Mitochondrial genome sequence diversity of Indian Plasmodium falciparum isolates

We have analysed the whole mitochondrial (mt) genome sequences (each ~6 kilo nucleotide base pairs in length) of four field isolates of the malaria parasite Plasmodium falciparum collected from different locations in India. Comparative genomic analyses of mt genome sequences revealed three novel India-specific single nucleotide polymorphisms. In general, high mt genome diversity was found in Indian P. falciparum, at a level comparable to African isolates. A population phylogenetic tree placed the presently sequenced Indian P. falciparum with the global isolates, while a previously sequenced Indian isolate was an outlier. Although this preliminary study is limited to a few numbers of isolates, the data have provided fundamental evidence of the mt genome diversity and evolutionary relationships of Indian P. falciparum with that of global isolates.     

Inferring the evolutionary history of Indian Plasmodium vivax from population genetic analyses of multilocus nuclear DNA fragments

The human malaria parasite Plasmodium vivax is globally widespread, causing high malaria morbidity. As P. vivax is highly endemic to India, and previous reports indicate genetic homogeneity in population samples, we tested the hypothesis of no genetic structuring in Indian P. vivax. Further, based on the reports of increasing incidence of Plasmodium falciparum infection in comparison with P. vivax in recent years in India, it was important to understand whether reduction in population size has resulted in decrease in P. vivax infection rate in India. For this, we utilized recently developed putatively neutral markers from chromosome 13 of P. vivax to score single nucleotide polymorphisms in 126 P. vivax isolates collected from 10 different places in India. The overall results indicated that Indian P. vivax bears high nucleotide diversity within population samples but moderate amount of genetic differentiation between population samples. STRUCTURE analysis grouped 10 population samples into three clusters based on the proportion of the genetic ancestries in each population. However, the pattern of clustering does not correlate with sampling locations in India. Furthermore, analyses of past demographic events indicated reduction in population size in majority of population samples, but when isolates from all the 10 samples were considered as a single population, the data fit to the demographic equilibrium model. All these observations clearly indicate that Indian P. vivax presents complex evolutionary history but possesses several features of being a part of ancestral distribution range of this species.     

Ancient mitochondrial genomes from Chinese cave hyenas provide insights into the evolutionary history of the genus Crocuta

Cave hyenas (genus Crocuta) are extinct bone-cracking carnivores from the family Hyaenidae and are generally split into two taxa that correspond to a European/Eurasian and an (East) Asian lineage. They are close relatives of the extant African spotted hyenas, the only extant member of the genus Crocuta. Cave hyenas inhabited a wide range across Eurasia during the Pleistocene, but became extinct at the end of the Late Pleistocene. Using genetic and genomic datasets, previous studies have proposed different scenarios about the evolutionary history of Crocuta. However, causes of the extinction of cave hyenas are widely speculative and samples from China are severely understudied. In this study, we assembled near-complete mitochondrial genomes from two cave hyenas from northeastern China dating to 20 240 and 20 253 calBP, representing the youngest directly dated fossils of Crocuta in Asia. Phylogenetic analyses suggest a monophyletic clade of these two samples within a deeply diverging mitochondrial haplogroup of Crocuta. Bayesian analyses suggest that the split of this Asian cave hyena mitochondrial lineage from their European and African relatives occurred approximately 1.85 Ma (95% CI 1.62–2.09 Ma), which is broadly concordant with the earliest Eurasian Crocuta fossil dating to approximately 2 Ma. Comparisons of mean genetic distance indicate that cave hyenas harboured higher genetic diversity than extant spotted hyenas, brown hyenas and aardwolves, but this is probably at least partially due to the fact that their mitochondrial lineages do not represent a monophyletic group, although this is also true for extant spotted hyenas. Moreover, the joint female effective population size of Crocuta (both cave hyenas and extant spotted hyenas) has sustained two declines during the Late Pleistocene. Combining this mitochondrial phylogeny, previous nuclear findings and fossil records, we discuss the possible relationship of fossil Crocuta in China and the extinction of cave hyenas.     

