the suppressivity of rho− mtDNA depends on the mitochondrial and nuclear genomes of the rho+ strain during crossing. Comparing two diverged laboratory strains, SK1 and W303, we measured suppressivity in crosses with four rho− strains. One rho− strain showed significantly higher suppressivity when crossed with SK1 than with W303. We then created cytoplasmic hybrids by swapping mtDNAs between these strains. Surprisingly, we found that the mtDNA of the rho+ strain, rather than its nuclear DNA, determines high suppressivity in crosses of SK1 rho+ with the rho− strain. Additionally, mtDNA replacement reduced respiration rate and growth rate on non-fermentable substrates while increasing the likelihood of functional mtDNA loss. Our data demonstrate that a mutant mtDNA variant’s ability to displace another mitochondrial DNA variant in a heteroplasmic cell depends more on mtDNA sequences than on the biochemical and structural context created by the nuclear genome background.

Population genetic structure of the bottlenose dolphin in the Turkish waters based on mtDNA sequences with implications for the Black Sea subspecies Tursiops truncatus ponticus

The bottlenose dolphin is a widely distributed species found in temperate waters, including the Mediterranean and Black Seas. The Black Sea population is recognized as a distinct subspecies, Tursiops truncatus ponticus, due to genetic and morphological differences. This study analyzed mitochondrial DNA (mtDNA) sequences of 73 bottlenose dolphin samples collected between 1999 and 2016 along the Turkish Black Sea coast, Turkish Straits System (TSS), Aegean, and Mediterranean Seas revealing 14 haplotypes, eight of which are reported here for the first time. The haplotype network shows two main nodes with star-like sub-networks, but no apparent geographic pattern. The lack of geographical groupings of haplotypes indicates the high mobility of the species within the sampled area. The northern Black Sea bottlenose dolphins (NBS) appear to be genetically differentiated from those in the southwestern Black Sea (SBS), Mediterranean, and Atlantic Ocean, but not from the TSS and Aegean Sea populations. Besides, SBS bottlenose dolphins were differentiated only from those in the western Mediterranean (WM) and the Atlantic. Therefore, NBS and SBS bottlenose dolphins should at least be categorized as different populations. However, contrary to some previous studies there was no evidence supporting the classification of Black Sea bottlenose dolphins as a separate subspecies. The TSS differs only from the Atlantic and the Aegean, and functions like a migratory ‘melting pot’ for surrounding populations. Despite the largest sample size, NBS bottlenose dolphins exhibited the lowest haplotype and nucleotide diversity. This low genetic diversity and potential isolation from adjacent populations highlight the need for prioritizing the conservation of NBS bottlenose dolphins.

Revalidation of Enteromius crocodilensis (Fowler, 1934) from synonymy with the disjunctly distributed Enteromius argenteus (Günther, 1868) based on molecular and morphological evidence.

The rosefin barb, Enteromius argenteus, as currently described, is a freshwater fish with a distribution that is geographically separated and divided into the northern population in the Kwanza River system in Angola, and the southern population in the Inkomati River system in South Africa and Eswatini. Due to this disjunct distribution pattern, it is likely that the two populations represent distinct species. mtDNA sequence data and detailed examination of morphometric characters revealed considerable genetic (2.8%-3.5%) and morphological differentiation of the two populations, suggesting their recognition as two distinct species. Enteromius crocodilensis is revalidated and redescribed for the southern population that is readily distinguished from E. argenteus by nonoverlapping circumpeduncular scale counts (12-13 vs. 14) and color pattern (presence of a conspicuous dark band along the midline vs. absence of characteristic melanin patterns). Phylogenetic analysis based on mtDNA cytochrome oxidase subunit I (COI) data places both E. argenteus and E. crocodilensis in a well-supported clade that includes a number of morphologically similar sawfin barb species from southern and central Africa.

Using eDNA to Supplement Population Genetic Analyses for Cryptic Marine Species: Identifying Population Boundaries for Alaska Harbour Porpoises.

