PHYLOGEOGRAPHIC STRUCTURE IN MITOCHONDRIAL DNA OF THE LAKE WHITEFISH (COREGONUS CLUPEAFORMIS) AND ITS RELATION TO PLEISTOCENE GLACIATIONS

Restriction-fragment length polymorphisms were employed to evaluate the phylogenetic relationships, the genetic diversity and the geographic structure in mitochondrial DNA (mtDNA) lineages of the lake whitefish, Coregonus clupeaformis. Thirteen restriction enzymes that produced 148 restriction fragments were used to assay mtDNAs of 525 specimens collected among 41 populations. The sampling covered the entire range of the species, from Alaska to Labrador. Four distinct phylogeographic assemblages were identified. The Beringian assemblage, confined to Yukon and Alaska, was phylogenetically distinct from other assemblages and exhibited the highest level of nucleotide diversity. The Acadian assemblage was confined to southeastern North America and composed of a unique mtDNA clade. The Atlantic assemblage was confined to southern Québec and the northeastern United States and was also observed among anadromous populations of northern Hudson Bay. This group was highly polymorphic and responsible for most of the mtDNA diversity observed outside Beringia. The Mississippian assemblage occupied most of the actual range of lake whitefish, from the Mackenzie delta to Labrador. Ninety-two percent of all whitefish of this proposed origin belonged to a single mtDNA haplotype. Overall, the diversity, the geographic structure and the times of divergence of mtDNA phylogenetic assemblages correlate with the Pleistocene glaciations classically assumed to have dramatically altered the genetic diversity of northern fishes in recent evolutionary times. Our results emphasize the dominant role of these catastrophic events in shaping the population genetic structure of lake whitefish.     

Use of cellular oncogene probes to identify Morone hybrids.

We tested the ability of cellular oncogene (c-onc) probes to identify F1 hybrids and the lineage of known backcrosses within the fish genus Morone. Total DNA was isolated from five to 14 individuals per North American Morone species (striped bass, white bass, white perch, and yellow bass). The DNA was digested with two restriction enzymes, Eco RI and Hin dIII, Southern blotted, and hybridized to six different c-onc probes including v-abl, v-erb B, c-myc, c-H-ras, c-K-ras, and v-src. We found fixed genotypic differences among the four species for all six probes in single restriction enzyme digests. The heritability of these nuclear DNA genotypes was evaluated in hatchery-produced F1 Morone hybrids (striped bass x white bass and striped bass x white perch) tested with the six informative single probe/restriction enzyme combinations. All F1 individuals exhibited heterozygosity in all diagnostic nuclear DNA fragments, confirming the Mendelian inheritance of these genotypes in these fish. Furthermore, analysis of these nuclear DNA genotypes in hatchery-produced backcrosses of F1 hybrids striped bass x (white bass x striped bass) detected both recombinant and parental genotypes at all six polymorphic c-onc sequences. The lineage of suspected Morone hybrids of unknown descent collected from Lewis Smith Lake, Alabama, and from the Occoquan River, Virginia, was determined using the c-onc probes. Our results suggest that c-onc probes are suitable markers to unequivocally identify F1 hybrids and backcrosses and to quantify introgression in natural populations of fishes. The addition of RFLP analysis of mtDNA provided a complete ancestral history of individual fish.     

Genetic variants of ribosomal DNA and mitochondrial DNA between swamp and river buffaloes

