The role of ultrasonography in the diagnosis of pyloric stenosis: a decision analysis

Sequence variants of the hypervariable region I of human mitochondrial DNA (mtDNA) of 125 individuals from three aboriginal population groups of northern Asia (Yakuts, Evens, and Koryaks) were analyzed. Unique types of mtDNA (mitotypes) were discovered in 80% of Koryaks, 78% of Evens, and 59% of Yakuts. The mitotypes observed were clustered into nine phylogenetically related groups, two of which, according to the data on the comparative analysis of Siberian and east Asian populations, were Koryak-specific, and one was Even-specific. Koryaks and Evens exhibited mtDNAs that were highly frequent in Ainu. The results are discussed in terms of genetic differentiation and the ethnogenesis of ethnic groups of northern Asia.     

Genetic variation and bill size dimorphism in a passerine bird, the reed bunting Emberiza schoeniclus

In passerine birds morphological differentiation in bill size within species is not commonly observed. Bill size is usually associated with a trophic niche, and strong differences in it may reflect the process of genetic differentiation and, possibly, speciation. We used both mitochondrial DNA (mtDNA) and nuclear microsatellites to study genetic variation between two subspecies of reed bunting, Emberiza schoeniclus schoeniclus and E.s. intermedia, along their distributional boundary in western Europe. These two subspecies are characterized by a high dimorphism in bill size and, although breeding populations of the two subspecies are found very close to each other in northern Italy, apparently no interbreeding occurs. The observed morphological pattern between the two subspecies may be maintained by geographically varying selective forces or, alternatively, may be the result of a long geographical separation followed by a secondary contact. MtDNA sequences of cytochrome b and ND5 (515 bp) showed little variation and did not discriminate between the two subspecies, indicating a divergence time of less than 500 000 years. The analysis of four microsatellite loci suggested a clear, although weak, degree of genetic differentiation in the large- and small-billed populations, as indicated by FST and RST values and genetic distances. The correlation between bill size and genetic distance between populations remained significant after accounting for the geographical distances between sampling localities. Altogether, these results indicate a very recent genetic differentiation between the two bill morphs and suggest that a strong selection for large bills in the southern part of the breeding range is probably involved in maintaining the geographical differentiation of this species.     

Species-specific segregation of gender-associated mitochondrial DNA types in an area where two mussel species (Mytilus edulis and M. trossulus) hybridize

In each of the mussel species Mytilus edulis and M. trossulus there exist two types of mtDNA, the F type transmitted through females and the M type transmitted through males. Because the two species produce fertile hybrids in nature, F and M types of one may introgress into the other. We present the results from a survey of a population in which extensive hybridization occurs between these two species. Among specimens classified as "pure" M. edulis or "pure" M. trossulus on the basis of allozyme analysis, we observed no animal that carried the F or the M mitotype of the other species. In most animals of mixed nuclear background, an individual's mtDNA came from the species that contributed the majority of the individual's nuclear genes. Most importantly, the two mtDNA types in post-F1 male hybrids were of the same species origin. We interpret this to mean that there are intrinsic barriers to the exchange of mtDNA between these two species. Because such barriers were not noted in other hybridizing species pairs (many being even less interfertile than M. edulis and M. trossulus), their presence in Mytilus could be another feature of the unusual mtDNA system in this genus.     

Molecular phylogeny and ecological diversification in a clade of New World songbirds (genus Vireo)

We constructed a molecular phylogeny for a clade of eye-ringed vireos (Vireo flavifrons and the V. solitarius complex) to examine existing hypotheses of speciation and ecological diversification. Complete sequences of the mtDNA cytochrome b gene were obtained from 47 individuals of this group plus four vireonid outgroups. Mean levels of sequence divergence in the clade varied from 0.29% to 5.7%. Differences were greatest between V. flavifrons and four taxa of 'V. solitarius'. The latter separated into three taxonomic, geographical and ecological groups: V. plumbeus plumbeus, V. cassinii cassinii, and V. solitarius solitarius plus V. solitarius alticola. These differed by an average of 2.6-3.2%. Populations within each group revealed low levels of sequence variation (x = 0.20%) and little geographical structuring. The mtDNA data generally corroborate results from allozymes. V. plumbeus shows a loss of yellow-green carotenoid pigmentation from the ancestral condition. The occupancy of relatively dry habitats by this species and V. cassinii represents a derived ecological shift from more-humid environments occupied by other species of vireonids. Ecological divergence in this clade occurred in allopatry and is associated with generic-level stability in morphometrics and foraging styles. Migratory behaviour and seasonal habitat shifts apparently evolved multiple times in vireos breeding in temperate environments. Present geographical and ecological distributions, and low levels of intrataxon genetic divergence, are hypothesized to be the result of postglacial regionalization of climate-plant associations and rapid northward expansion of breeding ranges.     

