Genetic divergence and relationships among smelts of the genus <Emphasis Type="Italic">Osmerus</Emphasis> from the Russian waters

Smelts of the genus Osmerus, O. eperlanus and O. mordax dentes, inhabiting the basins of the Atlantic, Arctic, and Pacific oceans were investigated using RFLP analysis of the mitochondrial DNA segments A8/A6/COIII/ND3 and ND3/ND4, and sequencing of the cytb and COI genes (mtDNA), and intron 1 of the rpS7 gene (nDNA). A total of 14 samples from the populations from most part of the Russian range were examined. The mean values of haplotype and nucleotide diversity constituted 0.5997 ± 0.11264 and 0.003201 for O. m. dentex, and 0.3086 ± 0.09892 and 0.000431 for O. eperlanus, respectively. The high level of interspecific diversity (12.94%) along with the low level of intraspercific diversity (0.049% for O. m. dentex, and 0.001% for O. eperlanus was observed. The dendrograms (UPGMA, NJ, MP, and BA) constructed using the data of RFLP analysis of mtDNA, along with the sequencing data of mitochondrial and nuclear genes were congruent. The representatives of O. eperlanus and O. m. dentex formed steady clusters in accordance with their species affiliation, albeit without subdivision into local populations depending on their geographic locality.     

Mitochondrial DNA variation in Pacific capelin (Mallotus villosus catervarius) from the Sea of Okhotsk, inferred from PCR-RFLP analysis

Four population samples of Pacific capelin Mallotus villosus catervarius (Pennant, 1784) from geographically distant localities in the Sea of Okhotsk, Tauy Bay and the eastern coast of the Sakhalin Island, were examined using PCR-RFLP analysis of three mitochondrial DNA regions (A8/A6/COIII/ND3, ND3/ND4L/ND4, and ND5/ND6). The nucleotide divergence of mtDNA sequences among the samples, as well as the analysis of geographic heterogeneity of the haplotype frequencies and quantitative estimation of genetic differentiation performed by means of AMOVA, showed that the samples examined belonged to one panmictic population. Genealogic analysis of the mtDNA variation structure was carried out. It was demonstrated that the high level of haplotype diversity (0.9639 +/- 0.00015) along with the low level of nucleotide diversity (0.003818 +/- 0.0000003) pointed to the exponential rate of population growth of the capelin from the Sea of Okhotsk, which rather recently in its evolutionary history faced the bottleneck effect.     

Patterns of polymorphism and gene flow of gender-associated mitochondrial DNA lineages in European mussel populations

Mussels of the genus Mytilus have two types of mitochondrial DNA (mtDNA). The M type is transmitted paternally and the F type is transmitted maternally. RFLP analysis is used to assess phylogenetic relationships and nucleotide diversity and divergence for both mtDNA genomes in European populations of M. edulis and Atlantic and Mediterranean forms of M. galloprovincialis. Ten restriction endonucleases were used to assay variation in regions of the ND2 and COIII genes for a total of 77 individuals. F and M genomes show a concordant phylogenetic split into two major divergent clades, one specific to Mediterranean M. galloprovincialis and the other containing haplotypes from the three taxa. For both genomes, the geographical distribution of mtDNA variation suggests: (i) extensive levels of mtDNA introgression; (ii) asymmetric mtDNA gene flow from Atlantic to Mediterranean populations; and (iii) recurrent historical hybridization events. Significantly higher mtDNA diversity and divergence are observed for the M than F genome in all three Mytilus taxa, although the evolutionary forces responsible for these differences cannot be resolved. The extensive mtDNA gene flow between European Mytilus taxa conflicts with the restricted mtDNA introgression observed in American mussels , implying geographical variation in the nature of nuclear/mtDNA interactions regulating biparental inheritance.     