Isolation by distance and biogeographical boundaries define patterns of population genetic structure for harbour porpoise along the Pacific coast from California to British Columbia. Until recently, inadequate sample sizes in many regions constrained efforts to characterise population genetic structure throughout the coastal waters of Alaska. Here, tissue samples from beachcast strandings and fisheries bycatch were supplemented with targeted environmental DNA (eDNA) samples in key regions of Alaska coastal and inland waters. Using a geographically explicit, hierarchical approach, we examined the genetic structure of Alaska harbour porpoises, using both mitochondrial DNA (mtDNA) sequence data and multilocus SNP genotypes. Despite a lack of evidence of genetic differentiation from nuclear SNP loci, patterns of relatedness and genetic differentiation from mtDNA suggest natal philopatry at multiple geographic scales, with limited gene flow among sites possibly mediated by male dispersal. A priori clustering of sampled areas at an intermediate scale (eastern and western Bering Sea, Gulf of Alaska and Southeast Alaska) best explained the genetic variance (12.37%) among regions. In addition, mtDNA differentiation between the Gulf of Alaska and eastern Bering Sea, and among regions within the Gulf of Alaska, indicated significant genetic structuring of harbour porpoise populations in Southeast Alaska. The targeted collection of eDNA samples from strata within Southeast Alaska was key for elevating the statistical power of our mtDNA dataset, and findings indicate limited dispersal between neighbouring strata within coastal and inland waters. These results provide evidence supporting a population boundary within the currently recognised Southeast Alaska Stock. Together, these findings will prove useful for ongoing management efforts to reduce fisheries conflict and conserve genetic diversity in this iconic coastal species.

Targeted nanopore sequencing using the Flongle device to identify mitochondrial DNA variants

Variants in mitochondrial genomes (mtDNA) can cause various neurological and mitochondrial diseases such as mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes (MELAS). Given the 16 kb length of mtDNA, continuous sequencing is feasible using long-read sequencing (LRS). Herein, we aimed to show a simple and accessible method for comprehensive mtDNA sequencing with potential diagnostic applications for mitochondrial diseases using the compact and affordable LRS flow cell “Flongle.” Whole mtDNA amplification (WMA) was performed using genomic DNA samples derived from four patients with mitochondrial diseases, followed by LRS using Flongle. We compared these results to those obtained using Cas9 enrichment. Additionally, the accuracy of heteroplasmy rates was assessed by incorporating mtDNA variants at equimolar levels. Finally, mtDNA from 19 patients with Parkinson’s disease (PD) was sequenced using Flongle to identify disease risk-associated variants. mtDNA variants were detected in all four patients with mitochondrial diseases, with results comparable to those obtained from Cas9 enrichment. Heteroplasmy levels were accurately detected (r2 > 0.99) via WMA using Flongle. A reported variant was identified in three patients with PD. In conclusion, Flongle can simplify the traditionally cumbersome and expensive mtDNA sequencing process, offering a streamlined and accessible approach to diagnosing mitochondrial diseases.

Enhancing Radiation-induced Reactive Oxygen Species Generation Through Mitochondrial Transplantation in Human Glioblastoma.

Glioblastoma (GBM) is the most aggressive primary brain malignancy in adults, with high recurrence rates and resistance to standard therapies. This study explores mitochondrial transplantation as a novel method to enhance the radiobiological effect (RBE) of ionizing radiation (IR) by increasing mitochondrial density in GBM cells, potentially boosting reactive oxygen species (ROS) production and promoting radiation-induced cell death. Using cell-penetrating peptides (CPPs), mitochondria were transplanted into GBM cell lines U3020 and U3035. Transplanted mitochondria were successfully incorporated into recipient cells, increasing mitochondrial density significantly. Mitochondrial chimeric cells demonstrated enhanced ROS generation post-irradiation, as evidenced by increased electron paramagnetic resonance (EPR) signal intensity and fluorescent ROS assays. The transplanted mitochondria retained functionality and viability for up to 14 days, with mitochondrial DNA (mtDNA) sequencing confirming high transfection and retention rates. Notably, mitochondrial transplantation was feasible in radiation-resistant GBM cells, suggesting potential clinical applicability. These findings support mitochondrial transplantation as a promising strategy to overcome therapeutic resistance in GBM by amplifying ROS-mediated cytotoxicity, warranting further investigation into its efficacy and mechanisms in vivo .