Comparative mitochondrial genome analysis reveals the evolutionary rearrangement mechanism in Brassica

The genus Brassica has many species that are important for oil, vegetable and other food products. Three mitochondrial genome types (mitotype) originated from its common ancestor. In this paper, a B. nigra mitochondrial main circle genome with 232,407 bp was generated through de novo assembly. Synteny analysis showed that the mitochondrial genomes of B. rapa and B. oleracea had a better syntenic relationship than B. nigra. Principal components analysis and development of a phylogenetic tree indicated maternal ancestors of three allotetraploid species in Us triangle of Brassica. Diversified mitotypes were found in allotetraploid B. napus, in which napus‐type B. napus was derived from B. oleracea, while polima‐type B. napus was inherited from B. rapa. In addition, the mitochondrial genome of napus‐type B. napus was closer to botrytis‐type than capitata‐type B. oleracea. The sub‐stoichiometric shifting of several mitochondrial genes suggested that mitochondrial genome rearrangement underwent evolutionary selection during domestication and/or plant breeding. Our findings clarify the role of diploid species in the maternal origin of allotetraploid species in Brassica and suggest the possibility of breeding selection of the mitochondrial genome.     

Whole Mitochondrial Genome Sequence of an Indian Plasmodium falciparum Field Isolate

Mitochondrial genome sequence of malaria parasites has served as a potential marker for inferring evolutionary history of the Plasmodium genus. In Plasmodium falciparum, the mitochondrial genome sequences from around the globe have provided important evolutionary understanding, but no Indian sequence has yet been utilized. We have sequenced the whole mitochondrial genome of a single P. falciparum field isolate from India using novel primers and compared with the 3D7 reference sequence and 1 previously reported Indian sequence. While the 2 Indian sequences were highly divergent from each other, the presently sequenced isolate was highly similar to the reference 3D7 strain.     

Evolutionary structure of Plasmodium falciparum major variant surface antigen genes in South America: Implications for epidemic transmission and surveillance

Strong founder effects resulting from human migration out of Africa have led to geographic variation in single nucleotide polymorphisms (SNPs) and microsatellites (MS) of the malaria parasite, Plasmodium falciparum. This is particularly striking in South America where two major founder populations of P. falciparum have been identified that are presumed to have arisen from the transatlantic slave trade. Given the importance of the major variant surface antigen of the blood stages of P. falciparum as both a virulence factor and target of immunity, we decided to investigate the population genetics of the genes encoding “Plasmodium falciparum Erythrocyte Membrane Protein 1” (PfEMP1) among several countries in South America, in order to evaluate the transmission patterns of malaria in this continent. Deep sequencing of the DBLα domain of var genes from 128 P. falciparum isolates from five locations in South America was completed using a 454 high throughput sequencing protocol. Striking geographic variation in var DBLα sequences, similar to that seen for SNPs and MS markers, was observed. Colombia and French Guiana had distinct var DBLα sequences, whereas Peru and Venezuela showed an admixture. The importance of such geographic variation to herd immunity and malaria vaccination is discussed.     

Complete mitochondrial genome sequence of the Himalayan Griffon,Gyps himalayensis (Accipitriformes: Accipitridae): Sequence,structure, and phylogenetic analyses

