Phylogenetic and evolutionary implications of nuclear ribosomal DNA variation in dwarf dandelions (Krigia,Lactuceae, Asteraceae)

Restriction site and length variations of nrDNA were examined for 51 populations of seven species ofKrigia. The nrDNA repeat ranged in size from 8.7 to 9.6 kilobase (kb). The transcribed region, including the two ITSs, was 5.35 kb long in all examinedKrigia populations. In contrast, the size of the nontranscribed IGS varied from 3.35 to 4.25 kb. Eight different types of length-variations were identified among the 51 populations, including distinct nrDNA lengths in the tetraploid and diploid populations of bothK. biflora andK. virginica. However, a few variations were detected among populations of the same species or within a cytotype. All populations ofKrigia sect.Cymbia share a 600 bp insertion in IGS near the 18 S gene, and this feature suggests monophyly of the section. AllKrigia spp. had a conjugated type of subrepeat composed of approximately 75 basepairs (bp) and 125 bp. Base modifications in the gene coding regions were highly conserved among species. Forty-five restriction sites from 15 enzymes were mapped, 24 of which were variable among populations. Only four of the variable sites occurred in the rRNA coding region while 20 variable sites were detected in the noncoding regions. Collectively, 25 enzymes generated about 66 restriction sites in each nrDNA; this amounts to about 4.3% of the nrDNA repeat. A total of 50 restriction sites was variable, 28 of which were phylogenetically informative. Phylogenetic analyses of site mutations indicated that two sections ofKrigia, sect.Cymbia and sect.Krigia, are monophyletic. In addition, relationships among several species were congruent with other sources of data, such as cpDNA restriction site variation and morphology. Both length and restriction site variation supported an allopolyploid origin of the hexaploidK. montana. The average sequence divergence value inKrigia nrDNA was 40 times greater than that of the chloroplast DNA. The rapid evolution of nrDNA sequences was primarily due to changes of the IGS sequences.     

Mitochondrial DNA analysis ofExophiala moniliae

Mitochondrial DNA(mtDNA) analysis with restriction enzymes, Hae III, Hind III and Msp I was performed in 17Exophiala moniliae strains. The results were as follows: (1)E. moniliae could be classified into 10 types based on restriction patterns, (2)E. moniliae is suggested to be a complex organism because of extensive mtDNA polymorphism among strains likeE. jeanselmei and (3) two types ofE. moniliae are identical with two types ofE. jeanselmei. These results suggest thatE. moniliae is not genetically defined fromE. jeanselmei and the taxonomical status ofE. moniliae requires reevaluation     

Mitochondrial DNA analysis of Sporothrix schenckii clinical isolates from China

Mitochondrial DNA (mtDNA) types based on restriction fragment length polymorphism (RFLP)patterns with HaeIII were investigated in clinical isolates of Sporothrix schenckii in China. In addition to 23 mtDNA types (Types 1–23) so far reported, a new mtDNA type (Type 24) was found in this study. Type 24 was divided into two subtypes, Subtype 24A and 24B based on RFLP with EcoRV. Sixty-seven isolates in China consisted of 58 isolates of Type 4, 5 of Type 6, 1 of Type 5, 1 of Type 20 and 2 of Type 24. Based on the phylogeny of the mtDNA types (Types 1–24) constructed by estimating sequence divergences of mtDNA, mtDNA types clustered into two groups: Group A (Types 1–3, Type 11, Types 14–19 and Types 22–23) and Group B (Types 4–10, Types 12–13,Types 20–21 and Type 24). These results suggest that mostS. schenckii isolates in China belong to Group B.This revised version was published online in October 2005 with corrections to the Cover Date.     

Genetic variation and differentiation of the two river sculpins,Cottus nozawae andC. amblystomopsis,deduced from allozyme and restriction enzyme-digested mtDNA fragment length polymorphism analyses