PDLLA microspheres containing steroids: spray-drying, o/w and w/o/w emulsifications as preparation methods

Sea urchins of the genus Arbacia (order Stirodonta) have discontinuous allopatric distributions ranging over thousands of kilometers. Mitochondrial DNA (mtDNA) sequences were used to reconstruct phylogenetic relationships of four Arbacia species and their geographic populations. There is little evidence of genetic structuring of populations within species, except in two cases at range extremes. The mtDNA sequence differentiation between species suggests that divergence occurred about 4-9 MYA. Gene sequences encoding the sperm protein bindin and its intron were obtained and compared with the mtDNA phylogeny. Sea urchins among the well-studied echinoid order Camarodonta, with degrees of mtDNA divergence similar to those of Arbacia species, are known to have remarkable variation in bindin. However, in Arbacia, little variation in deduced amino acid sequences of bindin was found, indicating that purifying selection acts on the protein. In contrast, bindin intron sequences showed much differentiation, including numerous insertion/deletions. Fertilization experiments performed between a divergent pair of Arbacia species from the Atlantic and Pacific Oceans revealed no evidence of blocks to gamete recognition. In Arbacia, fertilization specificities may have evolved relatively slowly as a result of extensive gene flow within species, greater functional constraint on the bindin polypeptide, or reduced selective pressure for species recognition in singly occurring species.     

Intraspecific phylogeography of the gopher tortoise, Gopherus polyphemus: RFLP analysis of amplified mtDNA segments

The slow rate of mtDNA evolution in turtles poses a limitation on the levels of intraspecific variation detectable by conventional restriction fragment surveys. We examined mtDNA variation in the gopher tortoise (Gopherus polyphemus) using an alternative restriction assay, one in which PCR-amplified segments of the mitochondrial genome were digested with tetranucleotide-site endonucleases. Restriction fragment polymorphisms representing four amplified regions were analysed to evaluate population genetic structure among 112 tortoises throughout the species' range. Thirty-six haplotypes were identified, and three major geographical assemblages (Eastern, Western, and Mid-Florida) were resolved by UPGMA and parsimony analyses. Eastern and Western assemblages abut near the Apalachicola drainage, whereas the Mid-Florida assemblage appears restricted to the Brooksville Ridge. The Eastern/Western assemblage boundary is remarkably congruent with phylogeographic profiles for eight additional species from the south-eastern U.S., representing both freshwater and terrestrial realms.     

Plasmon analyses of Triticum (wheat) and Aegilops: PCR-single-strand conformational polymorphism (PCR-SSCP) analyses of organellar DNAs

To investigate phylogenetic relationships among plasmons in Triticum and Aegilops, PCR-single-strand conformational polymorphism (PCR-SSCP) analyses were made of 14.0-kb chloroplast (ct) and 13. 7-kb mitochondrial (mt)DNA regions that were isolated from 46 alloplasmic wheat lines and one euplasmic line. These plasmons represent 31 species of the two genera. The ct and mtDNA regions included 10 and 9 structural genes, respectively. A total of 177 bands were detected, of which 40.6% were variable. The proportion of variable bands in ctDNA (51.1%) was higher than that of mtDNA (28. 9%). The phylogenetic trees of plasmons, derived by two different models, indicate a common picture of plasmon divergence in the two genera and suggest three major groups of plasmons (Einkorn, Triticum, and Aegilops). Because of uniparental plasmon transmission, the maternal parents of all but one polyploid species were identified. Only one Aegilops species, Ae. speltoides, was included in the Triticum group, suggesting that this species is the plasmon and B and G genome donor of all polyploid wheats. ctDNA variations were more intimately correlated with vegetative characters, whereas mtDNA variations were more closely correlated with reproductive characters. Plasmon divergence among the diploids of the two genera largely paralleled genome divergence. The relative times of origin of the polyploid species were inferred from genetic distances from their putative maternal parents.     

