Geographically specific heteroplasmy of mitochondrial DNA in the seaweed, Fucus serratus (Heterokontophyta: Phaeophyceae, Fucales)

The presence of more than one type of mitochondrial DNA within the same organism (mtDNA heteroplasmy) has been reported in vertebrates, invertebrates, basidiomycetes and some angiosperms, but never in marine (macro)algae. We examined sequence differences in a 135‐base pair (bp) region of the nad11 gene in mitochondria of the intertidal rockweed, Fucus serratus, using single‐strand conformation polymorphism (SSCP). Each of 70 and 22 individuals from Blushøj (Denmark) and Oskarshamn (Sweden), respectively, displayed haplotypes 2, 3, and 4 (= mtDNA heteroplasmy), whereas only haplotype 2 was found in each of 24 individuals from locations in Spain, France, Ireland, Iceland and Norway. As Blushøj and Oskarshamn were among the last areas to emerge from ice cover during the Last Glacial Maximum (18 000–20 000 years bp ), the geographically specific heteroplasmy may represent a founder effect and therefore, a valuable marker for understanding the role of post‐Ice Age recolonization. Geographically specific heteroplasmy also has important implications in phylogeographical studies based on mtDNA sequences.     

Low resolution of mitochondrial COI barcodes for identifying species of the genus Larus (Charadriiformes: Laridae)

We evaluated whether cytochrome c oxidase subunit I (COI) barcodes that have been previously suggested for birds are useful for identifying species of the genus Larus, which are resident or migratory birds in Korea. We found 31 intra- or interspecific COI haplotypes from 12 of 13 Larus species in Korea. Haplotype analyses showed that the COI barcodes could not distinguish some interspecific haplotypes from 6 of 12 Larus species because there were no nucleotide substitutions among their COI haplotypes. The neighbor-joining tree formed shallow branches in the clades expected for L. saundersi, L. crassirostris, L. canus, L relictus, and L. ridbundus. In the nine Larus species, COI haplotypes were not grouped as distinct entities that were correctly assigned to their corresponding species, resulting in polytypic clades. These results indicate that the COI sequences need to be cautiously selected as a DNA barcode for identifying species of Korean Larus birds.     

Stable heteroplasmy at the single-cell level is facilitated by intercellular exchange of mtDNA

Eukaryotic cells carry two genomes, nuclear (nDNA) and mitochondrial (mtDNA), which are ostensibly decoupled in their replication, segregation and inheritance. It is increasingly appreciated that heteroplasmy, the occurrence of multiple mtDNA haplotypes in a cell, plays an important biological role, but its features are not well understood. Accurately determining the diversity of mtDNA has been difficult, due to the relatively small amount of mtDNA in each cell (<1% of the total DNA), the intercellular variability of mtDNA content and mtDNA pseudogenes (Numts) in nDNA. To understand the nature of heteroplasmy, we developed Mseek, a novel technique to purify and sequence mtDNA. Mseek yields high purity (>90%) mtDNA and its ability to detect rare variants is limited only by sequencing depth, providing unprecedented sensitivity and specificity. Using Mseek, we confirmed the ubiquity of heteroplasmy by analyzing mtDNA from a diverse set of cell lines and human samples. Applying Mseek to colonies derived from single cells, we find heteroplasmy is stably maintained in individual daughter cells over multiple cell divisions. We hypothesized that the stability of heteroplasmy could be facilitated by intercellular exchange of mtDNA. We explicitly demonstrate this exchange by co-culturing cell lines with distinct mtDNA haplotypes. Our results shed new light on the maintenance of heteroplasmy and provide a novel platform to investigate features of heteroplasmy in normal and diseased states.     