Unusual mitochondrial genome structure of the freshwater goby Odontobutis platycephala: rearrangement of tRNAs and an additional non-coding region

Mitochondrial DNA was isolated from the Korean freshwater gobioid fish Odontobutis platycephala by long-polymerase chain reaction with conserved primers and this mtDNA was sequenced by primer walking using flanking sequences as sequencing primers. The resultant O. platycephala mtDNA sequence was found to be 17 588 bp in size with a mostly conserved structural organization when compared with that of other teleost fish. Rearrangements of tRNAs (tRNA-Ser, tRNA-Leu, tRNA-His) and an additional non-coding region (533 bp) were present between the ND4 and ND5 genes. In the present paper, the basic characteristics of the O. platycephala mitochondrial genome is reported, including its structural organization, base composition of rRNAs, tRNAs and protein-encoding genes, characteristics of mitochondrial tRNAs and the peculiar rearrangement features of some parts of the mtDNA. Phylogenetic analysis performed using the cytochrome b gene sequences of 16 Korean freshwater fishes (15 gobioids) with the Bayesian algorithm showed that O. platycephala forms a clade (1·00 of posterior probability) with other species of Odontobutis . This suggests that the observed rearrangement between the ND4 and ND5 genes in the O. platycephala mitogenome reflects independent events.     

ND4 mutation in transitional cell carcinoma: Does mitochondrial mutation occur before tumorigenesis?

To investigate how mitochondrial mutation occurs in cancers, we analyzed ND4 mutation in 53 transitional cell carcinomas (TCCs) of the upper urinary tract and the normal counterpart (perirenal soft tissue). Three methods, i.e., DNA sequencing, restriction fragment length polymorphism (RFLP), and denaturing high-performance liquid chromatography (DHPLC), were employed because of their different sensitive of detecting mutation. The results of sequencing and RFLP showed that ND4 mutations were only found in 24.5% (13/53) of tumor. However, 11 of these mutations could also be identified in the normal tissue by DHPLC, indicating that most mitochondrial mutations identified in tumors preexist as minor components, which are too low in quantity to be detected by less sensitive methods such as DNA sequencing. The result suggests that mtDNA mutation occurs before tumorigenesis and become apparent in cancer cells.     

Population genetic structure of northern Dolly Varden char Salvelinus malma malma in Asia and North America

The level of genetic differentiation of northern Dolly Varden char Salvelinus malma malma from Asia and North America was evaluated using the data on mtDNA variation (regions ND1/ND2, ND5/ND6, and Cytb/D loop) obtained by means of PCR-RFLP analysis. For S. m. malma, the mean values of haplotype and nucleotide diversity were 0.5261 +/- 0.00388 and 0.001558, respectively. The mean estimate of the population nucleotide divergence constituted 0.055%. It was demonstrated that S. m. malma on the most part of the species range examined (drainages of the Beaufort Sea, Chukotka Sea, Bering Sea, and the Sea of Okhotsk) was characterized by the population genetic structure with the low level of genetic differentiation and divergence. At the same time, populations from the Pacific Ocean Gulf of Alaska demonstrated marked genetic differentiation, supported by the high pairwise phi(ST) values (from 0.4198 to 0.5211) and nucleotide divergence estimates (mean divergence, 0.129%), from Asian and North American populations. Nested analysis of molecular variance (AMOVA) showed that most of the mtDNA variation in S. m. malma fell in the intrapopulation component (72.5%). At the same time, the differences between the populations (21.1%) and between the regions (6.4%) made lower contribution to the total variation.     

Mitochondrial DNA variation and population genetic structure of the northern redbelly dace (Phoxinus eos)