Potential role of heteroplasmic mitochondrial DNA mutations in modulating the subtype-specific adaptation of oral squamous cell carcinoma to cisplatin therapy

Cancer cells are constantly evolving to adapt to environmental changes, particularly during exposure to drug treatment. In this work, we aimed to characterize genetic and epigenetic changes in mitochondrial DNA (mtDNA) that may increase the resistance of oral squamous cell carcinoma (OSCC) to cisplatin. We first derived drug-resistant cells from two human OSCC cell lines, namely SAS and H103, by continual cisplatin treatments for about 4 months. To determine mtDNA changes induced by cisplatin, we performed nanopore sequencing and quantitative polymerase chain reaction analysis of mtDNA extracted from the cells pre- and post-treatment. We also assessed the mitochondrial functions of the cells and their capacity to generate intracellular reactive oxygen species (ROS). We found that in the cisplatin-resistant cells derived from SAS, there was a reduction in mtDNA content and significant enrichment of a m.3910G > C mutation in the MT-ND1 gene. However, such changes were not detected in cisplatin-resistant H103 cells. The cisplatin treatment also altered methylation patterns in both SAS and H103 cells and decreased their sensitivity to ROS-induced cytotoxicity. We suggest that the sequence alterations and epigenetic changes in mtDNA and the reduction in mtDNA content could be key drivers of cisplatin resistance in OSCC. These mtDNA alterations may participate in cellular adaptation that serves as a response to adverse changes in the environment, particularly exposure to cytotoxic agents. Importantly, the observed mtDNA changes may be influenced by the distinct genetic landscapes of various cancer subtypes. Overall, this study reveals significant insights into cisplatin resistance driven by complex mtDNA dynamics, particularly in OSCC. This underscores the need for targeted therapies tailored to the genetic profiles of individual OSCC patients to improve disease prognosis.

A novel missense mutation in ISCA2 causes aberrant splicing and leads to multiple mitochondrial dysfunctions syndrome 4.

Iron-sulfur cluster assembly 2 (ISCA2) deficiency is linked to an autosomal recessive disorder known as multiple mitochondrial dysfunctions syndrome 4 (MMDS4). This disorder is characterized by leukodystrophy and neuroregression. Currently, most of the reported patients are from Saudi Arabia. All these patients carry a homozygous founder variant (NM_194279.2:c.229G>A:p.Gly77Ser) in ISCA2. We describe a patient who underwent full clinical evaluation, including metabolic, neurological, and radiological examinations. Standard genetic testing, including whole exome sequencing coupled with autozygome analysis, was undertaken, as were assessments of mitochondrial DNA (mtDNA) copy number and mtDNA sequencing on DNA extracted from blood and cultured fibroblasts. Functional workup consisted of splicing assessment of ISCA2 using RT-PCR, biochemical assessment of complex I status using dipstick assays, and mitochondrial respiration measurements using a Seahorse XFp analyzer. We present the clinical and functional characterization of a novel homozygous ISCA2 missense variant (NM_194279.3:c.70A>G:p.Arg24Gly), leading to aberrant splicing in a patient presenting with neuroregression, generalized spasticity with exaggerated deep tendon reflexes and head lag, and progressive loss of acquired milestones. The novel variant was fully segregated in a wider family and was absent in a large control cohort, ethnically matching in-house exomes, local databases such as CGMdb and Saudi Human Genome Program, and ClinVar. Our analyses revealed that the variant is pathogenic, disrupting normal ISCA2 splicing and presumably leading to a truncated protein that disturbs metabolic pathways in patient-derived cells.