A comparison of mtDNA restriction sites vs. control region sequences in phylogeographic assessment of the musk turtle (Sternotherus minor)

A total of nearly 800 base pairs of mitochondrial DNA sequence was assayed in each of 52 musk turtles (Sternotherus minor) collected across the species' range in the south-eastern USA. About one-half of the sequence information in effect was accessed by conventional recognition-site assays of the entire mtDNA molecule; the remainder came from direct sequence assays of a normally hypervariable 5' section of the noncoding control region. The two assay methods produced essentially nonoverlapping sets of variable character states that were compared with respect to magnitudes and phylogeographic patterns of mtDNA variation. The two assay procedures yielded nearly identical outcomes with regard to: (a) total levels of species-wide mtDNA genetic variation; (b) mean levels of within-locale variation; (c) extremely high population genetic structure; (d) a phylogenetically significant separation of samples from the north-western half of the species' range vs. those in the south-eastern segment; and (e) considerably lower genetic variability within the north-western clade. The micro- and macro-phylogeographic mtDNA patterns in the musk turtle are consistent with a low-dispersal natural history, and with a suspected longer-term biogeographic history of the species, respectively.     

Comparison between the complete mtDNA sequences of the blue and the fin whale, two species that can hybridize in nature

The sequence of the mitochondrial DNA (mtDNA) molecule of the blue whale (Balaenoptera musculus) was determined. The molecule is 16,402 bp long and its organization conforms with that of other eutherian mammals. The molecule was compared with the mtDNA of the congeneric fin whale (B. physalus). It was recently documented that the two species can hybridize and that male offspring are infertile whereas female offspring may be fertile. The present comparison made it possible to determine the degree of mtDNA difference that occurs between two species that are not completely separated by hybridization incompatibility. The difference between the complete mtDNA sequences was 7.4%. Lengths of peptide coding genes were the same in both species. Except for a small portion of the control region, disruption in alignment was usually limited to insertion/deletion of a single nucleotide. Nucleotide differences between peptide coding genes ranged from 7.1 to 10.5%, and difference at the inferred amino acid level was 0.0-7.9%. In the rRNA genes the mean transition difference was 3.8%. This figure is similar in degree to the difference (3.4%) between the 12S rRNA gene of humans and the chimpanzee. The mtDNA differences between the two whale species, involving both peptide coding and rRNA genes, suggest an evolutionary separation of > or = 5 million years. Although hybridization between more distantly related mammalian species may not be excluded, it is probable that the blue and fin whales are nearly as different in their mtDNA sequences as hybridizing mammal species may be.     

Cytochrome b gene haplotypes characterize chromosomal lineages of anoa, the Sulawesi dwarf buffalo (Bovidae: Bubalus sp.)

Partial mitochondrial cytochrome b gene sequences reveal two deeply differentiated mtDNA lineages in anoa dwarf buffaloes (Bubalus depressicornis) from the studbook herd in European zoos. Three matrilinear lineages of lowland anoas (depressicornis type) contributed three rather similar sequence haplotypes, but one remarkably distinct haplotype was observed exclusively in mountain anoas (quarlesi type) descended from one founder female. The carriers of the distinctive mtDNA haplotype were also distinguished by several chromosomal and phenotypic peculiarities too. The differentiation between the mtDNA lineages of anoa approached or even surpassed the genetic divergence between some uncontested species of wild cattle. The depth of this haplotype divergence in anoas is discussed against the background of the phylogenetic age of these paleoendemic inhabitants of a predator-free island refugium, Sulawesi, who are among the most plesiomorphic living bovines. The studbook breeding of captive anoas as a safeguard against extinction might profit from such population genetic markers. These cytochrome b gene sequences were unable to resolve the phylogeny of nine bovine taxa robustly, except the divergence of Bubalus, Synceros, Bison, and Bos (sensu lato) genera.     