Genetic differentiation of the two sibling species,Cottus nozawae andC. amblystomopsis, from the northern part of Japan (Hokkaido Island and the Tohoku District) was investigated using allozyme variations and restriction fragment length polymorphisms of mitochondrial DNA. Although the two species are morphologically very similar, previously being thought to be a single species, they have different life-cycles;C. nozawae has a fluvial life-cycle with a small number of large-sized eggs, whereasC. amblystomopsis is an amphidromous species with a large number of small-sized eggs. Four populations ofC. amblystomopsis from Hokkaido Island and 24 populations ofC. nozawae (22 from Hokkaido Island and 2 from the Tohoku District) were sampled and examined Intrapopulational differentiation in the two species was measured by examining several indexes, including proportion of polymorphic loci (P), mean heterozygosity (H) and nucleotide diversity (π). All measurements were higher in theC. amblystomopsis populations, suggesting that intrapopulational variation inC. nozawae was less than inC. amblystomopsis and reflecting the difference in effective population sizes between them. Cluster analyses were performed using the UPGMA method, based on the data matrices of genetic distance (D) and the net nucleotide difference (δ) between populations. TheC. nozawae andC. amblystomopsis populations from Hokkaido Island composed a large cluster (Hokkaido group), while theC. nozawae populations from the Tohoku District composed a different cluster (Tohoku group). Bootstrap probabilities deduced from 1000 bootstrap replications for presence or absence of restriction sites showed that the mtDNA haplotypes detected within the Tohoku Group occurred in 99.9% of the bootstrap replicates outside the mtDNA haplotypes of the Hokkaido group, while those within the Hokkaido group occurred in 3.5–64.9% of bootstrap replicates. Consequently, the Hokkaido populations of the two species (Hokkaido group) were genetically close to each other, whileC. nozawae from the Tohoku District (Tohoku group) were distant from the Hokkaido group. These results suggest that the ancestral populations of the two species on Hokkaido Island shared the same gene pool, even after becoming geographically isolated from the ancestral population ofC. nozawae in the Tohoku District by the formation of the Tsugaru Straits.     

The mycological and molecular study of <Emphasis Type="Italic">Hortaea werneckii</Emphasis> isolated from blood and splenic abscess

Hortaea werneckii is an environmental dematiaceous fungus found in the halophilic environment. It causes tinea nigra. We report the isolation of H. werneckii from blood and splenic abscess of two patients with acute myelomonocytic leukaemia. H. werneckii grew at room temperature but not at 37 °C, it was identified by biochemical tests, growth characteristics and the presence of conspicuous collarette intercalary on dividing yeast cells. The use of specific oligonucleotide primer Hor-F (5′-TGGACACCTTCA TAACTCTTG-3′) and Hor-R (5′-TCACAACGCTTAGAGACGG-3′) confirmed the two isolates were H. werneckii. The sequence for 281 nucleotide of HW299 and HW403 were 99% identical but differed only in one nucleotide. In vitro anti-fungal susceptibility testing showed that the isolates were resistant to amphotericin B and flucytosine.     

EVOLUTION AND MITOCHONDRIAL DNA IN NEUROSPORA CRASSA

We studied mitochondrial DNA variability in 19 natural Neurospora crassa isolates and one wild-type isolate to examine evolution of these fungi and their mitochondrial DNA (mtDNA). We combined restriction endonuclease analysis of natural isolate mtDNA with DNA-DNA hybridization to cloned EcoR I fragments of a wild-type genome to discriminate between length mutations and site changes due to nucleotide substitution. Most variability was due to length mutations (insertions and deletions); genome size could vary 25% between pairs of isolates. Length-mutation distribution was not random, nor simply explained by the presence of coding versus noncoding regions. Restriction-site changes were few; the estimated amount of nucleotide substitution per nucleotide between the most divergent pair of isolates was 0.78%. Evolutionary relationships among isolates based on both types of mutations were compatible, and suggest that geographically distinct populations of mitochondrial DNA exist in the biological species, N. crassa. In contrast, no such correlation was shown by the previously determined distribution of nuclear heterokaryon incompatibility genes in the same isolates (Mylyk, 1975, 1976).     

Differentiation of mitochondrial DNA types inMacaca fascicularis

Restriction fragment length polymorphism in the mitochondrial DNA ofMacaca fascicularis from four geographical regions, Indonesia, the Philippines, Malaysia, and Indochina, was analyzed. In total, 21 types of mitochondrial DNA were detected using five restriction enzymes. These types were divided into two main groups based on phylogenetic analyses, one of which corresponded to the types of continental (Malaysia/Indochina) populations and the other to the types of a insular (Philippine) population. The types in the Indonesian population belonged to both groups. In the phylogenetic tree for the four populations, two clusters were constructed, one for the continental populations and the other for the insular ones.     