To clarify the genetic relationship between Swamp and River buffaloes, the restriction fragment length polymorphisms (RFLPs) of nuclear genomic ribosomal DNA (rDNA) and cytoplasmic mitochondrial DNA (mtDNA) were analysed. Blood or liver samples from 73 Swamp and three River buffaloes were collected in East and South-east Asian countries. DNA samples from cattle, goats and sheep were used for comparisons. The analysis of rDNA allowed water buffaloes, cattle, goats and sheep to be characterized by four distinct repeat-types. However, swamp and river buffaloes showed the same repeat-type. Divergence of water buffalo and cattle is considered to have occurred approximately four to six million years ago. The RFLPs for mtDNA divided water buffaloes into three haplotypes, swamp-1, swamp-2 and river types. Swamp-1 accounted for 91% of all swamp buffaloes while swamp-2 was observed only in water buffaloes from Thailand (9%). All river buffaloes were of the same haplotype. No differences were observed between swamp and river buffaloes at the rDNA level. In contrast, a few distinct differences between them were found at the mtDNA level. Therefore, mtDNA polymorphisms provide an adequate means for classifying water buffaloes into either swamp or river buffaloes.     

The evolutionary history of Drosophila buzzatii. XXX. Mitochondrial DNA polymorphism in original and colonizing populations

Both original and colonizer populations of Drosophila buzzatii have been analyzed for mtDNA restriction polymorphisms. Most of the mtDNA nucleotide variation in original populations of NW Argentina can be explained by intrapopulation diversity and only a small fraction can be accounted for by between-population diversity. Similar results are obtained using either the estimated number of nucleotide substitutions per site or considering each restriction site as a locus. Colonizer populations of the Iberian Peninsula are monomorphic and show only the most common haplotype from the original populations. Under the infinite island model and assuming that populations are in equilibrium, fixation indices indicate enough gene flow to explain why the populations are not structured. Yet, the possibility exists that populations have not reached an equilibrium after a founder event at the end of the last Pleistocene glaciation. Tajima's test suggests that directional selection and/or a recent bottleneck could explain the present mtDNA differentiation. Considering the significant population structure found for the chromosomal and some allozyme polymorphisms, the among- population uniformity for mtDNA variability argues in favor of the chromosomal and some allozyme polymorphisms being adaptive.      

Homing behaviour facilitates subtle genetic differentiation among river populations of Alosa sapidissima: microsatellites and mtDNA

Significant but subtle differentiation was detected for both microsatellite DNA and mitochondrial DNA among four populations of American shad Alosa sapidissima . The data indicate that straying among rivers is sufficient to permit only marginal population differentiation in this species, but suggest that individual river populations should be managed as distinct stocks. Comparison of the Hudson and Columbia populations, the latter derived from the former over 100 years ago, revealed only a slight reduction in microsatellite DNA variation for the founded population but halving of mitochondrial DNA, consistent with the haploid maternal inheritance of the latter marker. The depleted and endangered James River (Virginia) population and two other Atlantic coast populations exhibited similar levels of microsatellite DNA variation, but mtDNA diversity in the James River was marginally lower than in other Atlantic populations, again consistent with the low effective population size of mtDNA.     

HLA-DR4-associated haplotypes are genotypically diverse within HLA

Biochemical diversity among products of class II HLA genes has been observed in individuals who appear to be HLA-D and DR-identical by cellular and serologic typing. We used techniques of restriction enzyme fragment analysis by Southern blotting to analyze this diversity at the level of cellular DNA. A panel of 17 HLA-DR4 homozygous cell lines (HCL) were investigated by using cDNA probes homologous to DQ beta, DQ alpha, and DR beta genes. Each probe was hybridized to cellular DNA digested with a series of different restriction endonucleases. Polymorphisms were observed with the use of the enzymes Pst I, Hind III, and Bam HI: Hybridization of cellular DNA digested with Hind III and Pst I with the DQ beta probe revealed specific polymorphisms, as did hybridization of the Pst I digest with the DQ alpha cDNA probe and the Bam HI digest with the DR beta probe. The observed differences fall into two categories: first, considerable diversity was seen between HLA-DR4 HCL that represent different HLA-D-defined haplotypes; second, diversity was also observed among HCL of the same DR4-associated HLA-D cluster. In contrast to the DQ cDNA probes, hybridization with the DR beta probe revealed relatively limited polymorphism by using a panel of different restriction endonucleases. Thus, although there is a general pattern of polymorphic restriction enzyme fragments homologous to DQ probes within an HLA-D cluster, the pattern seen for any particular cell line was not sufficiently distinct to assign an HLA-D or DR specificity.     