Genetic Identification of Putative Hybrids Between Grey Wolf and Golden Jackal

We describe the results of genetic analysis of 11 phenotypically deviant individuals of grey wolf (Canis lupus Linnaeus, 1758) sensu lato collected in Voronezh State Nature Biosphere Reserve (Chernozem zone of European Russia) and Dagestan (Northern Caucasus, Russia) putatively identified morphologically as hybrids between grey wolf and golden jackal (Canis aureus Linnaeus, 1758). By means of maternally inherited mtDNA (sequences of cytochrome b gene fragment) and paternal lineage markers ZfY no F1 wolf-jackal hybrids were identified. As well, possibility of classification of the studied individuals to next generation hybrids from crosses between different wolf-jackal F1s. However, attribution of these animals to complex hybrids such as various backcrosses cannot be rejected. From the results of analysis by a set of autosomal microsatellite loci we putatively diagnosed a single F2 hybrid. As well, we obtained data that can be considered as traces of hybridization between wolf and jackal in southern regions of European Russa, albeit direct indications of introgression between these species not found. At the same time, the results of both genetic and craniological studies could be interpreted as indication to hybridization between wolves and domestic dogs on the same territories.

Aberrant fragmentomic features of circulating cell-free mitochondrial DNA enable early detection and prognosis prediction of hepatocellular carcinoma.

Early detection and effective prognosis prediction in patients with hepatocellular carcinoma (HCC) provides an avenue for survival improvement, yet more effective approaches are greatly needed. We sought to develop the detection and prognosis models with ultra-sensitivity and low cost based on fragmentomic features of circulating cell free mtDNA (ccf-mtDNA). Capture-based mtDNA sequencing was carried out in plasma cell-free DNA samples from 1168 participants, including 571 patients with HCC, 301 patients with chronic hepatitis B or liver cirrhosis (CHB/LC) and 296 healthy controls (HC). The systematic analysis revealed significantly aberrant fragmentomic features of ccf-mtDNA in HCC group when compared with CHB/LC and HC groups. Moreover, we constructed a random forest algorithm-based HCC detection model by utilizing ccf-mtDNA fragmentomic features. Both internal and two external validation cohorts demonstrated the excellent capacity of our model in distinguishing early HCC patients from HC and high-risk population with CHB/LC, with AUC exceeding 0.983 and 0.981, sensitivity over 89.6% and 89.61%, and specificity over 98.20% and 95.00%, respectively, greatly surpassing the performance of alpha-fetoprotein (AFP) and mtDNA copy number. We also developed a HCC prognosis prediction model by LASSO-Cox regression to select 20 fragmentomic features, which exhibited exceptional ability in predicting 1-year, 2-year and 3-year survival (AUC = 0.8333, 0.8145 and 0.7958 for validation cohort, respectively). We have developed and validated a high-performing and low-cost approach in a large clinical cohort based on aberrant ccf-mtDNA fragmentomic features with promising clinical translational application for the early detection and prognosis prediction of HCC patients.

Changes in TP53 Gene, Telomere Length, and Mitochondrial DNA in Benign Prostatic Hyperplasia Patients.

Benign prostatic hyperplasia (BPH) is a growing issue due to an ageing population. Our study investigated the possible associations between BPH and ageing hallmarks, including the telomere length (TL) and mitochondrial genome copy number (mtDNA CN), along with genetic variations in the TP53 gene and mtDNA. Prostate tissue samples were obtained from 32 patients with BPH, together with 30 blood samples. As a healthy control group, age-matching blood DNA samples were used. For the comparison of mtDNA sequence data, 50 DNA samples of the general Latvian population were used. The full mtDNA genome was analyzed by using Next-Generation Sequencing (NGS), the TP53 gene by Sanger sequencing, and the mtDNA copy number (mtDNA CN) and telomere length (TL) byqPCR assay. The results showed that in BPH patients, telomeres in the prostate tissue were significantly longer than in blood cells, while the TL in blood cells of the healthy controls was the shortest. Also, the mtDNA amount in the prostate tissue of BPH patients was significantly greater in comparison with blood cells, and controls had the smallest mtDNA CN. We did not find any mutations in the TP53 gene that could be linked to BPH; however, in mtDNA, we found several unique mutations and heteroplasmic changes, as well as genetic changes that have been previously associated with prostate cancer. In conclusion, prolonged telomeres and changes in the mtDNA amount might be involved in the molecular mechanisms of BPH. Some of the heteroplasmic or homoplasmic mtDNA variants might also contribute to the development of BPH. Additional studies are needed to substantiate these findings.

