Restriction Site Variation Research Articles

Mitochondrial DNA sequence variation among pheromotypes of the dingy cutworm, Feltia jaculifera (Gn.) (Lepidoptera: Noctuidae)

The dingy cutworm, Feltia jaculifera, is a complex of at least four pheromonal races (pheromotypes). We examined mitochondrial DNA (mtDNA) sequence and restriction-site variation in the cytochrome oxidase subunit I and II genes. Among 74 specimens representing the four pheromotypes, we found three discrete mitochondrial lineages. The most divergent mtDNA within F. jaculifera, the γ lineage, was associated with pheromotype C. This result confirms an earlier allozyme survey, which also found that pheromotype C was the most genetically distinct race. Thus, pheromotype C probably represents a classic sibling species. Of the two most similar mitochondrial lineages, the α lineage was disproportionately associated with pheromotype A and the β lineage was disproportionately associated with pheromotypes B and D. Although the correspondence between mitochondrial lineages and pheromotypes was incomplete, mtDNA variation provides strong nonbehavioral evidence for genetic divergence between pheromotypes A and B + D. Allozymes revealed more minor differentiation among the A, B, and D races. For pheromotypes A and B, the presence of distinct mtDNA lineages within populations without perfect correspondence to pheromone polymorphism has several possible explanations. It may be due to retained ancestral mtDNA polymorphism, whether initial divergence of pheromone races occurred in allopatry or sympatry, or to divergence in isolation with introgression following secondary contact. The greater differences in allele frequencies of mtDNA than of allozymes may be due either to the susceptibility of mtDNA to bottleneck effects or to asymmetric selection against hybrid females.

Numerous chloroplast DNA polymorphisms are shared among different populations and species in the aureoid Senecio (Packera) complex

Species boundaries within the aureoid Senecio complex (Packera) are poorly defined, possibly as a result of hybridization and introgression. The results of restriction site analysis of cpDNA using 16 enzymes and hybridization with lettuce probes representing over 95% of the chloroplast genome reveal a high level of intrapopulational polymorphism combined with a relatively low level of interspecific variation. Forty-nine populations representing 23 different species were examined, and only 20 variable restriction sites were identified. Eleven of the 20 variable sites were polymorphic in at least one population, and 14 of the 23 species were polymorphic for at least one site. The polymorphisms are widespread and are found in species representing all of the previously recognized subgroups of the aureoid complex. Individuals analyzed separately were shown to possess only a single haplorype. Although the polymorphisms may be the result of hybridization and introgression, it is difficult to explain the geographical patterns based on the current distribution of species. It is possible that the aureoid complex represents a single, polymorphic biological species. Keywords: Asteraceae, Senecio, aureoid complex, Packera, cpDNA, molecular systematics.

Phylogenetic analysis of restriction-site variation in wild and cultivated Amaranthus species (Amaranthaceae)

Amaranthus includes approximately 60 species, of which three are cultivated as a grain source. Many wild Amaranthus species possess agriculturally desirable traits such as drought and salt tolerance, and pathogen resistance. We examined relationships among wild and cultivated Amaranthus species based upon restriction-site variation in two chloroplast DNA regions and in a nuclear DNA region. The chloroplast regions consisted of (1) an intergenic spacer in transfer RNA genes and (2) the ribulose-1,5-bisphosphate carboxylase gene with a flanking open reading frame. The nuclear region was the internal transcribed spacers ITS-1 and ITS-2 flanking the 5.8S gene in the ribosomal DNA. These regions were amplified by the polymerase chain reaction and digested with a total of 38 restriction endonucleases. We detected 11 potentially informative restriction-site mutations and seven length-polymorphisms among the 28 Amaranthus species. Parsimony analysis was used to find the shortest tree for each separate data set (chloroplast, nuclear, and length) and for two combined matrices (chloroplast/nuclear and all data sets). Overall, there was a low level of variation which generated poorly resolved trees among the 28 species. Congruence analyses revealed that the chloroplast and nuclear data sets were congruent with each other but not to the length data set. The congruence of the chloroplast and nuclear data sets suggested that cytoplasmic gene flow may not be a confounding factor in our analyses. The phylogeny also suggested that drought tolerance evolved independently several times. The molecular phylogeny provides a basis for selection of species pairs for crop development.