Matrilineal inheritance of complex I dysfunction in a multigenerational Parkinson's disease family

Recent data suggesting complex I dysfunction in Parkinson's disease (PD) arises from mitochondrial DNA (mtDNA) mutation does not conclusively answer whether the responsible genetic lesion is inherited (primary) or somatic (secondary). To address this question, we identified a family in which multiple members over three generations are affected with PD through exclusively maternal lines. Cytoplasmic hybrids (cybrids) were created for 15 family members over two generations by transferring each individual's mtDNA to mtDNA-depleted human neuroblastoma cells. Eight of the 15 cybrid lines contained mtDNA obtained from maternally descended family members and seven contained mtDNA from paternally descended family members. After 6 weeks of culture, cybrid cell lines were assayed for complex I activity and oxidative stress, and mitochondrial morphology was analyzed by electron microscopy. Compared with the cybrid lines containing mtDNA from paternal descendants, cybrid lines containing mtDNA from maternal descendants had lower complex I activity, increased reactive oxygen species production, increased radical scavenging enzyme activities, and more abnormal mitochondrial morphologic features. These findings were present in cybrid lines containing mtDNA from maternal descendants with PD as well as in currently asymptomatic young maternal descendants, and support a precedent for inherited mtDNA mutation in some persons with PD.     

Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales

Powerful analyses of population structure require information from multiple genetic loci. To help develop a molecular toolbox for obtaining this information, we have designed universal oligonucleotide primers that span conserved intron-exon junctions in a wide variety of animal phyla. We test the utility of exon-primed, intron-crossing amplifications by analyzing the variability of actin intron sequences from humpback, blue, and bowhead whales and comparing the results with mitochondrial DNA (mtDNA) haplotype data. Humpback actin introns fall into two major clades that exist in different frequencies in different oceanic populations. It is surprising that Hawaii and California populations, which are very distinct in mtDNAs, are similar in actin intron alleles. This discrepancy between mtDNA and nuclear DNA results may be due either to differences in genetic drift in mitochondrial and nuclear genes or to preferential movement of males, which do not transmit mtDNA to offspring, between separate breeding grounds. Opposing mtDNA and nuclear DNA results can help clarify otherwise hidden patterns of structure in natural populations.     

Mitochondrial DNAs of the fungus Armillaria ostoyae: restriction map and length variation

A restriction-enzyme-site map is presented for the 147-kb mtDNA of North American Armillaria ostoyae. The locations of five structural genes, atp6, atp8, coxI, coxIII, and cob, along with the location and orientation of the large and small ribosomal RNA genes, were determined through Southern hybridizations with cloned genes from other fungal mtDNAs. Based on this map, the variation in mtDNA suggested geographic structure at two different levels. On a large geographic scale, 17 mtDNA types from North America were distinct, with respect to both size and restriction maps, from three mtDNA types from Europe. At the local scale, identical mtDNA types were evident among several different genetic individuals located no more than 1 km apart at a site in Michigan. No mtDNA type occurred more than once among genetic individuals from different regions of North America, although the occurrence of similar mtDNAs in isolates from distant regions suggested that this may occur at a low frequency with large sample sizes. Among the North American mtDNA types, analysis of discrete length variants was inconsistent with the hypothesis that the mtDNA of A. ostoyae evolves as a clonal lineage in which each length mutation represents a unique event. The two remaining hypotheses, that similar mutational events have occurred independently and that genetic exchange and recombination occurs among mtDNAs in natural populations of this species, remain to be tested.     

Host movement and the genetic structure of populations of parasitic nematodes

Mitochondrial DNA (mtDNA) sequence data were used to compare the population genetic structures of five species of parasitic nematodes from three different hosts: Ostertagia ostertagi and Haemonchus placei from cattle, H. contortus and Teladorsagia circumcincta from sheep, and Mazamastrongylus odocoilei from white-tailed deer. The parasites of sheep and cattle showed a pattern consistent with high gene flow among populations. The parasite of deer showed a pattern of substantial population subdivision and isolation by distance. It appears that host movement is an important determinant of population genetic structure in these nematodes. High gene flow in the parasites of livestock also indicates great opportunity for the spread of rare alleles that confer resistance to anthelmintic drugs. All species, including the parasite of deer, had unusually high within-population diversities (averages of 0.019-0.027 substitutions per site between pairs of individuals from the same population). Large effective population sizes (Ne), perhaps in combination with rapid mtDNA evolution, appear to be the most likely explanation for these high within-population diversities.     

