A comparison of microsatellite polymorphism and heterozygosity among field and laboratory populations of Schistosoma mansoni

The genetic diversity of a field population (recently collected in Melquiades, Brazil) and two laboratory strains (LE and NMRI) of a human blood fluke, Schistosoma mansoni, were analysed using microsatellite markers. Data from the three groups showed an extreme and consistent discrepancy in the level of polymorphism at all microsatellite loci between the field population and laboratory populations. The numbers of alleles detected in LE and NMRI populations averaged only 14 and 10% of those found in the field population, respectively. Especially apparent was the abundance of rare alleles in the Melquiades population when compared with the laboratory strains. The reduction in allelic diversity in the laboratory strains is most likely due to the founder effect and potential bottlenecks that may have occurred during decades of laboratory maintenance. Surprisingly, a much less drastic difference was found when comparing the average heterozygosity of the field population with the laboratory strains. This apparent anomaly may be explained by observed population substructuring (and a potential resultant Wahlund effect) in the natural population. Our comparison of genetic diversity between laboratory and field populations of S. mansoni emphasizes the need for studies of representative populations in schistosome vaccine development.     

Mitochondrial inheritance in Schistosoma mansoni: mitochondrial variable number tandem repeat mutation produces noise on top of the signal.

The Schistosoma mansoni mitochondrial genome contains tandemly arrayed copies of a 62-base repeat motif. The tandem array is highly polymorphic with respect to number of repeats and commonly exhibits heteroplasmy. This study shows that a very high rate of mutation rapidly produces new repeat lengths (new haplotypes) for this mitochondrial variable number tandem repeat. A maternal inheritance pattern is also demonstrated for this repeat sequence, while the high mutation rate causes some offspring to exhibit nonmaternal haplotypes. Frequent generation of new haplotypes can be observed within samples of clonal cohorts taken from monomiracidial snail infections. These same clonal cercarial groups, when crossed, produce F1 generations that exhibit the maternal set of haplotypes, across all individuals, with the frequent addition of new mutant haplotypes. In each of 2 crosses, a subset of the recently arisen haplotypes match paternal haplotypes by chance (30.4% and 18.8%), thus giving the false appearance of partial paternal inheritance of mitochondria.     

Global patterns reveal strong population structure in Haemonchus contortus, a nematode parasite of domesticated ruminants

We have examined the global population genetic structure of Haemonchus contortus. The genetic variability was studied using both amplified fragment length polymorphism (AFLP) and nad4 sequences of the mitochondrial genome. To examine the performance and information content of the two different marker systems, comparative assessment of population genetic diversity was undertaken in 19 isolates of H. contortus, a parasitic nematode of small ruminants. A total of 150 individual adult worms representing 14 countries from all inhabited continents were analysed. Altogether 1,429 informative AFLP markers were generated using four different primer combinations. Also, the genetic variation was high, which agrees with results from previous AFLP studies of nematode parasites of livestock. The genetic structure was high, indicating limited gene flow between the different isolates and populations from each continent mostly formed monophyletic groups in the phylogenetic analysis. However, for isolates representing Australia, Greece and one laboratory strain that originated from South Africa (WRS), there was no clear genetic relationship between the isolates and the distance between their geographical origins. Basically the same pattern was observed for the mitochondrial marker, although the phylogenetic analysis was less resolved than for AFLP. In contrast with previous findings on the population genetic structure of H. contortus, the calculation of population structure gave high values (Nst=0.59). The strong structure was present also for the four Swedish isolates (Nst=0.16) representing a small geographical area.     

Concordant evolution of mitochondrial and nuclear genomes in the wheat pathogen Phaeosphaeria nodorum

We compared patterns of mitochondrial restriction fragment length polymorphism (RFLP) diversity with patterns of nuclear RFLP diversity to investigate the effects of selection, gene flow, and sexual reproduction on the population genetic structure and evolutionary history of the wheat pathogen Phaeosphaeria nodorum. A total of 315 fungal isolates from Texas, Oregon, and Switzerland were analyzed using seven nuclear RFLP probes that hybridized to discrete loci and purified mitochondrial DNA that hybridized to the entire mtDNA genome. Forty-two different mitochondrial haplotypes and 298 different nuclear haplotypes were detected. The two most frequent mtDNA haplotypes were present in every population and represented 32% of all isolates. High levels of gene flow, low levels of population subdivision, no evidence for either host specificity or cyto-nuclear disequilibrium were inferred from the analysis of both genomes. The concordance in estimates of these population genetic parameters from both genomes suggests that the two genomes experienced similar degrees of migration, genetic drift and selection.     