The phylogenetic position of East AsianSedumspecies (Crassulaceae) based on chloroplast DNA trnL (UAA)-trnF (GAA) intergenic spacer sequence variation

SUMMARY Phylogenetic relationships of 27 Crassulaceae species, including a broad sample of 19 Sedum species, were inferred from sequence variation of the chloroplast trnL (UAA)–trnF (GAA) intergenic spacer to assess the position of alkaloid containing Asian Sedum species. The overall topology of the phylogeny fully agrees with results based on chloroplast DNA restriction-site variation. The five alkaloidal, Asian Sedum species are included in the Acre lineage. These results support Steven's and co-workers (1995) hypothesis on the distribution of alkaloids in the Crassulaceae which states that the occurrence of alkaloids is restricted to the species of the Acre lineage. Although monophyly of the Acre lineage is strongly supported, relationships within this clade are only partially resolved, probably as a result of incomplete systematic sampling.

RECONSTRUCTION OF THE EVOLUTION OF REPRODUCTIVE CHARACTERS IN PONTEDERIACEAE USING PHYLOGENETIC EVIDENCE FROM CHLOROPLAST DNA RESTRICTION-SITE VARIATION.

We reconstructed the phylogenetic history of Pontederiaceae using chloroplast DNA restriction-site variation from approximately two-thirds of the species in this family of aquatic monocotyledons. The molecular phylogeny was used to evaluate hypotheses concerning the evolution of reproductive characters associated with the breeding system. The family has four main genera, two of which (Eichhornia and Pontederia) have tristylous, predominantly outcrossing species, while two (Monochoria and Heteranthera) have enantiostylous taxa. Self-incompatibility is restricted to some but not all tristylous species. In Eichhornia and Pontederia, predominantly selfing species with small monomorphic flowers (homostyly) have been hypothesized to result from the multiple breakdown of tristyly. Restriction-site variation provided a well supported phylogeny of ingroup taxa, enabling the mapping of reproductive characters onto trees. Two contrasting optimization schemes were assessed, differing in the relative weights assigned to shifts in character states. The reconstructed sequence of floral character-state change was used to assess competing hypotheses concerning the origin and breakdown of tristyly, and the relationships between tristylous and enantiostylous syndromes. Our results indicate that the class of optimization scheme used was the most critical factor in reconstructing character evolution. Despite some topological uncertainties and difficulty in reconstructing the primitive floral form in the family, several broad conclusions were possible when an unordered, unequally-weighted optimization scheme was used: (1) tristyly originated either once or twice, while the occurrence of enantiostyly in Monochoria and Heteranthera was always found to have independent origins; (2) tristyly has repeatedly broken down leading to selfing, homostylous taxa; and (3) self-incompatibility probably arose after the origin of floral trimorphism, a sequence of events that conflicts with some evolutionary models.

The stoned locus of Drosophila melanogaster produces a dicistronic transcript and encodes two distinct polypeptides.

The stoned gene of Drosophila melanogaster is required for normal neuronal function in both adult and larva. We have identified DNA sequences that lie within a genetic region that is known to include the stoned gene and that also reveal restriction site variations in two stoned lethal mutants. This genomic region contains a single transcription unit coding for an approximately 8.4-kb transcript. The transcript is preferentially expressed in the head of adult flies. The isolation and sequencing of cDNA and genomic clones reveals that stoned appears to encode a dicistronic mRNA, although the possible existence of other forms of mRNA cannot be excluded. Antibody cross-reactivity shows that two proteins are translated from the stoned locus in vivo. Both open reading frames (ORFs) encode novel proteins. The protein encoded by the first ORF contains four tandemly repeated motifs, and one domain of the protein encoded by the second ORF shows similarity to a family of proteins (AP50s) associated with clathrin assembly protein complexes.

