Well-supported Monophyletic Group Research Articles

Phylogenetic relationships in the iguanid lizard genus Liolaemus: multiple origins of viviparous reproduction and evidence for recurring Andean vicariance and dispersal

Phylogenetic relationships within the iguanid lizard genus Liolaemus are investigated using 1710 aligned base positions (785 phylogenetically informative) of mitochondrial DNA sequences, representing coding regions for eight tRNAs, ND2, and portions of ND1 and COL Sixty new sequences ranging in length from 1736 to 1754 bases are compared with four previously reported sequences. Liolaemus species form two well-supported monophyletic groups of subgeneric status, Liolaemus and Eulaemus. These subgenera appear to have separated at least 12.6 million years ago based on the amount of molecular evolutionary divergence between them. Hypotheses that species occurring in the Andes, west of the Andes, and east of the Andes, each comprise distinct monophyletic groups are independently rejected statistically. The shortest estimate of phylogeny suggests mat Liolaemus originated either in the Andes or the eastern lowlands. Numerous evolutionary shifts have occurred between the Andes, and the eastern and western lowlands, suggesting recurring vicariance and dispersal. Species occurring at high elevations or high latitudes usually have viviparous reproduction. Depending on whether parity mode is considered reversible in Liolaemus, the most parsimonious reconstruction supports at least six independent origins of viviparity or at least three gains followed by three losses of viviparity among die 60 Liolaemus lineages examined.

Molecular evidence for the phylogeny of Australian gekkonoid lizards

Partial sequences of the mitochondrial12S rRNAand nuclear c-mosgenes were determined for 12 species of gekkonoid lizards representing the four major taxa of the Australian region, the Diplodactylini and Carphodactylini (forming the subfamily Diplodactylinae), the Pygopodidae and the Gekkoninae. One further species represented a non-Australian gekkonoid lineage, the Eublepharinae. The combined sequence data were used to reconstruct the underlying molecular phylogeny. We used the molecular phylogeny to test the monophyly of the diplodactyline tribes and conflicting hypotheses of relationships of the pygopods and of the genusOedura. Monophyly of the Diplodactylinae is supported, while pygopods form a monophyletic sister lineage to all Diplodactylinae. The molecular data support the monophyly of the Diplodactylini, withOedurafirmly placed as a diplodactylin. Monophyly of the Carphodactylini is not supported. The four carphodactylin genera form a paraphyletic cluster at the base of the Diplodactylini. Pygopods are nested within the traditional Gekkonidae and pygopods plus diplodactylines form a well-supported monophyletic group with respect to the remaining gekkonoids, the gekkonines and eublepharines.

Molecular evidence for the phylogeny of Australian gekkonoid lizards

Abstract Partial sequences of the mitochondrial12S rRNAand nuclear c-mosgenes were determined for 12 species of gekkonoid lizards representing the four major taxa of the Australian region, the Diplodactylini and Carphodactylini (forming the subfamily Diplodactylinae), the Pygopodidae and the Gekkoninae. One further species represented a non-Australian gekkonoid lineage, the Eublepharinae. The combined sequence data were used to reconstruct the underlying molecular phylogeny. We used the molecular phylogeny to test the monophyly of the diplodactyline tribes and conflicting hypotheses of relationships of the pygopods and of the genusOedura. Monophyly of the Diplodactylinae is supported, while pygopods form a monophyletic sister lineage to all Diplodactylinae. The molecular data support the monophyly of the Diplodactylini, withOedurafirmly placed as a diplodactylin. Monophyly of the Carphodactylini is not supported. The four carphodactylin genera form a paraphyletic cluster at the base of the Diplodactylini. Pygopods are nested within the traditional Gekkonidae and pygopods plus diplodactylines form a well-supported monophyletic group with respect to the remaining gekkonoids, the gekkonines and eublepharines.

Ribes (Grossulariaceae) phylogeny as indicated by restriction-site polymorphisms of PCR-amplified chloroplast DNA

We surveyed exemplars from all 12 infrageneric taxa ofRibes (Grossulariaceae) for restriction site variation in two cpDNA regions, fromrbcL toaccD and fromrpoC1 torpoC2, in order to develop an explicit phylogenetic hypothesis and to assess the validity of infrageneric classifications. Maximum parsimony analysis resolves sect.Ribes (red currants), sect.Berisia (European alpine currants), sect.Symphocalyx (golden currants), sect.Grossularia plus sect.Grossularioides (true gooseberries and spiny currants), andHesperia, Lobbia, and probably sect.Robsonia (west North American gooseberries) as well-supported monophyletic groups. The clade of sectionsGrossularioides andGrossularia is unexpected, and suggests that subgenusGrossularia is not monophyletic. Alternatively, sect.Grossularioides may have acquired its cpDNA via hybridization and introgression. SectionsCoreosma (black currants) andHeritiera (dwarf currants) are apparently non monophyletic. Relationships among the well-supported lineages and the other sampled taxa remain unresolved. Maximum likelihood analysis is consistent with the parsimony results.