A preliminary study on detecting human DNA in aquatic environments: Potential of eDNA in forensics

Human environmental DNA (eDNA) application have not been fully applied or adequately considered in the fields of eDNA and forensics. Nonetheless, this technique holds great potential as a complementary tool for detecting human DNA in aquatic environments, particularly in cases involving crimes connect to such environments. However, the detectability or stability of eDNA can vary depending on several factors. Therefore, this preliminary study investigates the detection and degradation rates of human eDNA, as well as the recovery of nuclear short tandem repeat (STR) profiles and mitochondrial DNA (mtDNA) sequencing, using water samples from both saltwater and freshwater sources. To conduct the experiment, whole human blood was spiked into the water samples. Water samples were then filtered using a 5 µm pore size filter, and samples were collected at various time intervals up to 23 days. A human specific qPCR assay targeting HV1 region of human mtDNA was used to detect human eDNA. Results demonstrated that human eDNA remains detectable for up to 36 hours in freshwater samples and up 84 hours in saltwater samples. The limit of detection (LOD) of human eDNA, (205 copies/µl), was achieved after 60 hours in freshwater and 180 hours in saltwater samples. Partial STR profiles could be recovered up to 24 hours for freshwater and saltwater. Results from mtDNA sequencing indicate that full mtDNA profiles could be recovered from freshwater samples up to 48 hours and remained detectable up to 72 hours in saltwater. Overall, the findings of this study underscore the importance of considering and incorporating human eDNA analysis as a valuable tool in forensic practice. By harnessing the power of eDNA, law enforcement agencies can enhance their investigation capabilities, improve the accuracy of forensic reconstructions, and ultimately contribute to the resolution of cases involving aquatic environments. Further research and validation are needed to optimize and expand the utilization of eDNA techniques in forensic investigations.

Whole Mitochondrial DNA Sequencing Using Fecal Samples from Domestic Dogs

Medical care for domestic dogs is now respected worldwide as being at a similar level to that of humans. We previously established a test method to determine whole mitochondrial DNA (mtDNA) using oral mucosal DNA that may be useful for medical care and welfare. However, the sample types tested in dogs are not limited to those obtained from the oral mucosa. Therefore, in the present study, we attempted to establish a test method to determine whole mtDNA sequences using feces, which represents the least invasive specimen. Two Japanese domestic dogs were used in the present study. DNA was extracted from approximately 100 mg of fresh feces from each dog, and PCRs were performed using four primer pairs that can amplify whole mtDNA. Following PCR, amplicons were pooled to create a DNA library using an experimental robot with an original program. Data were then acquired via NGS and data analysis was performed. The results showed that the whole mtDNA sequence of the two dogs was determined with high accuracy. Our results suggest that feces can be adapted for mitochondrial disease and individual identification testing and could serve as a useful testing method for the future medical care and welfare of domestic dogs.

The Origin of H. s. denisovan and Their Dispersal Across Iran

Before the early 2000s, anthropologists spoke of two taxa existing in the Late Middle and Early Upper Pleistocene: anatomically modern humans in Africa and Neanderthals associated with the Mousterian industry in Eurasia. Therefore, all Eurasian Paleolithic sites dating to that period were believed to be Mousterian and were associated with Neanderthals. In 2010, owing to the sequencing of mtDNA from a fragment of the distal phalanx of the hand found in Denisova Cave, a third species was introduced, genetically different from both anatomically modern humans and Neanderthals. This new taxon was termed H. s. denisovan—or simply Denisovan. Further studies showed that this population dispersed in the Late Middle and Early Upper Pleistocene across vast territories of Central and Southeast Asia. A question arose as to where Denisovans had originated and which routes they had taken to get to the Altai. A series of articles forthcoming in this journal will address these questions. The first of them focuses on the origin of Denisovans on the basis of H. heidelbergensis and on their migration via Iran to Central Asia.

Mitochondrial genetic variations in leukemia: a comprehensive overview.