This is the first study to describe the mitochondrial genome of the Himalayan Griffon, Gyps himalayensis, which is an Old World vulture belonging to the family Accipitridae and occurring along the Himalayas and the adjoining Tibetan Plateau. Its mitogenome is a closed circular molecule 17,381 bp in size containing 13 protein‐coding genes, 22 tRNA coding genes, two rRNA‐coding genes, a control region (CR), and an extra pseudo‐control region (CCR) that are conserved in most Accipitridae mitogenomes. The overall base composition of the G. himalayensis mitogenome is 24.55% A, 29.49% T, 31.59% C, and 14.37% G, which is typical for bird mitochondrial genomes. The alignment of the Accipitridae species control regions showed high levels of genetic variation and abundant AT content. At the 5′ end of the domain I region, a long continuous poly‐C sequence was found. Two tandem repeats were found in the pseudo‐control regions. Phylogenetic analysis with Bayesian inference and maximum likelihood based on 13 protein‐coding genes indicated that the relationships at the family level were (Falconidae + (Cathartidae + (Sagittariidae + (Accipitridae + Pandionidae))). In the Accipitridae clade, G. himalayensis is more closely related to Aegypius monachus than to Spilornis cheela. The complete mitogenome of G. himalayensis provides a potentially useful resource for further exploration of the taxonomic status and phylogenetic history of Gyps species.     

The first complete mitochondrial genome sequence of Nanorana parkeri and Nanorana ventripunctata (Amphibia: Anura: Dicroglossidae), with related phylogenetic analyses

Members of the Nanorana genus (family Dicroglossidae) are often referred to as excellent model species with which to study amphibian adaptations to extreme environments and also as excellent keystone taxa for providing insights into the evolution of the Dicroglossidae. However, a complete mitochondrial genome is currently only available for Nanorana pleskei. Thus, we analyzed the complete mitochondrial genomes of Nanorana parkeri and Nanorana ventripunctata to investigate their evolutionary relationships within Nanorana and their phylogenetic position in the family Dicroglossidae. Our results showed that the genomes of N. parkeri (17,837 bp) and N. ventripunctata (18,373 bp) encode 13 protein‐coding genes (PCGs), two ribosomal RNA genes, 23 transfer RNA (tRNA) genes, and a noncoding control region. Overall sequences and genome structure of the two species showed high degree of similarity with N. pleskei, although the motif structures and repeat sequences of the putative control region showed clear differences among these three Nanorana species. In addition, a tandem repeat of the tRNA‐Met gene was found located between the tRNA‐Gln and ND2 genes. On both the 5′ and 3′‐sides, the control region possessed distinct repeat regions; however, the CSB‐2 motif was not found in N. pleskei. Based on the nucleotide sequences of 13 PCGs, our phylogenetic analyses, using Bayesian inference and maximum‐likelihood methods, illustrate the taxonomic status of Nanorana with robust support showing that N. ventripunctata and N. pleskei are more closely related than they are to N. parkeri. In conclusion, our analyses provide a more robust and reliable perspective on the evolutionary history of Dicroglossidae than earlier analyses, which used only a single species (N. pleskei).     

Few mitochondrial DNA sequences are inserted into the turkey (Meleagris gallopavo) nuclear genome: evolutionary analyses and informativity in the domestic lineage

Mitochondrial DNA (mtDNA) insertions have been detected in the nuclear genome of many eukaryotes. These sequences are pseudogenes originated by horizontal transfer of mtDNA fragments into the nuclear genome, producing nuclear DNA sequences of mitochondrial origin (numt). In this study we determined the frequency and distribution of mtDNA‐originated pseudogenes in the turkey (Meleagris gallopavo) nuclear genome. The turkey reference genome (Turkey_2.01) was aligned with the reference linearized mtDNA sequence using last . A total of 32 numt sequences (corresponding to 18 numt regions derived by unique insertional events) were identified in the turkey nuclear genome (size ranging from 66 to 1415 bp; identity against the modern turkey mtDNA corresponding region ranging from 62% to 100%). Numts were distributed in nine chromosomes and in one scaffold. They derived from parts of 10 mtDNA protein‐coding genes, ribosomal genes, the control region and 10 tRNA genes. Seven numt regions reported in the turkey genome were identified in orthologues positions in the Gallus gallus genome and therefore were present in the ancestral genome that in the Cretaceous originated the lineages of the modern crown Galliformes. Five recently integrated turkey numts were validated by PCR in 168 turkeys of six different domestic populations. None of the analysed numts were polymorphic (i.e. absence of the inserted sequence, as reported in numts of recent integration in other species), suggesting that the reticulate speciation model is not useful for explaining the origin of the domesticated turkey lineage.     

Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India

Inferring the origin and dispersal of the chloroquine-resistant (CQR) malaria parasite, Plasmodium falciparum, is of academic and public health importance. The Pfcrt gene of P. falciparum is widely known as the CQR gene and two major haplotypes of this gene (CVIET and SVMNT) occur widely across CQR-endemic regions of the globe. In India, studies to date of the Pfcrt gene have indicated the widespread prevalence of the SVMNT haplotype (prevalent in the South America and Papua New Guinea), whereas the CVIET haplotype, primarily found in southeast Asia, was not detected at a high frequency in India. This distribution pattern of the two most common CQR-Pfcrt haplotypes in India is quite surprising. Thus, in order to understand probable evolutionary and migration patterns of the CQR-Pfcrt haplotypes into India, we generated new sequence data of exon 2 of the Pfcrt gene and collected published information on the CQR-Pfcrt haplotype data from India, Papua New Guinea, southeast Asia and South America, and performed several population and evolutionary genetic analyses. Among several interesting findings, statistically significant longitudinal clines for the CVIET and SVMNT haplotypes (in opposite directions) in India, and the clustering of India and Papua New Guinea under the SVMNT-specific clade in the phylogenetic tree, are the two most remarkable aspects of the data. It also appears that both the SVMNT and CVIET haplotypes in India have migrated from southeast Asia. In particular, whereas the Indian CVIET haplotype has a southeast Asian origin, the SVMNT haplotype, prevalent in India, seems to have originated in Papua New Guinea and entered India through southeast Asia.     

Plasmodium falciparum: genetic polymorphism in apical membrane antigen-1 gene from Indian isolates

A number of stage-specific antigens have been characterized for vaccine development against Plasmodium falciparum malaria. This study presents a comprehensive analysis of the sequence polymorphism in Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) in population samples from the eastern and western parts of India. This is the first study of its kind for the nearly full length PfAMA-1 gene from these regions in India. Our observations confirmed that sequence diversity of PfAMA-1 confines only to point mutations and shows 4-8% variation as compared to the prototypes. As opposed to the previous studies on PfAMA-1, our study revealed a greater degree of polymorphism in the Domain II region of PfAMA-1 protein, though signature for diversifying selection is seen throughout the gene. Our present investigation also indicates a very high degree of variation in the reported T- and B-cell epitopes of PfAMA-1. Few noteworthy and unique observations made in this study are the substitution of Cysteine residues responsible for the disulfide bond structure of the protein and the presence of premature termination after 595 amino acids in 3 of the 13 isolates under consideration. These crucial findings add new perspectives to the future of AMA-1 research and could have major implications in establishing AMA-1 as a vaccine candidate.     

Plasmodium falciparum: genetic diversity of C-terminal region of MSP-1 in isolates from Indian sub-continent

Malaria parasites exhibit sequence diversity for a number of stage specific antigens. Several studies have proved that merozoite surface protein-1 (MSP-1) is an effective target eliciting a protective immune response. The MSP-1(42) region comprising two EGF-like domains is involved in generating protective immune response in humans and other experimental animals. Searching for point mutations in this region is essential in view of vaccine development. We have investigated the sequence variations in Plasmodium falciparum MSP-1 carboxy terminal region in field isolates from different regions in India. Our study reveals the presence of eight variant types of MSP-1(19) in the Indian sub-continent, which comprise of E-TSR-L, Q-TSR-L, E-TSG-L, Q-KNG-L, Q-KNG-F, E-KNG-L, E-KNG-F, and E-KYG-F. The last named allele is a novel variant being reported for the first time.     