Molecular Identification and Phylogenetic Grouping of Diaporthe phaseolorum and Phomopsis longicolla Isolates from Soybean

ABSTRACT Diaporthe phaseolorum and Phomopsis longicolla isolates from soybean were examined using traditional mycological characteristics and molecular methods. Cultural characteristics including types of fruiting bodies and conidia were assessed for isolates collected from soybean stems and seeds. Cultures were identified as P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, or D. phaseolorum var. sojae. Molecular markers for these groups were developed and analyzed using polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLP) and DNA sequencing in the internal transcribed spacer (ITS) and the 5.8S ribosomal DNA. The ITS(4) and ITS(5) primers amplified PCR products for all isolates studied. Gel electrophoresis of undigested PCR products and DNA sequencing produced various fragment lengths including 604 bp for P. longicolla, 602 and 603 bp for D. phaseolorum var. caulivora, 603 bp for D. phaseolorum var. meridionalis, and from 597 to 609 bp for D. phaseolorum var. sojae. Digestion of these PCR products with enzymes AluI, HhaI, MseI, RsaI, and ScrFI resulted in distinct bands for identification of P. longicolla and the varieties of D. phaseolorum I. All P. longicolla, D. phaseolorum var. caulivora, and D. phaseolorum var. meridionalis isolates were distinguished using AluI and HhaI with RsaI or ScrFI. The banding patterns of D. phaseolorum var. sojae isolates were complex and were separated into 11 subgroups after digestion with AluI, HhaI, MseI, RsaI, and ScrFI. Phylogenetic analysis of 20 isolates of D. phaseolorum and P. longicolla based on the DNA sequence of the ITS region resolved six clades termed A, B, C, D, E, and F. Clade A included all sequenced D. phaseolorum var. caulivora isolates, two from Italy and one from the United States. Isolates in clade B were exclusively associated with D. phaseolorum var. meridionalis. Clades A and B formed a well-supported monophyletic group. Isolates in clades C, D, E, and F were morphologically defined as isolates of P. longicolla, D. phaseolorum var. sojae, and Diaporthe spp. The ITS sequences similarity of seven geographically diverse P. longi-colla isolates illustrated that P. longicolla isolates have a similar genetic background, with some affiliations to some D. phaseolorum var. sojae isolates. Morphological characteristics of the isolates along with the terminal clades of the ITS phylogeny suggest that P. longicolla is an individual species, D. phaseolorum var. caulivora and D. phaseolorum var. meridionalis are varieties of D. phaseolorum, and D. phaseolorum var. sojae is either several varieties of D. phaseolorum or possibly several distinct species.

The genus Oidiodendron: species delimitation and phylogenetic relationships based on nuclear ribosomal DNA analysis

Nuclear ribosomal DNA sequences (ITS region) of fifteen species in the hyphomycete genus Oidiodendron and ten species from the 4 genera in the Myxotrichaceae, Byssoascus, Gymnostellatospora, Myxotrichum, and Pseudogymnoascus, were analysed to: (i) reveal the levels of intra- versus interspecific sequence variation within the genus Oidiodendron, clarify species delimitation and examine the usefulness of some morphological characters used for identification; (ii) assess the possible conspecificity of documented ericoid mycorrhizal strains of Oidiodendron; and (iii) test the hypothesis based on morphological inference that the genus Oidiodendron belongs with the genus Myxotrichum in the Myxotrichaceae (Onygenales). Comparison of molecular and morphological data for multiple strains of O. griseum, O. tenuissimum and O. maius revealed that conidiophore length and the production of a diffusing pigment are not reliable key characters for the genus. Several historically important ericoid mycorrhizal strains, documented as O. griseum, were reidentified as O. maius. Parsimony analyses of 23 Oidiodendron strains showed that three highly supported monophyletic groups, each one consisting of a pair of species, are resolved within the genus. A low level of sequence divergence between the species in these pairs suggests conspecificity for each pair. Other interspecific relationships were not well-supported by bootstrap values. Parsimony analysis of a second dataset composed of mitotic and meiotic taxa showed that Oidiodendron, Myxotrichum and Byssoascus form a well-supported monophyletic group within the Myxotrichaceae, and that this group has diverged significantly from the two other genera in the family. Relationships in the monophyletic group were correlated with anamorph state produced; three Myxotrichum spp., and B. striatosporus, all with distinct or reported Oidiodendron states, nested with anamorphic species, while species of Myxotrichum with other anamorph states were excluded. In addition, sequence divergence measures between the meiotic and mitotic species clustered in two monophyletic clades were found to be comparable to the intraspecific levels for Oidiodendron spp. These results support the initial hypothesis that Oidiodendron is closely related phylogenetically to the genus Myxotrichum and suggest that the generic concept of Byssoascus needs reexamination.