Molecular genetic evidence for the post-Pleistocene divergence of populations of the arctic-alpine ground beetle Amara alpina (Paykull) (Coleoptera: Carabidae)

Aim This study examines the hypothesis that the biogeographic history of a species is reflected in the distribution of molecular genetic diversity and the phylogenies of extant populations. Location Populations of arctic-alpine ground beetle Amara alpina were analysed from Beringia (Alaska and northernmost British Columbia), the Hudson Bay region, the northern Appalachian Mountains, and the central Rocky Mountains of North America. Methods Mitochondrial restriction site variation of specimens from twenty-two populations were assayed by using radioactively labelled mtDNA to probe Southern membranes containing restriction enzyme digested total DNA. Restriction sites were mapped and genetic distances were calculated by pairwise comparison of presence and absence of restriction sites. Genetic distances were used in a molecular analysis of variance and to construct a minimal spanning tree. Parsimony methods were used to investigate the phylogenetic relationships between the haplotypes. These results were compared to an existing model for postglacial dispersal based on fossil and modern occurrences of arctic-alpine beetles. Results Among the twenty-two populations, fifteen haplotypes were detected. Genetic variation within each of the four regions corresponded to that expected from the palaeontologically based model. Beringian populations were the most genetically diverse. In contrast, no restriction site variation was observed in populations from the Hudson Bay region. Intermediate amounts of variation were observed in alpine populations of the Rocky and Appalachian Mountains. Maximum parsimony and cluster analysis provide evidence that at least two ancestral haplotypes existed in the Southern refugium from which the Rocky and the Appalachian Mountains populations were founded. Main conclusions The genetic results are generally consistent with the palaeontologically based model. The diversity of Beringian populations is consistent with this region having been continuously inhabited by Amara alpina throughout the Pleistocene. The Hudson Bay region was not deglaciated until about 6000 years, and its populations have no restriction site variation. The molecular genetic data support the interpretation that the Hudson Bay region was colonized from Beringia based on the occurrence of the same haplotype in both regions.     

Incongruent estimates of population differentiation among brook charr, Salvelinus fontinalis, from Cape Race, Newfoundland, Canada, based upon allozyme and mitochondrial DNA variation

We determined the amount and temporal stability of genetic differentiation among brook cham sampled from five rivers on Cape Race, Newfoundland, with an electrophoretic analysis of 42 protein coding loci. Fish from four of these rivers were analysed for restriction fragment length polymorphisms in mitochondrial DNA (mtDNA). A single mtDNA clone was observed in all rivers sampled, except one, where 47% offish were from a different and relatively divergent clone (0.31 % sequence divergence). In contrast, Cape Race brook charr show large amounts of genetic differentiation at six enzyme coding loci; Nei's genetic distance ranged between 0,020 and 0.048. This differentiation is relatively stable as no significant differences in allele frequencies were detected between fish sampled from two rivers over two consecutive years. The most divergent population based on protein polymorphism is not that with two mtDNA clonal lineages. In contrast to the commonly held view, mtDNA analyses do not necessarily provide greater resolution of population structure than allozyme analyses.     

A rapid protocol for the purification of mitochondrial DNA suitable for studying restriction fragment length polymorphisms

When analyzing mitochondrial DNA (mtDNA) from various normal and malignant human tissues, it became necessary to enhance mtDNA isolation for improved yields and quality. The method described here consists of rapid and simple-to-perform steps, avoiding complicated instrumentation. It was designed for preparation of undegraded mtDNA and is highly useful when limited amounts of tissues, cells and unique biopsies of tumors (fresh or frozen) are available. The resulting mtDNA is sufficiently pure for restriction analysis, subcloning, labeling and various types of hybridization. Using Sau3A and MspI, restriction analysis revealed new restriction-fragment length polymorphisms for Caucasians, independent of the DNA source, and hence excluding tissue-specific DNA modifications.     