Global matrilineal population structure in sperm whales as indicated by mitochondrial DNA sequences

The genetic variability and population structure of worldwide populations of the sperm whale was investigated by sequence analysis of the first 5'L 330 base pairs in the mitochondrial DNA (mtDNA) control region. The study included a total of 231 individuals from three major oceanic regions, the North Atlantic, the North Pacific and the Southern Hemisphere. Fifteen segregating nucleotide sites defined 16 mtDNA haplotypes (lineages). The most common mtDNA types were present in more than one oceanic region, whereas ocean-specific types were rare. Analyses of heterogeneity of mtDNA type frequencies between oceans indicated moderate (GST = 0.03) but statistically significant (p = 0.0007) genetic differentiation on a global scale. In addition, strong genetic differentiation was found between potential social groups (GST = 0.03-0.6), indicating matrilineal relatedness within groups. The global nucleotide diversity was quite low (pi = 0.004) implying a recent common mtDNA ancestry (< 100,000) years ago) and a young global population structure. However, within this time period, female dispersal has apparently been limited enough to allow the development of global mtDNA differentiation. The results are consistent with those from observational studies and whaling data indicating stable social affiliations, some degree of area fidelity and latitudinal range limitations in groups of females and juveniles.     

Phylogeography of Bufo marinus from its natural and introduced ranges

The marine toad, Bufo marinus, has a broad natural distribution extending from the south-west of the USA to southern Peru and the central Amazon. It was introduced to several localities in the Caribbean and Pacific Oceans to control sugar cane pests. We sequenced 468 bp of mitochondrial DNA (mtDNA) containing the ND3 gene, and flanking tRNA genes from toads spanning the broad natural and introduced ranges. Consistent with the known history of introductions and expected effects of serial bottlenecks, mtDNA within introduced populations in Hawaii and Australia was uniform and most closely related to samples from eastern Venezuela and French Guiana. However, mtDNA nucleotide diversity in the geographic region spanning the source areas is also relative low (0.18-0.46%) and the absence of variation in the introduced populations precludes quantitative assessment of the reduction in genetic diversity. Unexpectedly, there was a large phylogeographic break (5.4% sequence divergence) within the natural range separating populations east and west of the Venezuelan Andes. We hypothesize that the two major lineages of B. marinus were isolated by the uplift of the eastern Andean cordillera which was completed approximately 2.7 Ma. Another species of the marinus group, B. paracnemis, had mtDNA paraphyletic, with marinus, being nested within the eastern lineage. Thus, at least one speciation event within the marinus group postdates the split within marinus. These findings suggest that the taxonomy of B. marinus should be re-evaluated and that the search for pathogens to control Australian populations should be conducted in populations from both lineages in the natural range.     

Representative and efficient cloning of satellite DNAs based on PFGE pre-fractionation of restriction digests of genomic DNA

The world's most endangered canid is the Ethiopian wolf Canis simensis, which is found in six isolated areas of the Ethiopian highlands with a total population of no more than 500 individuals. Ethiopian wolf populations are declining due to habitat loss and extermination by humans. Moreover, in at least one population, Ethiopian wolves are sympatric with domestic dogs, which may hybridize with them, compete for food, and act as disease vectors. Using molecular techniques, we address four questions concerning Ethiopian wolves that have conservation implications. First, we determine the relationships of Ethiopian wolves to other wolf-like canids by phylogenetic analysis of 2001 base pairs of mitochondrial DNA (mtDNA) sequence. Our results suggest that the Ethiopian wolf is a distinct species more closely related to gray wolves and coyotes than to any African canid. The mtDNA sequence similarity with gray wolves implies that the Ethiopian wolf may hybridize with domestic dogs, a recent derivative of the gray wolf. We examine this possibility through mtDNA restriction fragment analysis and analysis of nine microsatellite loci in populations of Ethiopian wolves. The results imply that hybridization has occurred between female Ethiopian wolves and male domestic dogs in one population. Finally, we assess levels of variability within and between two Ethiopian wolf populations. Although these closely situated populations are not differentiated, the level of variability in both is low, suggesting long-term effective population sizes of less than a few hundred individuals. We recommend immediate captive breeding of Ethiopian wolves to protect their gene pool from dilution and further loss of genetic variability.     