The Genus Oidiodendron: Species Delimitation and Phylogenetic Relationships Based on Nuclear Ribosomal DNA Analysis

Nuclear ribosomal DNA sequences (ITS region) of fifteen species in the hyphomycete genus Oidiodendron and ten species from the 4 genera in the Myxotrichaceae, Byssoascus, Gymnostellatospora, Myxotrichum, and Pseudogymnoascus, were analysed to: (i) reveal the levels of intra- versus interspecific sequence variation within the genus Oidiodendron, clarify species delimitation and examine the usefulness of some morphological characters used for identification; (ii) assess the possible conspecificity of documented ericoid mycorrhizal strains of Oidiodendron; and (iii) test the hypothesis based on morphological inference that the genus Oidiodendron belongs with the genus Myxotrichum in the Myxotrichaceae (Onygenales). Comparison of molecular and morphological data for multiple strains of O. griseum, O. tenuissimum and O. maius revealed that conidiophore length and the production of a diffusing pigment are not reliable key characters for the genus. Several historically important ericoid mycorrhizal strains, documented as O. griseum, were reidentified as O. maius. Parsimony analyses of 23 Oidiodendron strains showed that three highly supported monophyletic groups, each one consisting of a pair of species, are resolved within the genus. A low level of sequence divergence between the species in these pairs suggests conspecificity for each pair. Other interspecific relationships were not well-supported by bootstrap values. Parsimony analysis of a second dataset composed of mitotic and meiotic taxa showed that Oidiodendron, Myxotrichum and Byssoascus form a well-supported monophyletic group within the Myxotrichaceae, and that this group has diverged significantly from the two other genera in the family. Relationships in the monophyletic group were correlated with anamorph state produced; three Myxotrichum spp., and B. striatosporus, all with distinct or reported Oidiodendron states, nested with anamorphic species, while species of Myxotrichum with other anamorph states were excluded. In addition, sequence divergence measures between the meiotic and mitotic species clustered in two monophyletic clades were found to be comparable to the intraspecific levels for Oidiodendron spp. These results support the initial hypothesis that Oidiodendron is closely related phylogenetically to the genus Myxotrichum and suggest that the generic concept of Byssoascus needs reexamination.

Phylogenetic Distinctiveness of a Threatened Aquatic Turtle (Ster nother us depressus)