Two sections of the control region and the genes coding for NADH dehydrogenase sub-units 5 and 6 (ND-5/6) of mitochondrial DNA (mtDNA) were amplified from Phoxinus eos with the polymerase chain reaction. Both sections of the control region were sequenced directly while the ND-5/6 fragment was sequenced in from each end only. Additionally, the entire ND-5/6 fragment was examined for sequence variation using RFLP analysis. No sequence variation was detected in the control region among 70 individuals sampled from 18 populations across three Ontario regions (Spanish River, Madawaska R. and Cataraqui R.). To examine ND-5/6 variation, a total of 75 individuals were sampled from five populations representing two of the three regions (Madawaska River and Cataraqui R.). Six haplotypes were detected by direct sequencing and four by RFLP analysis. Estimates of population subdivision from RFLP data, sequence analysis, and the two data sets combined for the ND-5/6 fragment, suggest that gene flow is restricted within and between regions. However, estimates of sequence divergence for both sequence and RFLP analysis of this fragment suggested that populations were either founded by already differentiated populations or that populations were founded by a single stock and divergence between regions occurred prior to isolation of populations within regions. These estimates of population structure are much greater than those obtained from allozyme analysis. Additionally, high levels of heterozygosity in nuclear DNA, but low mtDNA diversity suggests that populations have experienced reductions in population size sufficient to reduce only mtDNA variation. Random lineage extinction and limited time for the accumulation of new mutations are likely responsible for low levels of mtDNA variation in ND-5/6 and the control region, while functional constraints may limit variation more than expected in the control region in dace and other fishes.     

Polymorphism of human mitochondrial DNA

To date, a large data set on the mitochondrial DNA (mtDNA) sequence variation in human populations has been accumulated. The use of direct sequencing of the main noncoding region of mtDNA along with the RFLP analysis provide performance of complex analysis of mtDNA polymorphism in human populations. This approach proved to be effective for obtaining molecular genetic portraits of the world populations, as well as for the elucidation of the human evolutionary history and past migrations.     

Polymorphism of Human Mitochondrial DNA

To date, a large data set on the mitochondrial DNA (mtDNA) sequence variation in human populations has been accumulated. The use of direct sequencing of the main noncoding region of mtDNA along with the RFLP analysis provide performance of complex analysis of mtDNA polymorphism in human populations. This approach proved to be effective for obtaining molecular genetic portraits of the world populations, as well as for the elucidation of the human evolutionary history and past migrations.     

Mitochondrial DNA variation in Pacific capelin (<Emphasis Type="Italic">Mallotus villosus catervarius</Emphasis>) from the Sea of Okhotsk,inferred from PCR-RFLP analysis

Four population samples of Pacific capelin Mallotus villosus catervarius (Pennat, 1784) from geographically distant localities in the Sea of Okhotsk, Tauy Bay and the eastern coast of the Sakhalin Island, were examined using PCR-RFLP analysis of three mitochondrial DNA regions (A8/A6/COIII/ND3, ND3/ND4L/ND4, and ND5/ND6). The nucleotide divergence of mtDNA sequences among the samples, as well as the analysis of geographic heterogeneity of the haplotype frequencies and quantitative estimation of genetic differentiation performed by means of AMOVA, showed that the samples examined belonged to one panmictic population. Genealogic analysis of the mtDNA variation structure was carried out. It was demonstrated that the high level of haplotype diversity (0.9639 ± 0.00015) along with the low level of nucleotide diversity (0.003818 ± 0.0000003) pointed to the exponential rate of population growth of the capelin from the Sea of Okhotsk, which rather recently in its evolutionary history faced the bottleneck effect.     

Comparison between mitochondrial and chloroplast DNA variation in the native range of Silene vulgaris

A detailed survey of mitochondrial and chloroplast diversity in eight populations of Silene vulgaris from Central Europe was conducted for comparison with previously published data on diversity from S. vulgaris populations in the introduced range. Mitochondrial DNA (mtDNA) variation around the coxI gene was assessed with Southern blotting/restriction fragment length polymorphism methods. Chloroplast variation was assessed by sequencing the intergenic spacer separating the trnH and psbA genes. Thirty mtDNA haplotypes and 24 chloroplast DNA (cpDNA) haplotypes were found within 86 individuals. The overall genetic diversity h (0.941 for mitochondrial, and 0.893 for chloroplast markers) and within-population diversity were higher than reported in previous population studies of S. vulgaris in the USA and Europe. The frequency of private alleles was surprisingly high - more than 90% for both kinds of markers. Most of our populations were large and located in relatively undisturbed meadows, whereas surveys in Virginia consisted of smaller roadside populations. The slow rate of population turnover in European populations is discussed as a factor responsible for the relatively high diversity of S. vulgaris in undisturbed areas of its native range. Association between mtDNA and cpDNA haplotypes was also demonstrated. Finally, gender and mtDNA haplotype were associated in the Alps populations, where females were very rare.     