Estimating African American admixture proportions by use of population-specific alleles

We analyzed the European genetic contribution to 10 populations of African descent in the United States (Maywood, Illinois; Detroit; New York; Philadelphia; Pittsburgh; Baltimore; Charleston, South Carolina; New Orleans; and Houston) and in Jamaica, using nine autosomal DNA markers. These markers either are population-specific or show frequency differences >45% between the parental populations and are thus especially informative for admixture. European genetic ancestry ranged from 6.8% (Jamaica) to 22.5% (New Orleans). The unique utility of these markers is reflected in the low variance associated with these admixture estimates (SEM 1.3%-2.7%). We also estimated the male and female European contribution to African Americans, on the basis of informative mtDNA (haplogroups H and L) and Y Alu polymorphic markers. Results indicate a sex-biased gene flow from Europeans, the male contribution being substantially greater than the female contribution. mtDNA haplogroups analysis shows no evidence of a significant maternal Amerindian contribution to any of the 10 populations. We detected significant nonrandom association between two markers located 22 cM apart (FY-null and AT3), most likely due to admixture linkage disequilibrium created in the interbreeding of the two parental populations. The strength of this association and the substantial genetic distance between FY and AT3 emphasize the importance of admixed populations as a useful resource for mapping traits with different prevalence in two parental populations.     

Community-based trauma systems in the United States: an examination of structural development

To resolve the population genetic structure and phylogeography of the West Indian manatee (Trichechus manatus), mitochondrial (mt) DNA control region sequences were compared among eight locations across the western Atlantic region. Fifteen haplotypes were identified among 86 individuals from Florida, Puerto Rico, the Dominican Republic, Mexico, Columbia, Venezuela, Guyana and Brazil. Despite the manatee's ability to move thousands of kilometers along continental margins, strong population separations between most locations were demonstrated with significant haplotype frequency shifts. These findings are consistent with tagging studies which indicate that stretches of open water and unsuitable coastal habitats constitute substantial barriers to gene flow and colonization. Low levels of genetic diversity within Florida and Brazilian samples might be explained by recent colonization into high latitudes or bottleneck effects. Three distinctive mtDNA lineages were observed in an intraspecific phylogeny of T. manatus, corresponding approximately to: (i) Florida and the West Indies; (ii) the Gulf of Mexico to the Caribbean rivers of South America; and (iii) the northeast Atlantic coast of South America. These lineages, which are not concordant with previous subspecies designations, are separated by sequence divergence estimates of d = 0.04-0.07, approximately the same level of divergence observed between T. manatus and the Amazonian manatee (T. inunguis, n = 16). Three individuals from Guyana, identified as T. manatus, had mtDNA haplotypes which are affiliated with the endemic Amazon form T. inunguis. The three primary T. manatus lineages and the T. inunguis lineage may represent relatively deep phylogeographic partitions which have been bridged recently due to changes in habitat availability (after the Wisconsin glacial period, 10 000 B P), natural colonization, and human-mediated transplantation.     

Genetic variability in mitochondrial DNA of the screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae), from Brazil

Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) was used to examine genetic variation and population structure of screwworm flies in four populations from S?o Paulo State, Brazil. The total DNA of 405 individuals was digested with 15 restriction endonucleases and probed with five cloned HindIII fragments representing the entire mitochondrial genome of Cochliomyia hominivorax. The survey revealed that four enzymes (HaeIII, HindIII, MspI, and PvuII) were suitable to detect mtDNA variation among all populations. Based on the fragment patterns obtained for these four enzymes, a total of 15 haplotypes in combination was detected. Heteroplasmic individuals for the PvuII pattern were obtained in one of the populations. The estimated average for nucleotide sequence divergence (delta) was 0.92%. The cladogram of the geographical distribution among the observed haplotypes suggests that the sampled screwworms probably belong to a single evolutionary lineage with populations interconnected by reduced gene flow.