Single-cell mtDNA heteroplasmy in colorectal cancer

Human mitochondria can be genetically distinct within the same individual, a phenomenon known as heteroplasmy. In cancer, this phenomenon seems exacerbated, and most mitochondrial mutations seem to be heteroplasmic. How this genetic variation is arranged within and among normal and tumor cells is not well understood. To address this question, here we sequenced single-cell mitochondrial genomes from multiple normal and tumoral locations in four colorectal cancer patients. Our results suggest that single cells, both normal and tumoral, can carry various mitochondrial haplotypes. Remarkably, this intra-cell heteroplasmy can arise before tumor development and be maintained afterward in specific tumoral cell subpopulations. At least in the colorectal patients studied here, the somatic mutations in the single-cells do not seem to have a prominent role in tumorigenesis.     

Interhomologue sequence variation of alpha satellite DNA from human chromosome 17: Evidence for concerted evolution along haplotypic lineages

Alpha satellite DNA is a family of tandemly repeated DNA found at the centromeres of all primate chromosomes. Different human chromosomes 17 in the population are characterized by distinct alpha satellite haplotypes, distinguished by the presence of variant repeat forms that have precise monomeric deletions. Pairwise comparisons of sequence diversity between variant repeat units from each haplotype show that they are closely related in sequence. Direct sequencing of PCR-amplified alpha satellite reveals heterogeneous positions between the repeat units on a chromosome as two bands at the same position on a sequencing ladder. No variation was detected in the sequence and location of these heterogeneous positions between chromosomes 17 from the same haplotype, but distinct patterns of variation were detected between chromosomes from different haplotypes. Subsequent sequence analysis of individual repeats from each haplotype confirmed the presence of extensive haplotype-specific sequence variation. Phylogenetic inference yielded a tree that suggests these chromosome 17 repeat units evolve principally along haplotypic lineages. These studies allow insight into the relative rates and/or timing of genetic turnover processes that lead to the homogenization of tandem DNA families. Correspondence to: H.F. Willard     

Heteroplasmy of Short Tandem Repeats in Mitochondrial DNA of Atlantic Cod, Gadus Morhua

The mitochondrial DNA of the Atlantic cod (Gadus morhua) contains a tandem array of 40-bp repeats in the D-loop region of the molecule. Variation among molecules in the copy number of these repeats results in mtDNA length variation and heteroplasmy (the presence of more than one form of mtDNA in an individual). In a sample of fish collected from different localities around Iceland and off George's Bank, each individual was heteroplasmic for two or more mtDNAs ranging in repeat copy number from two (common) to six (rare). An earlier report on mtDNA heteroplasmy in sturgeon (Acipenser transmontanus) presented a competitive displacement model for length mutations in mtDNAs containing tandem arrays and the cod data deviate from this model. Depending on the nature of putative secondary structures and the location of D-loop strand termination, additional mechanisms of length mutation may be needed to explain the range of mtDNA length variants maintained in these populations. The balance between genetic drift and mutation in maintaining this length polymorphism is estimated through a hierarchical analysis of diversity of mtDNA length variation in the Iceland samples. Eighty percent of the diversity lies within individuals, 8% among individuals and 12% among localities. An estimate of theta = 2N(eo) mu greater than 1 indicates that this system is characterized by a high mutation rate and is governed primarily by deterministic dynamics. The sequences of repeat arrays from fish collected in Norway, Iceland and George's Bank show no nucleotide variation suggesting that there is very little substructuring to the North Atlantic cod population.     