Phylogenetic relationship of Turkish Apis mellifera subspecies based on sequencing of mitochondrial cytochrome C oxidase I region

Mitochondrial DNA sequence variation can be used to infer honey bee evolutionary relationships. We examined DNA sequence diversity in the cytochrome C oxidase I (COI or Cox1) gene segment of the mitochondrial genome in 112 samples of Apis mellifera from 15 different populations in Turkey. Six novel haplotypes were found for the COI gene segment. There were eight variable sites in the COI gene, although only three were parsimony-informative sites. The mean pairwise genetic distance was 0.3% for the COI gene segment. Neighbor-joining (NJ) trees of the COI gene segment were constructed with the published sequences of A. mellifera haplotypes that are available in GenBank; the genetic variation was compared among the different honeybee haplotypes. The NJ dendogram based on the COI sequences available in GenBank showed that Eastern European races were clustered together, whereas the Mellifera and Iberian haplotypes were clustered far apart. The haplotypes found in this study were clustered together with A. mellifera ligustica and some of the Greek honey bees (accession Nos. GU056169 and GU056170) found in NCBI GenBank database. This study expands the knowledge about the mitochondrial COI region and presents the first comprehensive sequence analysis of this region in Turkish honeybees.     

Genetic structure and diversity of the Diachasmimorpha longicaudata species complex in Thailand: SSCP analysis of mitochondrial 16S rDNA and COI DNA sequences

Genetic variation among 20 populations of Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) in Thailand was investigated using single strand conformation polymorphism (SSCP) analysis of mitochondrial DNA sequences. From a total 641 individual parasitoids, seven distinct haplotypes containing a total of 32 polymorphic sites were observed from cytochrome oxidase subunit I (COI) sequences along with five distinct haplotypes containing a total 16 polymorphic sites from 16S rDNA sequences. Values obtained through pairwise F_ST comparisons and analysis of molecular variance (AMOVA) indicated significant genetic differentiation among D. longicaudata populations in Thailand. Congruent relationships showing separation of these populations into three groups were obtained from Neighbor joining and Bayesian phylogenetic tree analyses along with the use of haplotype networks. This SSCP analysis of populations of the D. longicaudata species complex is the first report using molecular population genetic methods to analyze the structure of this parasitoid species in Thailand. This may provide useful information for release of parasitoid strains to maximize their benefit in biological control programs.     

Extensive mtDNA diversity in horses revealed by PCR–SSCP analysis

The hypervariable D-loop region of mitochondrial DNA (mtDNA) was amplified with the polymerase chain reaction using total horse DNA samples. Analysis of single strand conformation polymorphism (SSCP) of denatured amplification products was carried out by native polyacrylamide (8%) gel electrophoresis followed by silver staining. As many as 15 distinct SSCP variants were revealed when screening a total of 78 maternally unrelated horses representing five different breeds. All breeds showed a high degree of polymorphism and the estimated probability (PI_mt) that two maternally unrelated individuals have, by chance, identical SSCP variants varied between 0.14 and 0.30. We detected no heteroplasmy or deviations from strict and stable maternal inheritance when examining four maternal lineages, each represented by six to eight horses, separated by up to five generations from a common ancestral mare. The study establishes a simple screening method for detecting equine mtDNA types, which can be applied for tracing maternal genealogies and for association studies.     

Selection against Heteroplasmy Explains the Evolution of Uniparental Inheritance of Mitochondria

Why are mitochondria almost always inherited from one parent during sexual reproduction? Current explanations for this evolutionary mystery include conflict avoidance between the nuclear and mitochondrial genomes, clearing of deleterious mutations, and optimization of mitochondrial-nuclear coadaptation. Mathematical models, however, fail to show that uniparental inheritance can replace biparental inheritance under any existing hypothesis. Recent empirical evidence indicates that mixing two different but normal mitochondrial haplotypes within a cell (heteroplasmy) can cause cell and organism dysfunction. Using a mathematical model, we test if selection against heteroplasmy can lead to the evolution of uniparental inheritance. When we assume selection against heteroplasmy and mutations are neither advantageous nor deleterious (neutral mutations), uniparental inheritance replaces biparental inheritance for all tested parameter values. When heteroplasmy involves mutations that are advantageous or deleterious (non-neutral mutations), uniparental inheritance can still replace biparental inheritance. We show that uniparental inheritance can evolve with or without pre-existing mating types. Finally, we show that selection against heteroplasmy can explain why some organisms deviate from strict uniparental inheritance. Thus, we suggest that selection against heteroplasmy explains the evolution of uniparental inheritance.     