Molecular Phylogenetic Analysis of the Hawaiian Endemics Schiedea and Alsinidendron (Caryophyllaceae)

Schiedea and Alsinidendron (Caryophyllaceae), which represent the fifth or sixth largest endemic radiation of species in the angiosperm flora of the Hawaiian Islands, exhibit striking diversity in morphology, breeding system, and habitat. To gain a historical perspective on this diversity, we conducted a phylogenetic analysis using restriction site variation in chloroplast DNA and nuclear ribosomal DNA. In addition, we compared, and ultimately combined, the molecular data with a recently published morphologi- cal data set. Within the Schiedea-Alsinidendron lineage, DNA variation is limited, and relationships are generally poorly resolved. These results raise the possibility that, following the initial colonization of the Hawaiian archipelago and the early diversification of the complex, much of the complex radiated rapidly and relatively recently. Phylogenetic analyses of DNA data revealed three clades within the complex (the S. membranacea, S. nuttallii, and S. adamantis clades), in agreement with results of a morphologically-based analysis. Molecular data do not, however, support the S. globosa clade, a weakly-supported clade based on morphology. A combined analysis of morphological and molecular data provided both greater resolution and stronger internal support than either data set did individually. The molecular and combined topologies suggest nearly identical patterns of the evolution of sexual dimorphism, habitat shifts, and biogeography within the complex. However, the greater resolution in trees derived from the combined analysis suggests simpler patterns of breeding-system evolution and habitat shifts. Sexual dimorphism may have evolved twice in the complex, with a single reversal to hermaphroditism in one species, or perhaps only once, with three reversals to hermaphroditism. Although the habitat occupied by the ancestor of the complex remains uncertain, it appears that a single shift to dry habitats more or less accompanied the evolution of dimorphic breeding systems, followed by a single shift back to a mesic environment in one species. Alternatively, two parallel shifts to dry habitats may have occurred. Molecular data are consistent with an origin on Kaua'i of the S. membranacea, S. adamantis, and S. nuttallii clades, as suggested by previous morphological analyses. However, both the molecular and combined trees suggest it is equally likely that the complex originated on O'ahu.

Chloroplast DNA phylogeny of subtribe Dendrobiinae (Orchidaceae): Insights from a combined analysis based onrbcL sequences and restriction site variation

Phylogenetic analyses using two chloroplast DNA data sets, derived from variation of the ribulose-bisphosphate carboxylase gene (rbcL) and restriction sites, were performed to examine relationships among 13 taxa in subtribe Dendrobiinae, one of the most taxonomically complicated groups in Orchidaceae, and its putative sister groups. Owing to a limited number of informative substitutions, therbcL data set did not provide conclusive evidence in itself. The data set combiningrbcL and restriction site mutations, however, provided the following insights: (1)Pseuderia belongs with tribe Podochileae rather than tribe Dendrobieae. (2) Subtribe Dendrobiinae is monophyletic ifPseuderia is excluded. (3) ExcludingPseuderia, Dendrobiinae comprises three major clades: Clade 1 (Dendrobium sectionSpatulata, Cadetia, Diplocaulobium, andFlickingeria); Clade 2 (Dendrobium sectionsDendrobium andCallista); and Clade 3 (Epigeneium). (4)Epigeneium diverged early from the lineage including Clades 1 and 2. (5) Relative toCadetia, Diplocaulobium, andFlickingeria, Dendrobium is shown to be para-/polyphyletic. (6)Diplocaulobium andFlickingeria constitute a monophyletic clade, from which cladeDendrobium sectionSpatulata andCadetia form succesive sister groups. Among these results, (1) and (5) are especially stable in view of the congruence between the separate and combined analyses as well as robust internal support.

INTRASPECIFIC CHLOROPLAST DNA VARIATION AND BIOGEOGRAPHY OF NORTH AMERICAN LIRIODENDRON L. (MAGNOLIACEAE).

Restriction site variation in chloroplast DNA (cpDNA) was surveyed to analyze population dynamics in Liriodendron tulipifera L., a woody angiosperm found in eastern North America. Two cpDNA haplotypes, differing by the presence or absence of five restriction site changes (nucleotide sequence divergence estimated as approximately 0.15%) are geographically structured; 61 widespread populations possess the "northern" haplotype and three isolated populations of central Florida possess the "southern" haplotype. This geographic break in cpDNA distribution corresponds to patterns of geographic distribution revealed by a previous survey of allozyme variation, with the exception that analyses of allozyme data further divided the populations containing the northern cpDNA haplotype into two groups, a widespread upland group and a coastal intermediate group. Analyses of these two independent data sets together support the hypothesis that L. tulipifera survived the glacial advances of the Pleistocene in two distinct refugia, possibly as different taxa, and the intermediate coastal group was putatively formed from recent hybridizations between these entities.