Genetic variations ofCottus nozawae populations from five tributaries of the Shiribetsu River of southern Hokkaido,Japan

To estimate genetic differentiation and heterogeneity in the landlocked river sculpin,Cottus nozawae, between tributary populations in the same river-system, 107 specimens were captured from 5 tributaries of the Shiribetsu river (course length 128 km), Hokkaido Island and surveyed for allozyme variations and restriction fragment length polymorphisms of mitochondrial DNA (mtDNA). Three and two alleles were seen at theIdh-2 andPgm loci, respectively, but only one locus,Idh-2, out of twenty loci examined was regarded as polymorphic, since the frequency of the most common allele did not exceed 0.95. Three different mtDNA haplotypes were detected, there being replacement of them between the tributary populations. Heterogeneities of allele and haplotype frequencies were significant between some tributary populations, suggesting that genetic differentiation has occurred between them.     

Molecular genetic characterization of bacterial isolates causing brown blotch on cultivated mushrooms in Japan

The polymerase chain-reaction (PCR) was used to amplify 16S ribosomal DNA (16S rDNA) from bacteria, identified asPseudomonas tolaasii orP. fluorescens, causing brown blotch on cultivated mushrooms in Japan. PCR-amplified 16S rDNA was analyzed on the basis of nucleotide sequence and restriction fragment length polymorphisms (RFLP) to determine the specific identity of isolates. Banding patterns obtained through PCR using primers corresponding to repetitive extragenic palindromic sequences of enteric bacteria (REP-PCR) were used to determine the relatedness of conspecific isolates. AllP. tolaasii isolates and a mushroom pathogen identified asP. fluorescens had identical RFLP patterns and partial 16S sequences, and are considered conspecific. An isolate ofP. fluorescens from creamery wastes (IFO 3507) differed slightly from isolates ofP. tolaasii in both 16S sequence (0.8%) and RFLP patterns (d=0.08), and had almost entirely different REP-PCR bands (d=0.88–1.0). Phylogenetic analyses based on 16S sequences indicated thatP. tolaasii andP. fluorescens are close members ofPseudomonas sensu stricto. REP-PCR shows promise in characterizing isolates pathogenic on different mushroom crops. Two isolates ofP. tolaasii pathogenic onPleurotus ostreatus had identical banding patterns, but three isolates fromLentinula edodes showed the greatest diversity. Contribution No. 312 of the Tottori Mycological Institute, Totori, Japan.     

Genetic diversity of Ustilago maydis strains

Thirty wild isolates belonging to five different locations in Mexico plus two laboratory strains of Ustilago maydis were characterized by restriction fragment length polymorphism (RFLP) analysis using 23 different clones as probes derived from a PstI library and two restriction enzymes. All loci analysed presented a high level of polymorphism, including one locus with thirty one different alleles. Geographical grouping of the populations was based on Nei's genetic distance and there was no correlation between genetic and geographic distances among these isolates. Our results suggest that DNA fingerprinting is a useful method for detecting genetic variation in populations of U. maydis. This work demonstrated that considerable genetic variation may be present within field populations of U. maydis.     

DNA evidence for multiple origins of intergeneric allopolyploids in annualMicroseris (Asteraceae)

Seventy populations of North American annualMicroseris, Stebbinsoseris, andUropappus species were examined for chloroplast and nuclear ribosomal DNA restriction site variability to determine the origin of the allotetraploid speciesS. heterocarpa andS. decipiens. Previously identified chloroplast DNA restriction site variants were used in concert with restriction site variation forNco I in the nuclear-encoded ribosomal DNA repeat. The presence of two, mutually exclusive restriction site gains were observed in diploid populations ofM. douglasii; these same variants were also found in populations of allotetraploidS. heterocarpa, indicating mutiple origins of this species from different maternal diploid populations ofM. douglasii. Variation in the rDNA repeat between the diploid annual species and the putative paternal genome ofU. lindleyi was found to be additive inS. heterocarpa. A similar relationship was observed for the origin ofS. decipiens; cpDNA restriction site variants found inM. bigelovii andM. douglasii were present inS. decipiens. The rDNANco I variants also were additive in this purported allotetraploid. These results confirm the reticulate evolutionary pattern inStebbinsoseris and provide another example of multiple origins of intergeneric allopolyploids.     