Activation of c-myc and c-K-ras oncogenes in primary rat tumors induced by ionizing radiation.

An activated K-ras oncogene was detected by transfection in NIH 3T3 cells and by Southern blot analysis in 6 of 12 rat skin tumors induced by ionizing radiation. The DNA from 10 of the 12 tumors also showed c-myc gene amplification and restriction polymorphisms. Evidence for tissue specificity was observed in patterns of oncogene activation, with each of three clear cell carcinomas exhibiting activation of both c-myc and K-ras oncogenes.     

Mitochondrial restriction fragment length polymorphisms in wild Phaseolus vulgaris L.: insights on the domestication of the common bean

Summary Previous examination of intraspecific mitochondrial DNA (mtDNA) diversity in common bean, Phaseolus vulgaris, showed that five restriction fragment length polymorphisms (RFLPs) distinguish the mitochondrial genomes of the two major gene pools of cultivated beans, the Mesoamerican and the Andean. In the study presented here, mtDNA was used to compare the amount of diversity in cultivated beans to that in collections of wild beans to gain an understanding of how and when the mitochondrial genomes of the gene pools became distinct. The mtDNA of six wild bean accessions from Central and South America were digested with nine restriction endonucleases and analyzed by Southern hybridization. A total of twenty RFLPs were detected demonstrating a significantly higher amount of mtDNA variability in wild beans than in cultivated ones. All of the wild beans had the same mtDNA pattern for four out of the five inter-gene pool RFLPs, indicating that the polymorphism arose soon after domestication: two in the gene pool of the cultivated Mesoamerican beans and two in the gene pool of the cultivated Andean beans. The fifth RFLP must have occurred before domestication since the locus was also polymorphic in the wild beans. Wild beans from the south Andes were distinct and less variable than wild accessions of the north Andes and Mesoamerica. The distribution of mtDNA RFLPs among the wild beans supports the concept of two distinct domestication events for P. vulgaris.     

DNA fingerprints from hypervariable mitochondrial genotypes

Conventional surveys of restriction-fragment polymorphisms in mitochondrial DNA of menhaden fish (Brevoortia tyrannus/patronus complex) and chuckwalla lizards (Sauromalus obesus) revealed exceptionally high levels of genetic variation, attributable to differences in mtDNA size as well as in restriction sites. The observed probabilities that any two randomly drawn individuals differed detectably in mtDNA genotype were 0.998 and 0.983 in the two species, respectively. Thus, the variable gel profiles provided unique mtDNA "fingerprints" for most conspecific animals assayed. mtDNA fingerprints differ from nuclear DNA fingerprints in several empirical respects and should find special application in the genetic assessment of maternity.      

Mitochondrial DNA variation in populations of the chum salmon Oncorhynchus keta (Walbaum) from rivers of the Prymorye and Sakhalin

Mitochondrial DNA (mtDNA) variation was studied using restriction fragment length polymorphism (RFLP) in chum salmon populations from three rivers in southern Primorye and one river in Sakhalin Island. Significant differences were detected between the samples from Primorye and Sakhalin Island. No differences were found between the samples from the rivers of Primorye, which can be explained by a high rate of gene flow due to transplantation of spawn from one river to another. The effect of fish breeding on the chum salmon populations correlated with the indices of haplotype and nucleotide diversity (h and pi, respectively). The lowest diversity was found in the completely artificial population from the Ryazanovka River; the highest, in natural populations from the Narva and Naiba rivers. Frequencies of haplotypes in consecutive generations were significantly different, which confirms the effects of genetic drift on the small-size chum salmon populations of Primorye.     