Detection and isolation of nuclear haplotypes by PCR-SSCP

SSCP (single-strand conformational polymorphism) is used widely in the field of human biomedicine, but its potential as a population genetics tool for the recovery of nuclear gene genealogies remains to be realized. We describe and illustrate a use for SSCP in the physical isolation of nuclear haplotypes that circumvents several difficulties associated with more conventional cloning procedures. The DNA sequence can be determined directly from the isolated haplotypes and used for phylogenetic inference. SSCP provides a convenient first step toward generating nuclear genealogies for population studies.     

Nucleotide sequence variation in mitochondrial COI gene among 147 silkworm (Bombyx mori) strains from Japanese, Chinese, European and moltinism classes

We characterized the nucleotide sequences of PCR-amplified mitochondrial COI fragments of 147 silkworm (Bombyx mori) strains that have been maintained in the National Institute of Agrobiological Sciences. Coding sequences (714 bp) of the 147 COI fragments were classified into eight haplotypes based on nucleotide differences at eight segregating sites. No length variation was identified in this region. The 5'-noncoding region showed different features, wherein changes in the number of Ts in the T-stretch, together with two base substitutions, were observed. As a result, the 147 COI noncoding sequences were classified into six haplotypes. Combining the coding and noncoding regions, we identified 14 haplotypes. One of the 14 haplotypes, Hap1A was exclusively abundant in the Japanese native strain class, while this haplotype was less frequent in the other three native strain classes. This finding suggests that the Japanese strain class underwent significant genetic differentiation from the Chinese, European, and moltinism classes, when the each class is regarded as a population. Comparison of the nucleotide sequences to those of B. mandarina (which inhabits Japan) revealed changes that are significantly larger than those within either B. mori or B. mandarina. Furthermore, we detected no common haplotypes between them, which suggests the concept of suppressed gene flow between the two species.     

Cryptic divergence and strong population structure in the colonial invertebrate Pycnoclavella communis (Ascidiacea) inferred from molecular data

We studied sequence variation in the mitochondrial gene cytochrome c oxidase subunit I (COI) for 135 individuals from eight Mediterranean populations of the colonial ascidian Pycnoclavella communis across most of its presently known range of distribution in the Mediterranean. Three haplotypes from Atlantic locations were also included in the study. Phylogenetic, phylogeographic and population genetic analyses were used to unravel the genetic variability within and between populations. The study revealed 32 haplotypes for COI, 29 of them grouped within two Mediterranean lineages of P. communis (mean nucleotide divergence between lineages was 8.55%). Phylogenetic and network analyses suggest the possible existence of cryptic species corresponding to these two lineages. Population genetic analyses were restricted to the five populations belonging to the main genetic lineage, and for these localities we compared the information gleaned from COI sequence data and from eight microsatellite loci. A high genetic divergence between populations was substantiated using both kinds of markers (COI, global Fst=0.343; microsatellite loci, global Fst=0.362). There were high numbers of private haplotypes (COI) and alleles (microsatellites) in the populations studied. Restricted gene flow and inbreeding occur in the present range of distribution of the species. Microsatellite loci showed a strong incidence of failed amplifications, which we attribute to the marked intraspecies variability that hampered the application of these highly specific markers. Our results show important genetic variability at all levels studied, from within populations to between basins, possibly coupled to speciation processes. This variability is attributable to restricted gene flow among populations due to short-distance dispersal of the larvae.     

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.     

Heteroplasmic point mutations in the human mtDNA control region.