Genetic diversity assessed by amplified fragment length polymorphism analysis of the parasitic nematode Dictyocaulus viviparus the lungworm of cattle

We have examined the population genetic structure in a collection of nine isolates of the parasitic lungworm Dictyocaulus viviparus. Eight of the isolates were sampled from cattle in geographically separated farms throughout south-central Sweden, and one isolate was a laboratory strain that has been maintained in experimentally infected calves for almost four decades. A total of 72 worms were examined, with eight individual worms from the same individual host representing each isolate. The genetic variation as revealed by amplified fragment length polymorphism analysis using four selective primer combinations was high. Depending on the primer combination a total of 66-79 restriction fragments were amplified, with 26-44 peaks of similar complexity from each of the isolates. The heterozygosity within populations was relatively small, as were the population mutation and immigration rates, which seemed to be in neutral equilibrium. The genetic diversity was therefore reasonably well structured in the field; and the laboratory isolate was quite distinct from the field samples. There was no relationship between the patterns of genetic diversity and the geographical proximity of the farms. The estimates of heterozygosity were much larger and more consistent than those previously estimated for this nematode species using mitochondrial sequencing, and the genetic structuring was thus much less pronounced and the gene flow greater. We attribute these differences in estimation to the broader sampling of loci available using amplified fragment length polymorphism markers, which may therefore constitute a superior technique for the study of patterns of lungworm diversity. Furthermore, the data estimating gene flow for D. viviparus was less than previously reported for closely related species in North America. This might be related to different rates of movements of infected hosts. It seems likely that lungworm infections are rather persistent on different farms, and the sudden outbreaks of disease that can be observed with host movements are most likely to be related to the introduction of susceptible stock.     

Transmitochondrial differences and varying levels of heteroplasmy in nuclear transfer cloned cattle

To assess the extent of cytoplasmic genetic variability in cloned cattle produced by nuclear transplantation procedures, we investigated 29 individuals of seven male cattle clones (sizes 2–6) from two different commercial sources. Restriction enzyme and direct sequence analysis of mitochondrial DNA (mtDNA) detected a total of 12 different haplotypes. Transmitochondrial individuals (i.e., animals which share identical nuclei but have different mitochondrial DNA) were detected in all but one of the clones, demonstrating that mtDNA variation among cloned cattle is a very common phenomenon which prevents true genetic identity. The analyses also showed that the cytoplasmic genetic status of some investigated individuals and clones is further complicated by heteroplasmy (more than one mtDNA type in an individual). The relative proportions of different mtDNA‐types in two animals with mild heteroplasmy were estimated at 2:98% and 4:96% in DNA samples derived from blood. This is in agreement with values expected from karyoplast‐cytoplast volume ratios. In contrast, the mtDNA haplotype proportions observed in six other heteroplasmic animals of two different clones ranged from 21:79% to 57:43%, reflecting a marked increase in donor blastomere mtDNA contributions. These results suggest that mtDNA type of donor embryos and recipient oocytes used in nuclear transfer cattle cloning should be controlled to obtain true clones with identical nuclear and cytoplasmic genomes. Mol. Reprod. Dev. 54:24–31, 1999. © 1999 Wiley‐Liss, Inc.     

Molecular evolution of a plastid tandem repeat locus in an orchid lineage

The molecular evolution of a chloroplast minisatellite locus in the Anacamptis palustris (Orchidaceae) lineage and haplotype variation in two Italian A. palustris populations were investigated. A phylogenetic analyses of the chloroplast tRNA(LEU) intron, where the minisatellite locus is located, revealed that a deletion in the ancestor of the A. palustris lineage led to the formation of two noncontiguous, complementary sequence motifs. We propose a model to explain the initial formation of the minisatellite repeat motif, starting with the two noncontiguous, complementary sequence motifs. A survey of minisatellite variation in four species of the A. palustris lineage revealed several haplotypes that differed not only in repeat number, but also in repeat organization. A haplotype network suggests that three different minisatellite loci evolved independently at the same position in the tRNA(LEU) intron. A secondary structure model revealed that the A. palustris minisatellite repeat forms a stem region of the tRNA(LEU) intron, which allows its notable expansion without negatively affecting splicing. Minisatellite variation was high in the two examined A. palustris populations where 20 haplotypes were detected, whereas no length variation was detected in a neighboring poly (A) microsatellite locus. We estimated a chloroplast minisatellite mutation rate of 3.2 x 10(-3) mutations per generation. Southern blot analyses did not find evidence for chloroplast heteroplasmy. Based on the analysis of the largest known, extant A. palustris population, a stepwise mutation model (SMM) was inferred.     