Mitochondrial DNA differentiation between two forms of trout Salmo letnica, endemic to the Balkan Lake Ohrid, reflects their reproductive isolation

Mitochondrial haplotype diversity in sympatric populations of Ohrid trout, Salmo letnica was investigated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mtDNA control region and ND1, ND3/4, ND5/6 segments. A 310 bp fragment at the 5' end, and a 340-572 bp fragment at the 3' end of the control region were sequenced from representatives of the populations studied. Based on pairwise comparison of the sequences, five new haplotypes were identified plus one identical with the brown trout Andalusian haplotype from the southern Iberian Peninsula. The combination of both RFLP and sequence data sets yielded a total of 10 composite haplotypes. A high degree of genetic subdivision between S. letnica typicus and S. letnica aestivalis populations was observed. The notion of a sympatric origin for the two morphs is discussed. Length variation of the mtDNA control region due to the presence of an 82 bp unit, tandemly repeated one to four times, in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA is reported. Further, we demonstrate that a single duplication of the approximately 82 bp repeat unit is a common element of the salmonid mitochondrial control region. The unique genetic structure of Ohrid trout represents a highly valuable genetic resource that deserves appropriate management and conservation.     

Ovine mitochondrial DNA: restriction enzyme analysis, mapping and sequencing data

Restriction endonuclease fragment patterns of mitochondrial DNA (mtDNA) in sheep were analysed with 11 enzymes. Four breeds (Merinolandschaf, Rhoenschaf, Schwarzkoepfiges Fleischschaf and Skudde) of domestic sheep and European Mouflon were examined. A restriction map with 28 cleavage sites of seven enzymes was established. KpnI and PstI do not cut ovine mtDNA. Two EcoRI fragments of Merinolandschaf, Rhoenschaf and Mouflon each were cloned and partially sequenced. Intraspecific nucleotide sequence differences within 1.101 kb ranged from 0.09 to 0.27%. Hybridization analysis with a fragment of porcine mtDNA along with sequencing data from cloned fragments was used for orientation of the restriction map along the bovine sequence. Ovine mtDNA sequences encompassing parts of the Cyt.b-, ND5-, CoIII- and ATPase6 genes were compared with the corresponding sequences of the bovine mtDNA.     

Genetic diversity in the North American captive African elephant collection

One hundred and eight mitochondrial DNA (mtDNA) sequences were used in combination with multilocus genotypes generated from 19 nuclear microsatellite loci to assess the genetic diversity in the North American captive African elephant collection. The analysis of 4488 bp of mitochondrial nicotinamide adenine dinucleotide hydrogenenase subunit 4-cytochrome b (ND4-cyt b ) sequence revealed the existence of three distinct mtDNA lineages (Haplogroups I–III). The genetic distance between Haplogroups I/II and Haplogroup III was determined to be 0.046. The nucleotide diversity for the elephant mtDNA ND4-cyt b was <0.004. Analysis of multilocus genotypes indicated an average observed heterozygosity of 0.53 compared with the expected heterozygosity of 0.64. No subdivision in the data was detected including data partitioned by mtDNA haplogroup. Relatedness among the 108 individuals did not differ from that expected in random mating populations.     