As part of an investigation of the fixation mechanisms of mtDNA mutations in humans, we sequenced the first hypervariable segment of the control region in 180 twin pairs and found evidence of site heteroplasmy in 4 pairs. Significant levels of two mitochondrial haplotypes differing by a single point mutation were found in two MZ pairs, and within each pair, both members had similar levels of heteroplasmy. Two DZ pairs were found in which the predominant mitochondrial haplotype differed within the pair. We measured proportions of mitochondrial haplotypes within two twin pairs and their maternal relatives, using primer extension. In both maternal lineages, most family members were heteroplasmic, and the proportions of each genotype varied widely in different individuals. We used the changes in haplotype proportions within mother-offspring pairs to calculate the size range of potential bottlenecks in mitochondrial numbers occurring during development of the offspring. In most individuals, the most likely effective bottleneck sizes ranged from 3 to 20 segregating units, though in two individuals a small bottleneck was very unlikely and there was no upper limit on its possible size. We also used the data from this study, together with unpublished data from other populations, to estimate the frequency of site heteroplasmy in normal human populations. From this, we calculated that the rate of mutation and fixation in the first hypervariable segment of the human mtDNA control region is between 1.2 x 10(-6) and 2.7 x 10(-5) per site per generation. This range is in good agreement with published estimates calculated by other methods.     

Mitochondrial nucleotide variability in invasive populations of the root weevil Diaprepes abbreviatus (Coleoptera: Curculionidae) of Florida and preliminary assessment of Diaprepes sp. from Dominica

Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) is a root weevil introduced into the United States from the Caribbean in 1964. It is associated with >300 plants, including citrus, sugarcane, and potatoes. D. abbreviatus is widespread in Florida, and it has recently been detected in limited areas of California and Texas. The purpose of this research is to evaluate the utility of 16S ribosomal (16S rRNA) and cytochrome oxidase I (COI) mitochondrial markers for the delineation of genetic populations of D. abbreviatus in Florida and for the characterization of patterns of dispersion among these populations. We also assessed these markers as genetic tools for the clarification of taxonomic uncertainties in specimens from Dominica (Lesser Antilles). We analyzed 111 weevils from six Florida populations and six specimens from Dominica. In Florida, we found three haplotypes with only one haplotype in each population. Florida haplotypes differed by one to three nucleotide substitutions, possibly the result of a recent divergence from one source population or three different introductions from closely related populations from the Caribbean. In contrast, specimens from Dominica showed a high genetic variability with three 16S haplotypes and six unique COI haplotypes, delineating two mitochondrial clades. We show that these mitochondrial markers are useful for phylogeographic studies of D. abbreviatus.     

Temperature-related activity of <Emphasis Type="Italic">Gomphiocephalus hodgsoni</Emphasis> (Collembola) mitochondrial DNA (COI) haplotypes in Taylor Valley,Antarctica

Gomphiocephalus hodgsoni (Collembola) is the most common and widely distributed arthropod in the Dry Valleys of southern Victoria Land, and is genetically diverse with over 70 mitochondrial cytochrome c oxidase subunit I (COI) haplotypes. There is also considerable physiological variation among G. hodgsoni individuals in their cold tolerance and metabolic activity. Here, we assessed genetic haplotypes of G. hodgsoni relative to the environmental conditions during which individuals were active. We sequenced the COI region of 151 individuals collected in pitfall traps from three sites within Taylor Valley and found 19 unique haplotypes that separated into two distinct lineages (1.6 % divergence), with one lineage comprising 80 % of the sequenced population. During two-hourly sampling, air temperature was the strongest predictor of activity between the two lineages (R ² = 0.56), and when combined with subsurface soil temperature, relative humidity and photosynthetically active radiation, explanatory power increased to R ² = 0.71. With steadily increasing air temperatures predicted for much of Antarctica, it is likely that some haplotypes will have a selective advantage and potentially result in decreased genetic variability within populations. We suggest that temporal monitoring of the relative proportions of COI haplotypes or other appropriate genetic markers may provide a subtle measure of biological responses to environmental changes within Antarctic terrestrial ecosystems.     