Leukemias are a group of heterogeneous hematological malignancies driven by diverse genetic variations, and the advent of genomic sequencing technologies facilitates the investigation of genetic abnormalities in leukemia. However, these sequencing-based studies mainly focus on nuclear DNAs. Increasing evidence indicates that mitochondrial dysfunction is an important mechanism of leukemia pathogenesis, which is closely related to the mitochondrial genome variations. Here, we provide an overview of current research progress concerning mitochondrial genetic variations in leukemia, encompassing gene mutations and copy number variations. We also summarize currently accessible mitochondrial DNA (mtDNA) sequencing methods. Notably, somatic mtDNA mutations may serve as natural genetic barcodes for lineage tracing and longitudinal assessment of clonal dynamics. Collectively, these findings enhance our understanding of leukemia pathogenesis and foster the identification of novel therapeutic targets and interventions.

Recognizing Leber's Hereditary Optic Neuropathy to avoid delayed diagnosis and misdiagnosis.

Leber's Hereditary Optic Neuropathy (LHON) is a maternally inherited optic nerve disease primarily caused by mutations in mitochondrial DNA (mtDNA). The peak of onset is typically between 15 and 30 years, but variability exists. Misdiagnosis, often as inflammatory optic neuritis, delays treatment, compounded by challenges in timely genetic diagnosis. Given the availability of a specific treatment for LHON, its early diagnosis is imperative to ensure therapeutic appropriateness. This work gives an updated guidance about LHON differential diagnosis to clinicians dealing also with multiple sclerosi and neuromyelitis optica spectrtum disorders-related optic neuritis. LHON diagnosis relies on clinical signs and paraclinical evaluations. Differential diagnosis in the acute phase primarily involves distinguishing inflammatory optic neuropathies, considering clinical clues such as ocular pain, fundus appearance and visual recovery. Imaging analysis obtained with Optical Coherence Tomography (OCT) assists clinicians in early recognition of LHON and help avoiding misdiagnosis. Genetic testing for the three most common LHON mutations is recommended initially, followed by comprehensive mtDNA sequencing if suspicion persists despite negative results. We present and discuss crucial strategies for accurate diagnosis and management of LHON cases.

A comprehensive atlas of nuclear sequences of mitochondrial origin (NUMT) inserted into the pig genome

BackgroundThe integration of nuclear mitochondrial DNA (mtDNA) into the mammalian genomes is an ongoing, yet rare evolutionary process that produces nuclear sequences of mitochondrial origin (NUMT). In this study, we identified and analysed NUMT inserted into the pig (Sus scrofa) genome and in the genomes of a few other Suinae species. First, we constructed a comparative distribution map of NUMT in the Sscrofa11.1 reference genome and in 22 other assembled S. scrofa genomes (from Asian and European pig breeds and populations), as well as the assembled genomes of the Visayan warty pig (Sus cebifrons) and warthog (Phacochoerus africanus). We then analysed a total of 485 whole genome sequencing datasets, from different breeds, populations, or Sus species, to discover polymorphic NUMT (inserted/deleted in the pig genome). The insertion age was inferred based on the presence or absence of orthologous NUMT in the genomes of different species, taking into account their evolutionary divergence. Additionally, the age of the NUMT was calculated based on sequence degradation compared to the authentic mtDNA sequence. We also validated a selected set of representative NUMT via PCR amplification.ResultsWe have constructed an atlas of 418 NUMT regions, 70 of which were not present in any assembled genomes. We identified ancient NUMT regions (older than 55 million years ago, Mya) and NUMT that appeared at different time points along the Suinae evolutionary lineage. We identified very recent polymorphic NUMT (private to S. scrofa, with < 1 Mya), and more ancient polymorphic NUMT (3.5–10 Mya) present in various Sus species. These latest polymorphic NUMT regions, which segregate in European and Asian pig breeds and populations, are likely the results of interspecies admixture within the Sus genus.ConclusionsThis study provided a first comprehensive analysis of NUMT present in the Sus scrofa genome, comparing them to NUMT found in other species within the order Cetartiodactyla. The NUMT-based evolutionary window that we reconstructed from NUMT integration ages could be useful to better understand the micro-evolutionary events that shaped the modern pig genome and enriched the genetic diversity of this species.

Intraspecific Variation of Brown Rat Rattus norvegicus in Russia by D-Loop mtDNA Data.