Morphological and genetic differentiation of the African clupeid Limnothrissa miodon 34 years after its introduction to Lake Kivu

The evolutionary consequences of an artificial introduction of the clupeid Limnothrissa miodon from Lake Tanganyika into Lake Kivu, East Africa were examined. In 1959, 57 400 fry (mixture of Limnothrissa and the related clupeid, Stolothrissa tanganicae ), were released into Lake Kivu to boost fisheries production. Comparisons were made between respective source and transplant populations 34 years later (1993) using morphometrics ('truss' method), allozymes (29 enzyme-coding loci) and mitochondrial (mt) DNA variation (RFLP analysis of PCR-amplified ND5/6 genes). Significant morphological and genetic differentiation between source and transplant samples was detected, with a distinct clustering of Kivu Limnothrissa on respective dendrograms, especially at the morphometric and mtDNA levels. Differentiation within Lake Tanganyika was, however, consistently higher than that between lakes. Allozymic diversity was similar in samples from both lakes (Lake Tanganyika: heterozygosity = 0.0658, mean number of alleles=1.44; Lake Kivu: heterozygosity = 0.0655; mean number of alleles = 1.48), however, a significantly lower mtDNA haplotype diversity was detected in Lake Kivu (Lake Tanganyika: 0.905; Lake Kivu: 0.755). Data suggest that high post-introduction mortality and various demographic factors reduced the effective population size of the introduced population to tens rather than thousands of individuals, resulting in a reduction in genetic diversity and founder effect.     

Phylogenetic relationships of Sakhalin taimen Parahucho perryi inferred from PCR-RFLP analysis of mitochondrial DNA

RFLP analysis of three amplified mtDNA fragments (Cytb/D-loop, ND1/ND2, and ND3/ND4L/ND4) was performed in the following taxa: Parahucho perryi, Hucho taimen, Brachymystax lenok, B. tumensis, Salmo salar, Salvelinus leucomaenis, and S. levanidovi. For mtDNA of P. perryi, a substantial decrease in the haplotype and nucleotide diversity was observed as a result of random genetic drift, caused by a reduction in the effective population size. Nucleotide divergence estimates between the mtDNA haplotypes were determined. Sakhalin taimen P. perryi was found to be approximately equally diverged from S. salar and from the charrs of the genus Salvelinus, by 11.0 and 10.0%, respectively. The divergence between P. perryi and H. taimen constituted 14.6%, between P. perryi and lenoks of the genus Brachymystax, 14.2%, and between H. taimen and Brachymystax, 7.7%. The analysis of possible phylogenetic relationships of the mtDNA from P. perryi among the group of taxa examined confirmed validity of the genus Parahucho. Phylogenetic reconstructions performed showed that robustness of the trees constructed for the complex of phylogenetically informative characters over three mtDNA fragments was considerably higher than that of the trees constructed for individual genes.     

Genetic structure of Amphioctopus fangsiao (Mollusca,Cephalopoda) in Chinese waters inferred from variation in three mtDNA genes (ATPase 6, ND2, and ND5)

Amphioctopus fangsiao is an economically important resource found in Chinese coastal waters. Three mitochondrial DNA genes were used to assess the genetic structure among nine populations spanning the northern to southern regions of the Chinese coast. Fragments of 575, 639, and 640 bp in length representing three genes (ATPase 6, ND2, and ND5) were amplified from 183, 170, and 167 individuals, respectively. Overall, ATPase 6, ND2, and ND5 showed high haplotype diversity and low nucleotide diversity (HD: 0.683–0.896; −π: 0.021–0.033). All three mtDNA genes revealed high molecular variance among populations and low variance within populations. Φ_ST values obtained for ATPase 6 (Φ_ST = 0.000 to 0.997), ND2 (Φ_ST = 0.000 to 0.997), and ND5 (Φ_ST = 0.125 to 0.983) showed differentiation among the populations. The constructed phylogeographic tree and haplotype network separated the nine populations into two clades representing the northern and southern populations. Low salinity in the Changjiang River estuary may act as a barrier to promote the differentiation between the two clades. These results enhance our understanding of the genetic structure of A. fangsiao and can promote the management of its genetic resources.