Mitochondrial and Allozyme Genetics of Incipient Speciation in a Landlocked Population of Galaxias Truttaceus (Pisces: Galaxiidae)

Galaxias truttaceus is found in coastal rivers and streams in south-eastern Australia. It spawns at the head of estuaries in autumn and the larvae spend 3 months of winter at sea before returning to fresh water. In Tasmania there are landlocked populations of G. truttaceus in a cluster of geologically young lakes on the recently glaciated Central Plateau. These populations have no marine larval stage and spawn in the lakes in spring. Speciation due to land locking is thought to be a frequent occurrence within Galaxias. To investigate the nature of the speciation event which may be occurring within lake populations of G. truttaceus we studied the mitochondrial DNA (mtDNA) and allozyme diversity of both lake and stream populations. Using the presence or absence of restriction sites recognized by 13 six-base restriction endonucleases, we found 58 mtDNA haplotypes among 150 fish collected from 13 Tasmanian and one south-east Australian mainland stream populations. The most parsimonious network relating the haplotypes by site loss or gain was starlike in shape. We argue that this arrangement is best explained by selection upon slightly beneficial mutations within the mitochondrial genome. Gene diversity analysis under Wright's island model showed that the populations in each drainage were not genetically subdivided. Only two of these stream haplotypes were found among the 66 fish analyzed from four lake populations. Despite the extreme lack of mtDNA diversity in lake populations, the observed nuclear DNA heterozygosity of 40 lake fish (0.10355) was only slightly less than that of 82 stream fish (0.11635). In the short time (3000-7000 years) that the lake fish have been landlocked, random genetic drift in a finite, stable-sized population was probably not responsible for the lack of mtDNA diversity in the lake populations. We infer the lake populations have probably experienced at least one, severe, but transitory bottleneck possibly induced by natural selection for life-history characters essential for survival in the lacustrine habitat. If speciation is occurring in the landlocked populations of G. truttaceus, then it may be driven by genetic transilience.     

Distribution of mitochondrial DNA variation in lake sturgeon (Adpenser fulvescens) from the Moose River basin, Ontario, Canada

We analysed mitochondrial DNA (mtDNA) variation of lake sturgeon ( Adpenser fulvescens ) from the Moose River basin. Our objective was to address various proximate and ultimate factors which may influence the distribution of lake sturgeon mtDNA haplotype lineages in this watershed. The lake sturgeon sampled were characterized by only two mtDNA hapiotypes based on a restriction fragment length polymorphism analysis with 40 restriction endonucleases and direct sequencing of 275 nucleotides in the mtDNA control region. We detected no heterogeneity in the mtDNA haplotype frequencies of lake sturgeon captured from different sites within rivers including those separated by major hydroelectric installations. However, lake sturgeon from one tributary had significantly different haplotype frequencies than those from other tributaries suggesting that they composed a discrete genetic stock. These results suggest that gene flow among most sites is significant and is an important factor affecting the distribution of mtDNA variation in this species. The genetic structuring and diversity are discussed in relation to lake sturgeon management and conservation.     

Genetic differentiation among chum salmon, Oncorhynchus keta (Walbaum), from Primorye and Sakhalin

Mitochondrial DNA (mtDNA) was examined in six wild populations of chum salmon ( Oncorhynchus keta Walbaum) distributed over the Primorye Region which extends approximately 1000 km along the Sea of Japan coast, and six populations from Sakhalin, using restriction enzyme analysis. By means of two restriction enzymes ( Bam HI and Eco 81I) eight mtDNA clonal lines were revealed in the 346 chum salmon studied. Mitochondrial DNA variants grouped the fish into two major clusters representing the Primorye and Sakhalin regions. Analysis of different chum salmon generations showed stability in the temporal population genetic structure in the three Primorye populations studied, but instability in the Sakhalin population of the Naiba River. We succeeded in detecting four major mtDNA clonal variants in Primorye chum salmon. The geographic distribution of clonal frequencies in Primorye populations has a clinal U-shape associated with the north-south axis of the Primorye region. On the whole, Sakhalin populations are less heterogeneous than Primorye ones. Two hatchery-seeded stocks from south west Sakhalin showed no mtDNA clonal variation.     