A Feldmannia Algal Virus Has Two Genome Size-Classes

Persistent viruses occur intracellularly in brown algae, specifically the Ectocarpales, and as reported here in the genusFeldmannia. Feldmanniaspecies are small (1 mm–several cm), filamentous forms with single-celled meiotic sporangia that normally produce haploid zoospores. In the isolate reported here, spores were not observed in the sporangia but rather numerous (∼106per cell) polyhedral viruses are formed in their place. Two dsDNA genome classes of 158 and 178 kbp, with two restriction site variants of each, are described. The individual abundance of each genome in viral preparations is affected by culture temperature. A cosmid library was used to generate circular restriction enzyme (BamHI,NotI, andPstI) site maps.

Evolutionary Genetics of Hawaiian Freshwater Fish

We surveyed mitochondrial (mt)DNA restriction site variation within each of the five native species of Hawaiian freshwater fishes: Lentipes concolor, Stenogobius hawaiiensis, Sicyopterus stimpsoni, Awaous guamensis, and Eleotris sandwicensis. MtDNA haplotypes did not vary geographically except among samples of S. hawaiiensis, where haplotypes from Maui clustered together. One haplotype in this cluster also was found on Kauai and on Oahu, and one haplotype was found only on Kauai. MtDNA data suggest gene flow among islands, consistent with interisland dispersal through amphidromous larvae. We hypothesize that dispersal has prevented further speciation in these fishes and that colonization of island freshwater habitats by larvae could be independent of natal island.

Low Levels of Intraspecific Genetic Variation at a Rapidly Evolving Chloroplast DNA Locus in North American Duckweeds (Lemnaceae)

Although most previous studies on chloroplast (cp) DNA variation in plants have concentrated on systematics and evolution above the species level, intraspecific variation in cpDNA is common and has provided useful insights into population-level evolutionary processes. Polymerase chain reaction methods were used to examine restriction site and sequence variation in the chloroplast rpLI6 gene within and among populations of duckweed species (Spirodela and Lemna) from the southern and eastern United States. To our knowledge, the rpL16 region has not previously been used to investigate cpDNA variation in nature. While considerable restriction site and sequence variation were detected among species, no variation was found within populations of either of the two species (S. punctata and L. minor) selected for sequence analysis, and S. punctata showed no interpopulational variation. Two cpDNA haplotypes were identified in L. minor, with one haplotype restricted to three sites in Louisiana and the other found in all other populations sampled. This paucity of variation cannot be readily explained as the result of a low mutation rate. In general, group II introns appear to be subject to very little functional constraint, and extensive sequence differences have been found between species in the chloroplast rpL16 intron in particular. However, factors such as historical range expansions and contractions, founding effects, fluctuations in local population size, and natural selection may play a role in reducing cpDNA sequence variability in these species.

Low levels of intraspecific genetic variation at a rapidly evolving chloroplast dna locus in North American duckweeds (Lemnaceae)

Although most previous studies on chloroplast (cp) DNA variation in plants have concentrated on systematics and evolution above the species level, intraspecific variation in cpDNA is common and has provided useful insights into population‐level evolutionary processes. Polymerase chain reaction methods were used to examine restriction site and sequence variation in the chloroplast rpLI6 gene within and among populations of duckweed species (Spirodela and Lemna) from the southern and eastern United States. To our knowledge, the rpL16 region has not previously been used to investigate cpDNA variation in nature. While considerable restriction site and sequence variation were detected among species, no variation was found within populations of either of the two species (S. punctata and L. minor) selected for sequence analysis, and S. punctata showed no interpopulational variation. Two cpDNA haplotypes were identified in L. minor, with one haplotype restricted to three sites in Louisiana and the other found in all other populations sampled. This paucity of variation cannot be readily explained as the result of a low mutation rate. In general, group II introns appear to be subject to very little functional constraint, and extensive sequence differences have been found between species in the chloroplast rpL16 intron in particular. However, factors such as historical range expansions and contractions, founding effects, fluctuations in local population size, and natural selection may play a role in reducing cpDNA sequence variability in these species.