Assessment of Plasmodium falciparum PfMDR1 transport rates using Fluo‐4

Mutations in the multidrug resistance transporter of Plasmodium falciparum PfMDR1 have been implicated to play a significant role in the emergence of worldwide drug resistance, yet the molecular and biochemical mechanisms of this transporter are not well understood. Although it is generally accepted that drug resistance in P. falciparum is partly associated with PfMDR1 transport activity situated in the membrane of the digestive vacuole, direct estimates of the pump rate of this transport process in the natural environment of the intact host–parasite system have never been analysed. The fluorochrome Fluo‐4 is a well‐documented surrogate substrate of PfMDR1 and has been found to accumulate by actively being transported into the digestive vacuole of several parasitic strains. In the present study, we designed an approach to use Fluo‐4 fluorescence uptake as a measure of compartmental Fluo‐4 concentration accumulation in the different compartments of the host–parasite system. We performed a ‘reverse Fluo‐4 imaging' approach to relate fluorescence intensity to changes in dye concentration rather than Ca²⁺ fluctuations and were able to calculate the overall rate of transport for PfMDR1 in Dd2 parasites. With this assay, we provide a powerful method to selectively measure the effect of PfMDR1 mutations on substrate transport kinetics. This will be of high significance for future compound screening to test for new drugs in resistant P. falciparum strains.     

Characterization of the first mitochondrial genome of a catenulid flatworm: Stenostomum leucops (Platyhelminthes)

The first complete mitochondrial genome of a catenulid, Stenostomum leucops, was characterized. Illumina sequencing and 90 813 reads were utilized in the assembly, producing one contig with an average coverage of 1118×. The length of this genome is 15 742 bp with 12 protein‐coding, two rRNA and 22 tRNA genes. Although the atp8 gene is absent in other Platyhelminthes, a highly divergent putative atp8 gene was found in S. leucops. In contrast to other Platyhelminthes, the mitochondrial genes of S. leucops are encoded on both strands. The gene order in the S. leucops mitogenome is very divergent from those observed in other Platyhelminthes, showing only small blocks of synteny. With AAA as the codon for lysine S. leucops shows the probable plesiomorphic condition, whereas Rhabditophora possess the derived GAA. This evolutionary transition is correlated with changes in the respective anticodons in trnK. It remains unclear whether the absence of the D arm and loop in trnS1 is a convergence in Catenulida and Neodermata.     

The effects of allospecific mitochondrial genome on the fitness of northern redbelly dace (Chrosomus eos)

Instantaneous mitochondrial introgression events allow the disentangling of the effects of hybridization from those of allospecific mtDNA. Such process frequently occurred in the fish Chrosomus eos, resulting in cybrid individuals composed of a C. eos nuclear genome but with a C. neogaeus mtDNA. This provides a valuable model to address the fundamental question: How well do introgressed individuals perform in their native environment? We infer where de novo production of cybrids occurred to discriminate native environments from those colonized by cybrids in 25 sites from two regions (West‐Qc and East‐Qc) in Quebec (Canada). We then compared the relative abundance of wild types and cybrids as a measure integrating both fitness and de novo production of cybrids. According to mtDNA variation, 12 introgression events are required to explain the diversity of cybrids. Five cybrid lineages could not be associated with in situ introgression events. This includes one haplotype carried by 93% of the cybrids expected to have colonized West‐Qc. These cybrids also displayed a nearly complete allopatric distribution with wild types. We still inferred de novo production of cybrids at seven sites, that accounted for 70% of the cybrids in East‐Qc. Wild‐type and cybrid individuals coexist in all East‐Qc sites while cybrids were less abundant. Allopatry of cybrids restricted to the postglacial expansion suggests the existence of higher fitness for cybrids in specific conditions, allowing for the colonization of different environments and expanding the species’ range. However, allospecific mtDNA does not provide a higher fitness to cybrids in their native environment compared to wild types, making the success of an introgressed lineage uncertain.     