SNP frequency and allelic haplotype structure of Beta vulgaris expressed genes

Based on EST sequences, fragments of 37 genes have been amplified and sequenced in two inbred lines of sugar beet. The rate of single nucleotide polymorphisms (SNP) corresponded to 1 every 130 bp, with an average (nucleotide diversity) value of 7.6×10^–3. When extrapolated to the whole sugar beet genome, randomly compared lines differ at 5.4×10⁶ SNPs in the genetic pool considered. In a wider search for SNP-related polymorphisms, 96 fragments of expressed genes were scanned with SSCP (single-strand conformation polymorphism) and heteroduplex (HA) analyses in 8 inbred lines. One SSCP or HA polymorphism was found every 1,470 bp of amplified DNA, corresponding to 5×10⁵ SSCP or HA loci in the whole genome. This frequency, 11 times lower than the SNP rate, was attributed to the high frequency of base pair substitution along the amplified fragment analysed electrophoretically. Therefore nucleotide variability was further studied by sequencing fragments of 10 genes in the same 8 lines. The results indicate that sugar beet alleles of expressed genes are very frequently organized as robust intragene haplotypes. In the 8 lines analysed, two haplotypes were identified for each of three gene fragments, three haplotypes for six gene fragments and four haplotypes for one gene fragment which is in good correspondence with the number of alleles detected by SSCP and HA analysis. In a cross between two lines, SSCP or HA alleles of expressed genes have 54% probability to be different.     

Opening a can of worms: unprecedented sympatric cryptic diversity within British lumbricid earthworms

Earthworms play a major role in many aspects of soil fertility, food web ecology and ecosystem functioning, and hence are frequently the subjects of, for example, ecological and toxicological research. Our aim was to examine the genetic structure of common earthworm species, to identify cryptic lineages or species that may be distinct ecotypes or biotypes (and hence confound current research based upon morphotypes) and to try to explain the massive cryptic diversity that eventually emerged. We demonstrated that species such as Allolobophora chlorotica, Aporrectodea longa, Aporrectodea rosea and Lumbricus rubellus all comprise highly divergent lineages with species-level divergence at the mitochondrial cytochrome oxidase I (COI) gene. In Allo. chlorotica alone, we found 55 haplotypes for COI, with 35 of these being found in pink and 20 in green morph worms. There were no cases of the two colour morphs sharing COI haplotypes. Phylogenetic analyses of mitochondrial COI and 16S genes showed the presence of five highly divergent lineages, suggesting the presence of multiple cryptic species within Allo. chlorotica. There was no clear geographical pattern to lineage distribution and many populations were polymorphic for both mitochondrial DNA lineage and colour morph. Amplified fragment length polymorphism results, based on two primer combinations, were broadly congruent with mitochondrial DNA results with one significant exception. Despite showing over 14% divergence at COI, amplified fragment length polymorphism markers showed that the two green morph lineages may be interbreeding and therefore represent a single taxon. The cryptic diversity revealed by these results has profound consequences for all areas of earthworm research.     

Sscp analysis of variations in haplotypes of citrus tristeza virus isolated from yuzu (Citrus junos) in geographically separate regions of Korea

Yuzu (Cittus junos) trees were examined from six geographically separate provinces in the Republic of Korea, including four islands (Geoje, Namhae, Wan, and Jeju), 1 peninsula (Goheung), and 1 inland area (Boseong). The population of sequence variants of citrus tristeza virus (CTV) was isolated and analyzed by single-strand conformation polymorphism (SSCP) analysis of cDNA from thep20 gene. SSCP profiles of 65 PCR products showed different band patterns but with similar intensities. Sixteen haplotypes were subgrouped according to their SSCP profiles and severity of symptoms. Their genomes were sequenced and compared. DNA analysis of thep20 genes revealed nucleotide identities ranging from 88-99.8%. Based on SSCP analysis, the pathologically mild isolates of CTV yielded two to three DNA bands, whereas the most virulent isolates contained more than two bands. Comparisons of these physically separate haplotypes suggest that CTV isolates with multiple SSCP profiles could have arisen as a result of a mixed infection and genetic recombination of two divergent isolates. Plants with severe disease symptoms, such as stem pitting, closely corresponded to a CTV strain showing typical SSCP profiles in Florida (USA) and Japan.     