Phylogeny of Temperate Gentianaceae: A Morphological Approach

The intrafamilial relationships of the Gentianaceae are investigated by means of a cladistic analysis based on morphological and to a lesser extent on chemical data. The 21 genera that are selected for the analysis represent all tribes and subtribes except Leiphaimeae, Rusbyantheae and Voyrieae. The large genus Gentiana is represented by three of its sections. The former loganiaceous genera Anthocleista and Fagraea are used as outgroups. Standard parsimony analyses and analyses using weights that are based on the cladistic reliability of the characters give congruent results as far as the global relationships are concerned. The best supported clade contains Eustoma (Tachiinae) and all included Gentianinae, Erythraeinae and Chironiinae. The basal division in this clade is between Ixanthus and the other genera. In this way Ixanthus, an endemic of the Canary Islands, connects the mostly woody tropical and the mostly herbaceous temperate taxa. Subtribe Gentianinae (excluding Ixanthus) is monophyletic, unlike Erythraeinae and Chironiinae. In most analyses, however, both subtribes together (and including Eustoma) are the sister-group of Gentianinae. Possibly Erythraeinae, Chironiinae and Eustoma should be merged. The basal parts of the cladograms, involving the woody tropical representatives and Exacum, are poorly resolved. More extensive sampling, especially among the tropical representatives, is necessary to elucidate these basal relationships. The tropical ancestry of the family, the switch from a woody to a herbaceous life form, and the position of critical taxa, such as Swertia and Halenia or Tripterospermum, are discussed. The Gentianaceae is a cosmopolitan family of medium size, with 76 genera (Brummitt 1992) and about 1200 species (Mabberley 1990; see Table 1). Its oldest known fossils are from the Eocene of North and Central America (Crepet and Daghlian 1981; Graham 1984). Recent cladistic analyses based on rbcL sequence data (Olmstead et al. 1993; Bremer et al. 1994), restriction site variation of the chloroplast genome (Downie and Palmer 1992) and morphological, anatomical, embryological and chemical data (Struwe et al. 1994) indicate that Gentianaceae are one of the principal families of the monophyletic order Gentianales. Results in Bremer et al. (1994) and Struwe et al. (1994) are consistent with the hypothesis (e.g. Downie and Palmer 1992; Bremer and Struwe 1992) that Loganiaceae sensu Leeuwenberg and Leenhouts (1980) are a paraphyletic assemblage with members showing closest relationships to other families both within and outside of the Gentianales. As far as Gentianaceae is concerned, Struwe et al.'s (1994) main conclusion is to formally include Potalia Aubl., Fagraea Thunb. and Anthocleista Afzel. ex R. Br. (tribe Potalieae of Loganiaceae sensu Leeuwenberg and Leenhouts 1980) in the Gentianaceae. This transfer had already been proposed by Bureau (1856) in the previous century and more recently by Fosberg and Sachet (1980) on the basis of gross morphology (although monographers of the Loganiaceae disagreed, e.g. Leeuwenberg and Leenhouts 1980) and by Jensen (1992) on the basis of the presence of advanced iridoid glucosides. It should be noted that the inclusion of Anthocleista and Fagraea increases the woody paleotropic representation of the family, that is otherwise restricted to Gentianothamnus Humbert (Humbert 1937). While a consensus seems to be emerging about the monophyly of the Gentianales and the inclusion of Potalieae in Gentianaceae, much work remains to be done concerning the interfamilial relationships within the order, including the relationships of the smaller families often included in Gentianales (e.g. Saccifoliaceae, Dialypetalanthaceae) and concerning the infrafamilial relationships of the bigger Gentianales families. We focus on the Gentianaceae. Because the broad-based cladistic analyses (e.g. Downie and Palmer 1992; Olmstead et al. 1993; Bremer et al. 1994; Struwe et al. 1994) to date