For the first time in Russia, the intraspecific structure of the brown rat Rattus norvegicus was studied using the mitochondrial control region (D-loop) as a molecular marker. The mtDNA sequence was determined in brown rats from eight regions of European and Asian Russia. Three rat clades were identified, including one in European Russia and two in Asian Russia. The synanthropic subspecies R. n. norvegicus was found to have two, European and Asian, lineages. The European lineage included haplotypes from central and southern regions of Russia, and the Asian lineage included haplotypes from Eastern Siberia and the Russian Far East. The exoanthropic subspecies R. n. caraco from the Russian Far East formed a separate genetic lineage.

Forensic efficiency evaluation of a mtDNA whole genome sequencing system constructed with long fragment amplification strategy on DNA nanoball sequencing platform

Mitochondrial DNA (mtDNA) is an important genetic marker for degraded biological sample identification, maternal pedigree tracing, and population genetic structure study owing to its characteristics of high copy number, anti-degradable ring structure, and maternal inheritance. Whole mtDNA genome sequencing is an optimal method for the analysis of mtDNA polymorphism and heterogeneity because it allows for the comprehensive use of maternal genetic information. However, because of lacking quantitative evaluations for sequencing data, the scientific interpretation standards for mtDNA sequencing results of the previously used sequencing systems are often different, and false positive or false negative results are prone to occur when faced with the interference of nuclear genomic DNA, or the heterogeneities of mtDNA sequence and structure. In this study, we evaluated a novel mtDNA whole genome sequencing system using long fragment amplification strategy on the DNA nanoball (DNB) sequencing platform. This system demonstrated high sequencing quality and specific mtDNA sequencing efficiencies on positive control DNA and FTA bloodstain samples, as the average Q20 and Q30 values of the corresponding samples were 97.17 % and 91.93 %; 97.37 % and 92.48 %, respectively. The mean mapping percentages for the reference sequences of whole genome DNA (wgDNA), mtDNA, and nuclear genomic DNA (ngDNA) in the corresponding samples were 99.98 %, 99.97 %, 0.03 %, and 99.91 %, 99.40 %, 0.60 %; respectively. The average error calling rates for the bases A, C, G, and T of the whole mtDNA genome were 0.2519 %, 0.2550 %, 0.2906 %; and 0.2392 %, respectively. The efficacy of heteroplasmy identification was assessed using a set of theoretical sites with predetermined rates. These sites were created by combining the samples with known mtDNA haplotypes in certain proportions. The absolute errors between observed and theoretical heteroplasmy values were 89.59 %, 74.68 %, 50.20 %, 12.65 %, 8.31 %, and 4.85 %, while the theoretical heteroplasmy values were 5 %, 10 %, 20 %, 80 %, 90 %, and 95 %, respectively. The absolute error exhibited relative stability when the mtDNA sequencing depth exceeded 500×. Furthermore, the system sequencing efficiency was also confirmed among different kinds of samples, and these samples included natural samples (e.g., peripheral blood samples preserved on FTA cards for 2 and 11 years, and on filter paper for 6 and 9 years), degraded samples, sensitivity samples, samples derived from various bodily fluids, and maternal pedigree samples. In summary, the whole mtDNA genome sequencing system used for forensic identification demonstrated high performance in analyzing mtDNA sequence information, and showed significant prospects for forensic application and maternal genetic research.

Morphological and molecular evidence support four new Liberonautes Bott, 1955 (Decapoda, Potamonautidae, Liberonautinae) species of freshwater crabs from Sierra Leone, West Africa

Abstract Four new Liberonautes species of freshwater crabs are described from material collected during an expedition to the rainforests of Sierra Leone, West Africa. Liberonautes kasonko sp. nov., L. njala sp. nov., L. schubarti sp. nov. and L. sugarloaf sp. nov. are recognized based on a combination of morphological characters including the carapace, adult male chelipeds, and first gonopods, and on genetic evidence involving analyses of partial segments of the mitochondrial genes of COI, 16S rRNA and 12S rRNA. Diagnoses, illustrations, and a tree of phylogenetic relationships based on mtDNA sequences, as well as a map showing the distributions of the new species are provided.