Chloroplast DNA variation of Panax (Araliaceae) in Nepal and its taxonomic implications

The restriction site and size variation of five PCR amplified fragments of noncoding chloroplast DNA (cpDNA) was examined in material from 13 populations ofPanax from Nepal and China. Fourteen restriction endonucleases produced 81 restriction site and length variations from the large single-copy region of cpDNA, 27 of which are polymorphic. The cpDNA dataset suggests two distinct groups ofPanax from Nepal (clades I and II). Clade I consists of two populations ofP. pseudoginseng subsp.pseudoginseng, and clade II is composed of material referable toP. pseudogingeng subsp.himalaicus (vars.himalaicus, angustifolius, andbipinnatifidus). The three accessions ofP. pseudoginseng subsp.japonicus andP. ginseng studied from China had cpDNA characters that differed from the HimalayanPanax. The highly distinctive cpDNA profile and morphology ofP. pseudoginseng subsp.pseudoginseng sensu Hara (1970) from central Nepal support its status as a separate species, which has an extremely restricted distribution.     

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 São Paulo State, Brazil. The total DNA of 405 individuals was digested with 15 restriction endonucleases and probed with five clonedHindIII fragments representing the entire mitochondrial genome ofCochliomyia hominivorax. The survey revealed that four enzymes (HaeIII,HindIII,MspI, andPvuII) 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 thePvuII pattern were obtained in one of the populations. The estimated average for nucleotide sequence divergence (δ) 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.     

Genetic variability among <Emphasis Type="Italic">Pholiota aurivella</Emphasis> isolates from a small natural population

Genetic differences between 36 Pholiota aurivella wild isolates collected from 13 decayed logs of Salicaceae trees distributed along about 1200 m of a streambed in a forest were characterized by somatic incompatibility and mating tests, and by restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA). There was a perfect correlation between somatic incompatibility and mating type groups, and isolates could be divided into 15 genets (genetically identical clones). Because the mtDNAs of the 36 wild isolates have 14 different EcoRI RFLP patterns, they likely originated from at least 14 distinct wild strains, indicating that multiple wild strains with distinct genetic compositions coexist in the forest investigated in this study. mtDNA variation of P. aurivella is apparently very high despite the close proximity of sample collection sites within the forest. The territories of single P. aurivella genets within a host log are apparently larger than other nonpathogenic wood-decaying basidiomycetes reported previously, such as Flammulina velutipes and Lentinula edodes.     

Polymorphism of the Noncoding Region of the Mitochondrial Genome in Three Kazakh Populations Inhabiting Different Areas of Kazakhstan and in DNA Samples from Ancient Populations of the Kazakhstani Altai

The polymorphism of the major noncoding region of mitochondrial DNA (mtDNA D loop, 528 bp) has been studied in samples from three modern Kazakh populations (from Almaty, the Semipalatinsk Region, and the Altai Mountains) and in DNA samples of ancient human populations of the Kazakhstani Altai. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis for 13 restriction sites, including BamHI, EcoRV, Sau3AI (one site each), KpnI (two sites), HaeIII (three sites), and RsaI (five sites) were used. The frequency distributions of all sites have been determined. The gene diversity (h) and the genetic distances between different Kazakh populations and other populations of the world have been calculated. The RFLP analysis of the mtDNA control region of fossil samples has been performed similarly to the analysis of modern mtDNA samples. Two fossil mtDNA samples from burial mound 11 are monomorphic with respect to all restriction sites analyzed.     

Intraspecific variability and exopolysaccharide production inAureobasidium pullulans

Forty seven strains of the black yeasts,Aureobasidium pullulans andHormonema dematioides, and the type strain ofHormonema macrosporum were examined using PCR-ribotyping and universally primed PCR with subsequent hybridization. Four groups (populations) were distinguished withinA. pullulans with PCR-ribotyping, which largely coincided with UP-PCR/hybridization groups. The UP-PCR technique revealed a greater degree of heterogeneity between the groups studied. Five strains identified asHormonema dematioides on the basis of physiological and morphological data formed a group recognizable with PCR-ribotyping and UP-PCR/hybridization, which also includedH. macrosporum. Aureobasidium pullulans is characterized by the absence of RsaI restriction sites in rDNA amplified with primers 5.8S-R and LR7, whileHormonema species possessed several bands after RsaI digestion. For analysis of distance between populations, PCR-ribotyping with AluI and MspI is sufficient. Strains ofA. pullulans produce exopolysaccharides in liquid media with different nitrogen sources, while the strains ofHormonema synthesize minor amounts of polysaccharides in media with peptone. Populations ofA. pullulans differ slightly from each other in their optimal, medium-dependent production of polysaccharides.     