YAC clone contigs covering 5 Mb of a repeat sequence island on the mouse X Chromosome

We have initiated work towards the construction of YAC clone contigs across a repeat sequence island region on the mouse X Chromosome (Chr). The repeat sequence island region-the 141 island-located at band A3 contains 50 copies of a localized long complex repeat unit (LCRU). We have isolated 87 YAC clones from the 141 island and have used a dual faceted approach towards the construction of contigs across the repeat sequence island. First, we have identified YAC clones originating from the same region of the island by the identification of commonly held LCRU restriction site variants. Second, we have constructed rare cutter restriction maps of each YAC clone. Taken together, we have been able to assemble one large contig of 2.8 Mb and a number of smaller contigs. In total, contigs covering 5Mb of the island region have been identified. The island region would appear to represent a major component of the A3 Giemsa dark band on the mouse X Chr.

Genetics ofCronartium ribicola. II. Variation in the ribosomal gene cluster

The ribosomal gene repeat in Cronartium ribicola J.C. Fisch is highly variable among spore samples from British Columbia, Canada. Both restriction site variation and length variation occur. Length heterogeneity results from differences in the number of subrepeats in the intergenic spacer (IGS). The number of IGS size classes in haploid cultures is limited but is very large and highly variable in aeciospores from single cankers. The proportions of different size classes vary among cankers on different trees, and among subsamples taken around the periphery of large old cankers. The results are consistent with the fungus having a haploid infective mycelium that produces functional pycnia that result in localized dikaryotic areas following fusion between flexuous hyphae and pycnia. Restriction site variation appears lower than has been reported in range-wide samples of endemic fungal species, consistent with the hypothesis that introduction of C. ribicola to western North America was limited and does not represent the full genetic range of the species. No particular restriction site variants or IGS size classes characterize samples from particular geographic areas. No evidence for geographic races of the fungus was obtained. Keywords: rusts, rust races, ribosomal DNA, intergenic spacer, population structure, RFLP.

Phylogenetic relationships of Thlaspi s.l. (subtribe Thlaspidinae, Lepidieae) and allied genera based on chloroplast DNA restriction-site variation

Chloroplast DNA restriction-site variation was analyzed in 30 accessions representing 20 species from the major lineages in Thlaspi s.l. (previously described as genera by Meyer 1973, 1979) and allied genera from the subtribe Thlaspidinae (Peltaria, Teesdalia, Cochlearia, Ionopsidium, Aethionema). A total of 161 variable restriction sites were detected. Phylogenetic analyses indicated a division of Thlaspi s.l. into three groups consistent with Meyer's genera Thlaspi s. str., Microthlaspi and Noccaea/Raparia. The genus Thlaspi s.l. as currently described proved to be paraphyletic because one of its major lineages, i.e. Thlaspi s. str., appeared to be more closely related to other genera (Peltaria, Teesdalia) than to the remaining lineages of Thlaspi s.l., i.e. Noccaea/Raparia and Microthlaspi. Sequence divergence values (100 x p) between the Thlaspi s.l. lineages were similar to values between these groups and related genera (Teesdalia, Peltaria), respectively. Chloroplast DNA variation was also used to assess subtribal classification of the genera studied. The cpDNA data were inconsistent with the controversial taxonomic classifications based on morphology. The molecular data would suggest that (1) the subtribe Thlaspidinae, as traditionally described, is not monophyletic; (2) the Thlaspidinae should be reduced to a group consisting of Thlaspi s. str., Peltaria, Teesdalia, Microthlaspi, Noccaea/Raparia, and that Aethionema should be excluded from the Thlaspidinae; and (3) Cochlearia and Ionopsidium represent the subtribe Cochleariinae.