Evaluation of DNA barcoding and identification of new haplomorphs in Canadian deerflies and horseflies

This paper reports the first tests of the suitability of the standardized mitochondrial cytochrome c oxidase subunit I (COI) barcoding system for the identification of Canadian deerflies and horseflies. Two additional mitochondrial molecular markers were used to determine whether unambiguous species recognition in tabanids can be achieved. Our 332 Canadian tabanid samples yielded 650 sequences from five genera and 42 species. Standard COI barcodes demonstrated a strong A + T bias (mean 68.1%), especially at third codon positions (mean 93.0%). Our preliminary test of this system showed that the standard COI barcode worked well for Canadian Tabanidae: the target DNA can be easily recovered from small amounts of insect tissue and aligned for all tabanid taxa. Each tabanid species possessed distinctive sets of COI haplotypes which discriminated well among species. Average conspecific Kimura two‐parameter (K2P) divergence (0.49%) was 12 times lower than the average divergence within species. Both the neighbour‐joining and the Bayesian methods produced trees with identical monophyletic species groups. Two species, Chrysops dawsoni Philip and Chrysops montanus Osten Sacken (Diptera: Tabanidae), showed relatively deep intraspecific sequence divergences (～10 times the average) for all three mitochondrial gene regions analysed. We suggest provisional differentiation of Ch. montanus into two haplotypes, namely, Ch. montanus haplomorph 1 and Ch. montanus haplomorph 2, both defined by their molecular sequences and by newly discovered differences in structural features near their ocelli.     

Mitochondrial DNA diversity of <Emphasis Type="Italic">Cleruchoides noackae</Emphasis> (Hymenoptera: Mymaridae): a potential biological control agent for <Emphasis Type="Italic">Thaumastocoris peregrinus</Emphasis> (Hemiptera: Thaumastocoridae)

Thaumastocoris peregrinus Carpintero and Dellapé (Hemiptera: Thaumastocoridae) is a native Australian Eucalyptus sap-feeding insect that has become invasive and seriously damaging to commercially grown Eucalyptus in the Southern Hemisphere. Cleruchoides noackae Lin and Huber (Hymenoptera: Mymaridae) was recently discovered as an egg parasitoid of the Thaumastocoridae in Australia. Mitochondrial DNA (mtDNA; cytochrome c oxidase subunit I, COI) sequence diversity amongst 104 individuals from these native C. noackae populations revealed 24 sequence haplotypes. The COI haplotypes of individuals collected from the Sydney and Southeast Queensland clustered in distinct groups, indicating limited spread of the insect between the regions. Individuals collected from Perth in Western Australia were represented by four COI haplotypes. Although this population is geographically more isolated from other populations, two COI haplotypes were identical to haplotypes found in the Sydney region. The results suggest that C. noackae has recently been introduced into Perth, possibly from the Sydney area. The high mtDNA diversity and limited spread that is suggested for C. noackae is in contrast to the lack of geographic associated mtDNA diversity and extensive spread of T. peregrinus. If implemented as a biological control agent, this factor will need to be considered in collecting and releasing C. noackae.     

Mitochondrial D-loop sequence variation among the 16 maternal lines of the Lipizzan horse breed

Mitochondrial DNA from 49 Lipizzan horses representing 16 maternal lines from the original stud at Lipica was used for SSCP analysis and DNA sequencing. The SSCP analysis of the 444 bp long fragment of the D-loop region extending from the tRNA(Pro) gene to the central conserved sequence block revealed three distinct groups of SSCP patterns. Both ends of the D-loop region (378 bp and 310 bp), which are considered as the most variable regions within the mammalian mitochondrial DNA, were sequenced. According to 49 polymorphic sites identified within the both parts of the D-loop region, the 16 maternal lines were grouped into 13 distinct mitochondrial haplotypes. The minimal difference between two different haplotype DNA sequences was one nucleotide and the maximal 24 nucleotides. The inheritance of mitochondrial haplotypes was stable and no sequence variation potentially attributable to mutation within maternal line was observed. Considerable DNA sequence similarity of Lipizzan mitochondrial haplotypes with the haplotypes from other breeds was observed. Phylogenetic analysis of the sequence data revealed a dendrogram with three separated branches, supporting the historical data about the multiple origin of the Lipizzan breed.