     

Topological conflicts in phylogenetic analysis of different regions of the sable ( Martes zibellina L.) mitochondrial genome

Phylogenetic analysis of different regions of the mitochondrial genome of the sable showed the presence of several topologies of phylogenetic trees, but the most statistically significant topology is A-BC, which was obtained as a result of the analysis of the mitochondrial genome as a whole, as well as of the individual CO1, ND4, and ND5 genes. Analysis of the intergroup divergence of the mtDNA haplotypes (D xy) indicated that the maximum D xy values between A and BC groups were accompanied by minimum differences between B and C groups only for six genes showing the A-BC topology (12S rRNA, CO1, CO2, ND4, ND5, and CYTB). It is assumed that the topological conflicts observed in the analysis of individual sable mtDNA genes are associated with the uneven distribution of mutations along the mitochondrial genome and the mitochondrial tree. This may be due to random causes, as well as the nonuniform effect of selection.     

Analysis of the mitochondrial DNA from patients with Wolfram (DIDMOAD) syndrome

Wolfram or DIDMOAD (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy and Deafness) syndrome, which has long been known as an autosomal-recessive disorder, has recently been proposed to be a mitochondrial-mediated disease with either a nuclear or a mitochondrial genetic background. The phenotypic characteristics of the syndrome resemble those found in other mitochondrial (mt)DNA mediated disorders such as Leber's hereditary optic neuropathy (LHON) or maternally inherited diabetes and deafness (MIDD). Therefore, we looked for respective mtDNA alterations in blood samples from 7 patients with DIDMOAD syndrome using SSCP-analysis of PCR-amplified fragments, encompassing all mitochondrial ND and tRNA genes, followed by direct sequencing. Subsequently, we compared mtDNA variants identified in this disease group with those detected in a group of LHON patients (n = 17) and in a group of 69 healthy controls. We found that 4/7 (57%) DIDMOAD patients harbored a specific set of point mutations in tRNA and ND genes including the so-called class II or secondary LHON mutations at nucleotide positions (nps) 4216 and 4917 (haplogroup B). In contrast, LHON-patients were frequently (10/17, 59%) found in association with another cluster of mtDNA variants including the secondary LHON mutations at nps 4216 and 13708 and further mtDNA polymorphisms in ND genes (haplogroup A), overlapping with haplogroup B only by variants at nps 4216 and 11251. The frequencies of both haplogroups were significantly lower in the control group versus the respective disease groups. We propose that haplogroup B represents a susceptibility factor for DIDMOAD which, by interaction with further exogeneous or genetic factors, might increase the risk for disease. (Mol Cell Biochem 174: 209–213, 1997)     

Molecular characterization of clonal population structure and biogeography of arctic apomictic Daphnia from Greenland and Iceland

As part of a continuing international project to characterize the population genetic (clonal) structure of arctic members of the Daphnia pulex complex, 34 populations from western Iceland (N= 1373), and 76 populations from western Greenland (N= 2917), were surveyed for allozymic variation at six polymorphic enzyme loci. Mean clonal richness (±SE) was 1.91±0.19 and 1.50±0.12 for Iceland and Greenland populations, respectively. Mean clonal diversity (±1 SE) was 1.20±0.07 and 1.13±0.04 for Iceland and Greenland populations, respectively. Four widely distributed clones constituted 92.2% of the total animals surveyed from Iceland, while three locally abundant clones constituted 80.1% of the total animals collected primarily from Disko Island, western Greenland. Selected populations were screened for mitochondrial (mt)DNA variation using PCR-based RFLP analysis of a 2100 bp fragment containing part of the ND4 and ND5 genes. One mtDNA haplotype was very widespread in both western Greenland and western Iceland, although, a number of mutational derivatives were also detected. These data indicate the potential for long-distance dispersal of mtDNA lineages, of the order of hundreds or thousands of kilometers across the arctic. Phylogenetic analysis of the sequence of a 254 base pair (bp) fragment of the control region of the mtDNA molecule revealed two major clades one of which consisted solely of non-melanic lineages, and the other of which consisted almost exclusively of melanic lineages (i.e. one non-melanic lineage also clustered in this clade). Sequence divergence between the two clades averaged 7.3%. Both mitochondrial analyses did not reveal any distinct intraregional clustering of lineages. We discuss our results in reference to previous molecular work done on this arctic Daphnia complex, and we attempt to infer phylogeographic patterning based on geological/glaciological historical events in this region of the arctic.