Origin and diversification of the human parasite Schistosoma mansoni

Schistosoma mansoni is the most widespread of the human-infecting schistosomes, present in 54 countries, predominantly in Africa, but also in Madagascar, the Arabian Peninsula, and the Neotropics. Adult-stage parasites that infect humans are also occasionally recovered from baboons, rodents, and other mammals. Larval stages of the parasite are dependent upon certain species of freshwater snails in the genus Biomphalaria, which largely determine the parasite's geographical range. How S. mansoni genetic diversity is distributed geographically and among isolates using different hosts has never been examined with DNA sequence data. Here we describe the global phylogeography of S. mansoni using more than 2500 bp of mitochondrial DNA (mtDNA) from 143 parasites collected in 53 geographically widespread localities. Considerable within-species mtDNA diversity was found, with 85 unique haplotypes grouping into five distinct lineages. Geographical separation, and not host use, appears to be the most important factor in the diversification of the parasite. East African specimens showed a remarkable amount of variation, comprising three clades and basal members of a fourth, strongly suggesting an East African origin for the parasite 0.30-0.43 million years ago, a time frame that follows the arrival of its snail host. Less but still substantial variation was found in the rest of Africa. A recent colonization of the New World is supported by finding only seven closely related New World haplotypes which have West African affinities. All Brazilian isolates have nearly identical mtDNA haplotypes, suggesting a founder effect from the establishment and spread of the parasite in this large country.     

Phylogenetic relationships of rat lungworm, Angiostrongylus cantonensis, isolated from different geographical regions revealed widespread multiple lineages

We conducted a pilot survey of genetic variation of A. cantonensis using small subunit (SSU) ribosomal (r) RNA and mitochondrial cytochrome c oxidase subunit I (coxI) gene sequences. Two distinct SSU genotypes (G1 and G2) were identified among 17 individual A. cantonensis worms from 17 different geographical localities in Japan, Mainland China, Taiwan, and Thailand. The partial coxI sequences were determined for 83 worms from 18 different geographical localities from Japan, Mainland China, Taiwan, and Thailand. Phylogenetic analysis showed eight distinct coxI haplotypes (ac1 to ac8). In 16 out of 18 localities, only a single coxI haplotype was found. However, in two localities, two coxI haplotypes coexisted. The common haplotypes found were: haplotype ac1 (Tokyo, Chiba, Kanagawa, Amamioshima Island, and Taichung), haplotype ac2 (Ishikawa, Shenzhen, and Lianjiang), haplotype ac5 (the Okinawa and the Ogasawara Islands), and haplotype ac7 (Miyagi, Aichi, and Kanagawa). Each of these regions is separated from the others by high mountain ranges or oceans. In addition, the lower genetic variation and particular geographical distribution of A. cantonensis in each location could indicate a founder effect, which may have resulted from multiple independent origins, and suggests that haplotypes migrated from endemic areas via human-related transportation.     

A model to explain the origin of a parasite sex-specific population structure

A discrete time model was built to understand the origin of the sex-specific population structure of the human blood fluke, Schistosoma mansoni. We have estimated both male/female individual ratio and male/female genotype ratio of this parasite taking into account all the experimental published values on differential male and female life-history traits all along the life cycle. We considered in our model male and female life-history traits when both separated and together. The model showed that both male/female individual ratio and male/female genotype ratio of S. mansoni adults are biased toward males in each combination. This bias was more important in male/female genotype ratio than in the male/female individual ratio for the same initial values of cercarial development success. This model could explain the sex specific population structure of this parasite. Firstly, we showed that the male-biased individual ratio finds its origin in the vertebrate host. Secondly, we showed that the male-biased genotype ratio originates prior to any interrelationship between adult worms and could generate by itself a sex-specific genetic structure.     

Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis

In a search for specific molecular markers for population analysis of Puccinia striiformis f. sp. tritici, the ribosomal DNA (rDNA) intergenic spacer (IGS) 1 region (rDNA-IGS1, between the 28S and the 5S rDNA genes) was amplified, cloned, and sequenced. It was found to exhibit multiple bands and length polymorphism. Surprisingly, single isolates were found to possess between three to five different IGS1 haplotypes. Bands were cloned and sequenced, and two highly variable regions (α and β) were found between conserved regions, with repeat units interspersed in both types of regions. There were 14 different repeat units, and these were sometimes grouped further into four combinations of repeat units, with a few individual nucleotides (A or C) inserted between the repeats. Among three geographically dispersed isolates, the variable region α was divided into eight types, and the variable region β was divided into two types based on repeat units. Most of the 14 repeat units were shared by the variable and the conserved regions. Among the three isolates, there were a total of 12 IGS1 haplotypes, but some of these were shared between isolates such that there were only eight unique haplotypes. The occurrence of multiple haplotypes within single isolates may be useful for analyzing the population structure, tracking the origin of annual epidemics and providing insights into evolutionary biology of this pathogen.     

Genetic diversity of the arbuscular mycorrhizal fungus Glomus intraradices as determined by mitochondrial large subunit rRNA gene sequences is considerably higher than previously expected

Glomus intraradices is a widespread arbuscular mycorrhizal fungus (AMF), which has been found in an extremely broad range of habitats, indicating a high tolerance for environmental factors and a generalist life history strategy. Despite this ecological versatility, not much is known about the genetic diversity of this fungal species across different habitats or over large geographic scales. A nested polymerase chain reaction (PCR) approach for the mitochondrial rRNA large subunit gene (mtLSU), distinguished different haplotypes among cultivated isolates of G. intraradices and within mycorrhizal root samples from the field. From analysis of 16 isolates of this species originating from five continents, 12 mitochondrial haplotypes were distinguished. Five additional mtLSU haplotypes were detected in field-collected mycorrhizal roots. Some introns in the mtLSU region appear to be stable over years of cultivation and are ancestral to the G. intraradices clade. Genetic diversity within G. intraradices is substantially higher than previously thought, although some mtLSU haplotypes are widespread. A restriction fragment length polymorphism approach also was developed to distinguish mtLSU haplotypes without sequencing. Using this molecular tool, intraspecific genetic variation of an AMF species can be studied directly in field plants.     

The association between mitochondrial genetic variation and reduced colony fitness in an invasive wasp

Despite the mitochondrion's long‐recognized role in energy production, mitochondrial DNA (mtDNA) variation commonly found in natural populations was assumed to be effectively neutral. However, variation in mtDNA has now been increasingly linked to phenotypic variation in life history traits and fitness. We examined whether the relative fitness in native and invasive common wasp (Vespula vulgaris) populations in Belgium and New Zealand (NZ), respectively, can be linked to mtDNA variation. Social wasp colonies in NZ were smaller with comparatively fewer queen cells, indicating a reduced relative fitness in the invaded range. Interestingly, queen cells in this population were significantly larger leading to larger queen offspring. By sequencing 1,872 bp of the mitochondrial genome, we determined mitochondrial haplotypes and detected reduced genetic diversity in NZ. Three common haplotypes in NZ frequently produced many queens, whereas the four rare haplotypes produced significantly fewer or no queens. The entire mitochondrial genome for each of these haplotypes was sequenced to identify polymorphisms associated with fitness reduction. We found 16 variable sites; however, no nonsynonymous mutation that was clearly causing impaired mitochondrial function was detected. We discuss how detected variants may alter secondary structures, gene expression or mito‐nuclear interactions, or could be associated with nuclear‐encoded variation. Whatever the ultimate mechanism, we show reduced fitness and mtDNA variation in an invasive wasp population as well as specific mtDNA variants associated with fitness variation within this population. Ours is one of only a few studies that confirm fitness impacts of mtDNA variation in wild nonmodel populations.     