Microsatellite and mitochondrial DNA assessment of population structure and stocking effects in Arctic charr Salvelinus alpinus (Teleostei: Salmonidae) from central Alpine lakes

Despite geographical isolation and widespread phenotypic polymorphism, previous population genetic studies of Arctic charr, Salvelinus alpinus , have detected low levels of intra- and interpopulation variation. In this study, two approaches were used to test the generality of low genetic diversity among 15 Arctic charr populations from three major drainages of the central Alpine region of Europe. First, a representative subsample of each drainage was screened by PCR–RFLP analysis of mtDNA using 31 restriction enzymes. All individuals but one shared an identical haplotype. In contrast, microsatellite DNA variation revealed high levels of genetic diversity within and among populations. The number of alleles per locus ranged from six to 49, resulting in an overall expected heterozygosity from 0.72 ± 0.09 to 0.87 ± 0.04 depending on the locus. Despite evidence for fish transfers among Alpine charr populations over centuries, genetic diversity was substantially structured, as revealed by hierarchical Φ statistics. Eighteen per cent of total genetic variance was apportioned to substructuring among Rhône, Rhine, and Danube river systems, whereas 19% was due to partitioning among populations within each drainage. Cluster analyses corroborated these results by drainage-specific grouping of nonstocked populations, but also revealed damaging effects of stocking practices in others. However, these results suggest that long-term stocking practices did not generally alter natural genetic partitioning, and stress the importance of considering genetic diversity of Arctic charr in the Alpine region for sound management. The results also refute the general view of Arctic charr being a genetically depauperate species and show the potential usefulness of microsatellite DNAs in addressing evolutionary and conservation issues in this species.     

Phylogeographical lineages of Arctic grayling (Thymallus arcticus) in North America: divergence, origins and affinities with Eurasian Thymallus

The number and location of Arctic glacial refugia utilized by taxa during the Pleistocene are continuing uncertainties in Holarctic phylogeography. Arctic grayling (Thymallus arcticus) are widely distributed in freshwaters from the eastern side of Hudson Bay (Canada) west to central Asia. We studied mitochondrial DNA (mtDNA) and microsatellite DNA variation in North American T. arcticus to test for genetic signatures of survival in, and postglacial dispersal from, multiple glacial refugia, and to assess their evolutionary affinities with Eurasian Thymallus. In samples from 32 localities, we resolved 12 mtDNA haplotypes belonging to three assemblages that differed from each other in sequence by between 0.75 and 2.13%: a 'South Beringia' lineage found from western Alaska to northern British Columbia, Canada; a 'North Beringia' lineage found on the north slope of Alaska, the lower Mackenzie River, and to eastern Saskatchewan; and a 'Nahanni' lineage confined to the Nahanni River area of the upper Mackenzie River drainage. Sequence analysis of a portion of the control region indicated monophyly of all North American T. arcticus and their probable origin from eastern Siberian T. arcticus at least 3 Mya. Arctic grayling sampled from 25 localities displayed low allelic diversity and expected heterozygosity (H(E)) across five microsatellite loci (means of 2.1 alleles and 0.27 H(E), respectively) and there were declines in these measures of genetic diversity with distance eastward from the lower Yukon River Valley. Assemblages defined by mtDNA divergences were less apparent at microsatellite loci, but again the Nahanni lineage was the most distinctive. Analysis of molecular variance indicated that between 24% (microsatellite DNA) and 81% (mtDNA) of the variance was attributable to differences among South Beringia, North Beringia and Nahanni lineages. Our data suggest that extant North American Arctic grayling are more diverse phylogeographically than previously suspected and that they consist of at least three major lineages that originated in distinct Pleistocene glacial refugia. T. arcticus probably originated and dispersed from Eurasia to North America in the late to mid-Pliocene, but our data also suggest more recent (mid-late Pleistocene) interactions between lineages across Beringia.