Genomic organization of ribosomal RNA genes inBromus(Poaceae)

Restriction site maps of the rDNA genes of nine Bromus species are described. The rDNA repeat units ranged from 8.2 to 11.1 kbp in length. Intraspecific length variation was observed in the BamHI digestions in three of the nine species. Restriction site variation was observed mainly in the intergenic spacer (IGS) but was also detected in the coding region. A unique KpnI site was present in the IGS of Bromus tectorum and Bromus sericeus (subgenus Stenobromus); in addition, B. sericeus contained an extra EcoRI site. An additional DraI site was observed in the IGS of Bromus trinii (subgenus Neobromus). A BstEII site in the IGS, common to seven of the species, was absent in B. tectorum and B. sericeus. In the coding region, a 2.1-kbp BstEII fragment was present in four subgenera represented by Bromus inermis and Bromus erectus (subgenus Festucaria), Bromus marginatus and Bromus carinatus (subgenus Ceratochloa), B. tectorum and B. sericeus (subgenus Stenobromus), and B. trinii (subgenus Neobromus); a similar fragment of only 1.1 kbp was present in Bromus mollis and Bromus arvensis (subgenus Bromus). An additional BamHI site was present in the coding region of B. erectus. Ribosomal DNA data suggested that B. mollis and B. arvensis (subgenus Bromus) are genetically isolated from the other subgenera, which showed a derived relationship. Restriction site mapping of the rDNA genes could provide useful molecular data for species identification and population and evolutionary studies in Bromus. Key words : Bromus, ribosomal DNA, restriction maps, evolutionary relationships.

Hierarchical Analysis of Nucleotide Diversity in Geographically Structured Populations

Existing methods for analyzing nucleotide diversity require investigators to identify relevant hierarchical levels before beginning the analysis. We describe a method that partitions diversity into hierarchical components while allowing any structure present in the data to emerge naturally. We present an unbiased version of NEI's nucleotide diversity statistics and show that our modification has the same properties as WRIGHT's FST. We compare its statistical properties with several other FST estimators, and we describe how to use these statistics to produce a rooted tree of relationships among the sampled populations in which the mean time to coalescence of haplotypes drawn from populations belonging to the same node is smaller than the mean time to coalescence of haplotypes drawn from populations belonging to different nodes. We illustrate the method by applying it to data from a recent survey of restriction site variation in the chloroplast genome of Coreopsis grandiflora.

MOLECULAR GENETIC EVIDENCE FOR PARALLEL LIFE-HISTORY EVOLUTION WITHIN A PACIFIC SALMON (SOCKEYE SALMON AND KOKANEE, ONCORHYNCHUS NERKA).

The Pacific salmon Oncorhynchus nerka typically occurs as a sea-run form (sockeye salmon) or may reside permanently in lakes (kokanee) thoughout its native North Pacific. We tested whether such geographically extensive ecotypic variation resulted from parallel evolutionary divergence thoughout the North Pacific or whether the two forms are monophyletic groups by examining allelic variation between sockeye salmon and kokanee at two minisatellite DNA repeat loci and in mitochondrial DNA (mtDNA) Bgl II restriction sites. Our examination of over 750 fish from 24 populations, ranging from Kamchatka to the Columbia River, identified two major genetic groups of North Pacific O. nerka: a "northwestern" group consisting of fish from Kamchatka, western Alaska, and northwestern British Columbia, and a "southern" group consisting of sockeye salmon and kokanee populations from the Fraser and Columbia River systems. Maximum-likelihood analysis accompanied by bootstrapping provided strong support for these two genetic groups of O. nerka; the populations did not cluster by migratory form, but genetic affinities were organized more strongly by geographic proximity. The two major genetic groups resolved in our study probably stem from historical isolation and dispersal of O. nerka from two major Wisconsinan glacial refugia in the North Pacific. There were significant minisatellite DNA allele frequency differences between sockeye salmon and kokanee populations from different parts of the same watershed, between populations spawning in different tributaries of the same lake, and also between sympatric populations spawning in the same stream at the same time. MtDNA Bgl II restriction site variation was significant between sockeye salmon and kokanee spawning in different parts of the same major watershed but not between forms spawning in closer degrees of reproductive sympatry. Patterns of genetic affinity and allele sharing suggested that kokanee have arisen from sea-run sockeye salmon several times independently in the North Pacific. We conclude that sockeye salmon and kokanee are para- and polyphyletic, respectively, and that the present geographic distribution of the ecotypes results from parallel evolutionary origins of kokanee from sockeye (divergences between them) thoughout the North Pacific.