Genetic diversity in cocoa revealed by cDNA probes

The variability of the cocoa (Theobroma cacao) nuclear genome was investigated. A total of 203 cocoa clones was surveyed for restriction fragment length polymorphisms (RFLPs) using four restriction endonuclease and 31 seed cDNA probes. A high level of polymorphism has been found. This study points to a structuring of the species that fits with the distinction between the Criollo and Forastero populations. These results combined with previously obtained nuclear rDNA and mtDNA data allow us to propose new hypotheses on the origin and evolution of the different cocoa populations.     

Oenothera chloroplast DNA polymorphisms associated with plastome mutator activity

Summary Oenothera plants homozygous for a recessive allele at the plastome mutator (pm) locus show non-Mendelian mutation frequencies that are 1000-fold higher than spontaneous levels. Chloroplast DNA (cpDNA) was isolated from nine mutants and two green isolates of the plastome mutator line. cpDNA restriction patterns were compared to cpDNA from a representative of the progenitor Johansen strain, and cpDNAs from all eleven plastome mutator lines show changes of fragment mobility due to deletion events at five discrete regions of the plastome. Most of the mutants have cpDNA restriction patterns identical to that of one of the green isolates from the plastome mutator line, and therefore, most of the differences in fragment length are probably not responsible for the mutant phenotypes. In contrast to the plastome mutator line, cpDNA from several populations of a closely related wild-type Oenothera species have few restriction fragment length polymorphisms. This suggests that both mutation frequencies and site-specific cpDNA deletions are elevated in the plastome mutator line, and implicates a defect in the cpDNA repair or replication machinery.     

Genetic evidence for the existence of cryptic species in theAnopheles albitarsis complex in Brazil: Allozymes and mitochondrial DNA restriction fragment length polymorphisms

Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations ofAnopheles albitarsis in Brazil. Two sympatric species,An. deaneorum andAn. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (forHad-1, Pgi-1, Pep-1, Mpi-1, andIdh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species ofAn. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes.An. marajoara form CM had a higher variability (mean heterozygosity,H=0.22, and percentage of polymorphic loci,P=66.7) than did form IG and form MA (H=0.08 in both, andP=25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates ofF statistics, and genetic similarities, we propose thatAn. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1)An. deaneorum (currently one taxon) and (2) theAn. marajoara species complex, which further consists of at least three cryptic forms,marajoara form MA,marajoara form IG, andmarajoara form CM. The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense of the United States. This research was conducted when the senior author was on the staff of the USDA-ARS Laboratory in Gainesville, Florida.     

Detection and distribution of six linear mitochondrial plasmids in the shiitake mushroom,Lentinula edodes

The presence of plasmids was surveyed in 90 wild isolates ofLentinula edodes collected from geographically different world regions. DNA plasmids of different sizes were found in about 80% of the isolates. The plasmids detected were of six kinds, designated as pLE1 (9.0 kb), pLE2 (11.1 kb,=pLLE1 described by other authors), pLE3A (9.8 kb), pLE3B (10.8 kb), pLE3C (12.1 kb), and pLE3D (12.3 kb). Hybridization analysis suggested that pLE1 and pLE2 were distinct plasmid types of different homology groups to each other, and the four other plasmids were variant types belonging to a third homology group. These plasmids had no homology with their host's and non-host's nuclear and mitochondrial genome DNAs. Restriction analysis and electron microscopy indicated that the plasmids are linear in form. Since all six plasmids were transmitted uniparentally in sexual crosses and were consistently associated with the DNA preparations from mitochondria fractionated from mycelia of representative isolates, they were suggested to be located in mitochondria, similar to many other known fungal DNA plasmids. Geographically, pLE1 and pLE2 were widely distributed in natural populations ofL. edodes, while the remaining four plasmids were uniquely present in delimited natural populations. Contribution No. 322 from the Tottori Mycological Institute.