To Fire the Train: A Second Malignant-Hyperthermia Gene

During direct sequencing of the first hypervariable segment of the human mitochondrial control region, we identified one individual with a heteroplasmic point mutation at nt 16,256. We used primer extension to analyze the proportions of each mitochondrial haplotype in peripheral blood, buccal cells, and single hair roots from this individual and from eight members of his maternal lineage. Significant levels of heteroplasmy were found in only three individuals, and, in these cases, the proportions of each haplotype were similar in both blood and buccal cells. From the changes in mitochondrial haplotypes within mother-offspring pairs, we calculated that the most likely size of a mitochondrial bottleneck during development was 1-27 segregating units. However, highly variable levels of heteroplasmy were found in single hair roots, even among roots from the same individual. We analyzed a large number of hair roots from one individual and found that the proportion of one haplotype was within a range of 9% to > 99% in different roots. Roots originating from within a small patch of skin had haplotype proportions as variable as those from different areas of skin.     

Application of mtDNA d-loop region for the study of Russian sturgeon population structure from Iranian coastline of the Caspian Sea

The population structure of the Russian sturgeon ( Acipenser gueldenstaedtii ) was investigated using PCR amplification of the mtDNA D-loop region. Seven composite haplotypes were detected and average nucleotide and haplotype diversity over all samples were found to be 0.05 ± 0.00 and 0.75 ± 0.00 (mean ± SE) respectively. Restriction digest of the mtDNA D-loop region detected two genotypes A and B with a relative high frequencies of 0.5 and 0.36 respectively. These two genotypes (A and B) can be considered as potential genetic markers for different biological groups, stocks or seasonal races of Russian sturgeon. A maximum of 10.8% nucleotide diversity was found between haplotypes AAAAB and BBBBF. 100% heteroplasmy was found in Russian sturgeon and restriction digest of the mtDNA D-loop region also exhibited site heteroplasmy.     

Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes

The present study compared the genetic variation among 14 different geographical isolates of Opisthorchis viverrini sensu lato from Thailand and Lao PDR using sequence data for 2 mitochondrial DNA genes, the subunit 1 of NADH dehydrogenase gene (nad1) and cytochrome c oxidase gene (cox1). Four different nad1 haplotypes were detected among isolates, all of which were identical at the amino acid sequence level. Nucleotide sequence variation among 14 isolates ranged from 0 to 0.3% for nad1. Two different cox1 haplotypes were detected among isolates. These two haplotypes differed at 2 nucleotide positions, one of which resulted in a change in the amino acid sequence. Nucleotide sequence variation among isolates for cox1 ranged from 0 to 0.5%. Comparison of cox1 sequences of O. viverrini to those of other trematodes revealed nucleotide differences of 13-31%. A phylogenetic analysis of the cox1 sequence data revealed strong statistical support for a clade containing O. viverrini and 2 other species of opisthorchid trematodes; O. felineus and Clonorchis sinsensis.     

Clonal diversity and geographic structure in <Emphasis Type="Italic">Pleurochaete squarrosa</Emphasis> (Pottiaceae): different sampling scale approach

This paper describes our investigation of genetic variation and clonal structure of the Mediterranean moss Pleurochaete squarrosa (Brid.) Lindb. (Pottiaceae), using inter-simple sequence repeat (ISSR) molecular markers and trnL_UAA (intron of plastid gene for Leu tRNA) sequence, choosing different sampling strategies and scales on 16 European populations. Moreover, the intercontinental distribution of two trnL haplotypes, previously found over a large area and including 24 populations in three continents, was also investigated. Despite the prevalent asexual reproduction, P. squarrosa shows a high level of genetic diversity. Some site features seem to affect the clonal structure at the local scale, influencing the relocation of detached fragments and the level of intermingling, but they do not substantially affect genetic diversity. The peculiar vegetative reproduction coupled with somatic mutation could partly account for the genetic variation detected. Genetic distances highlight geographic isolation and limited gene flow among populations. We found only two trnL haplotypes in Europe due to length polymorphism, but, over an intercontinental scale, only non-delete trnL was found in Africa and the USA. ISSR analysis within each population detected a higher genetic distance between the samples with different trnL haplotypes, suggesting the presence of two different genetic lineages within this species, geographically overlapping in the Mediterranean Basin.