Phylogeography of var gene repertoires reveals fine‐scale geospatial clustering of Plasmodium falciparum populations in a highly endemic area

Plasmodium falciparum malaria is a major global health problem that is being targeted for progressive elimination. Knowledge of local disease transmission patterns in endemic countries is critical to these elimination efforts. To investigate fine‐scale patterns of malaria transmission, we have compared repertoires of rapidly evolving var genes in a highly endemic area. A total of 3680 high‐quality DBLα‐sequences were obtained from 68 P. falciparum isolates from ten villages spread over two distinct catchment areas on the north coast of Papua New Guinea (PNG). Modelling of the extent of var gene diversity in the two parasite populations predicts more than twice as many var gene alleles circulating within each catchment (Mugil = 906; Wosera = 1094) than previously recognized in PNG (Amele = 369). In addition, there were limited levels of var gene sharing between populations, consistent with local parasite population structure. Phylogeographic analyses demonstrate that while neutrally evolving microsatellite markers identified population structure only at the catchment level, var gene repertoires reveal further fine‐scale geospatial clustering of parasite isolates. The clustering of parasite isolates by village in Mugil, but not in Wosera was consistent with the physical and cultural isolation of the human populations in the two catchments. The study highlights the microheterogeneity of P. falciparum transmission in highly endemic areas and demonstrates the potential of var genes as markers of local patterns of parasite population structure.     

Structural and mechanistic insights into MICU1 regulation of mitochondrial calcium uptake

Mitochondrial calcium uptake is a critical event in various cellular activities. Two recently identified proteins, the mitochondrial Ca²⁺ uniporter (MCU), which is the pore‐forming subunit of a Ca²⁺ channel, and mitochondrial calcium uptake 1 (MICU1), which is the regulator of MCU, are essential in this event. However, the molecular mechanism by which MICU1 regulates MCU remains elusive. In this study, we report the crystal structures of Ca²⁺‐free and Ca²⁺‐bound human MICU1. Our studies reveal that Ca²⁺‐free MICU1 forms a hexamer that binds and inhibits MCU. Upon Ca²⁺ binding, MICU1 undergoes large conformational changes, resulting in the formation of multiple oligomers to activate MCU. Furthermore, we demonstrate that the affinity of MICU1 for Ca²⁺ is approximately 15–20 μM. Collectively, our results provide valuable details to decipher the molecular mechanism of MICU1 regulation of mitochondrial calcium uptake.     

Resequencing and comparison of whole mitochondrial genome to gain insight into the evolutionary status of the Shennongjia golden snub‐nosed monkey (SNJ R. roxellana)

Shennongjia Rhinopithecus roxellana (SNJ R. roxellana) is the smallest geographical population of R. roxellana. The phylogenetic relationships among its genera and species and the biogeographic processes leading to their current distribution are largely unclear. To address these issues, we resequenced and obtained a new, complete mitochondrial genome of SNJ R. roxellana by next‐generation sequencing and standard Sanger sequencing. We analyzed the gene composition, constructed a phylogenetic tree, inferred the divergence ages based on complete mitochondrial genome sequences, and analyzed the genetic divergence of 13 functional mtDNA genes. The phylogenetic tree and divergence ages showed that R. avunculus (the Tonkin snub‐nosed monkey) was the first to diverge from the Rhinopithecus genus ca. 2.47 million years ago (Ma). Rhinopithecus bieti and Rhinopithecus strykeri formed sister groups, and the second divergence from the Rhinopithecus genus occurred ca. 1.90 Ma. R. roxellana and R. brelichi diverged from the Rhinopithecus genus third, ca. 1.57 Ma. SNJ R. roxellana was the last to diverge within R. roxellana species in 0.08 Ma, and the most recent common ancestor of R. roxellana is 0.10 Ma. The analyses on gene composition showed SNJ R. roxellana was the newest geographic population of R. roxellana. The work will help to develop a more accurate protection policy for SNJ R. roxellana and facilitate further research on selection and adaptation of R. roxellana.