The musk turtle (Sternotherus minor) is common throughout the southeastern United States. In 1955 a morphologically atypical form confined to the Black Warrior River System in Alabama was discovered and accorded full species status as S. depressus, the “flattened musk turtle.” Questions remain about the taxonomic status and evolutionary history of the flattened musk turtle because (1) the geographic distribution of S. depressus is nested within the range of S. minor; (2) the flattened shell might be a recently evolved anti‐predator adaptation; and (3) reports exist of intergrades between S. depressus and S. minor. We generated and employed sequence data from mitochondrial DNA to address the phylogenetic distinctiveness and phylogeographic position of S. depressus relative to all other musk and mud turtles (Kinosternidae) in North America. The flattened musk turtle forms a well‐supported monophyletic group separate from S. minor. Genetic divergence observed between S. depressus and geographic populations of S. minor is no less than that distinguishing many kinosternid congeners from one another. These molecular genetic findings bolster rationale for the taxonomic recognition of S. depressus and, hence, for special efforts to protect this federally threatened species.Distintividad Filogenética de una Tortuga Acuática Amenazada (Sternotherus depressus)Las tortugas acuáticas (“musk turtles”) de la especie Sternotherus minor ( familia Kinosternidae) están ampliamente distribuidas en la región sudeste de los Estados Unidos de Norteamérica. En 1955, una variedad de musk turtle morfológicamente atípica, con el caparazón notoriamente comprimido y restringida a la cuenca del río Black Warrior en Alabama, fue descrita y asignada a una especie nueva, S. depressus (“flattened musk turtle”). Posteriormente, el origen evolutivo y el estatus taxonómico de esta nueva especie han sido cuestionados debido a: (1) su restringida distribución geográfica esta anidada dentro del rango de distribución de S. minor; (2) El caparazón aplanado podría ser una adaptación anti‐depredador recientemente evolucionada; y (3) existen reportes de morfologías intermedias entre S. depressus y S. minor. En el presente trabajo se emplean secuencias de ADN mitocondrial para evaluar la identidad genética, posición filogenética, y distribución filogeográfica de S. depressus en relación a las demás especies norteamericanas de la familia Kinosternidae. Los datos obtenidos confirman la identidad de S. depressus como una especie separada genéticamente de la poblaciones locales de S. minor, y de las otras especies de la familia Kinosternidae. Estos resultados sugieren el reconocimiento taxonómico de S. depressus y justifican los esfuerzos conservacionistas para proteger esta especie amenazada.

Long-branch distraction and the Strepsiptera.

Whiting and Wheeler (1994) proposed that Strepsiptera + Diptera (Halteria) were sister groups based on molecular and mor? phological data. Since that proposition, there has arisen a controversy as to wheth? er this sister-group relationship can be taken at face value, or whether it is an ar? tifact of long-branch attraction. Although there appears to be no disagreement that these taxa are sister groups in a parsimony analysis, the contention centers around whether we should accept the results of parsimony, or whether we are being pos? itively misled by long-branch attraction (Felsenstein, 1987). The original phylogenetic analysis of Whiting and Wheeler (1994) consisted of 18S rDNA sequenced from 23 exemplar taxa representing all holometabolous or? ders, with three sequences from the Dip? tera and four from the Strepsiptera, and an extensive morphological matrix. Whiting and Wheeler found support for a mono? phyletic Halteria and also found that the remaining ordinal relationships were high? ly congruent with morphology. These au? thors concluded that the Halteria appeared to be a well-supported monophyletic group, and discussed the implications these results have on homeotic evolution in

Phylogenetic relationships of Cornaceae and close relatives inferred from matK and rbcL sequences

Phylogenetic relationships were inferred using nucleotide sequences of the chloroplast gene matK for members of Cornales, a well-supported monophyletic group comprising Cornaceae and close relatives. The shortest trees resulting from this analysis were highly concordant with those based on previous phylogenetic analysis of rbcL sequences. Analysis of a combined matK and rbcL sequence data set (a total of 2652 bp [base pairs]) provided greater resolution of relationships and higher internal support for clades compared to the individual data sets. Four major clades (most inclusive monophyletic groups) of Cornales are indicated by both sets of genes: (1) Cornus-Alangium, (2) nyssoids (Nyssa-Davidia-Camptotheca)- mastixioids (Mastixia, Diplopanax), (3) Curtisia, and (4) Hydrangeaceae-Loasaceae. The combined evidence indicates that clades 2 and 3 are sisters, with clade 4 sister to the remainder of Cornales. These relationships are also supported by other lines of evidence, including synapomorphies in fruit and pollen morphology and gynoecial vasculature. Comparisons of matK and rbcL sequences based on one of the most parsimonious rbcL-matK trees indicate that matK has a much higher A-T content (66.9% in matK vs. 55.8% in rbcL) and a lower transition:transversion ratio (1.23 in matK vs. 2.21 in rbcL). The total number of nucleotide substitutions per site for matK is 2.1 times that of rbcL in Cornales. These findings are similar to recent comparisons of matK and rbcL in other dicots. Variable sites of matK are almost evenly distributed among the three codon positions (1.0:1.0:1.3), whereas variable sites of rbcL are mostly at the third position (1.8:1.0 :7.5). Among- lineages rates of nucleotide substitutions in rbcL are basically homogeneous throughout Cornales, but are more heterogeneous in matK.

Phytogeny and evolution of Caliciaceae, Mycocaliciaceae, and Sphinctrinaceae (Ascomycota), with notes on the evolution of the prototunicate ascus

Small subunit ribosomal DNA has been sequenced from seven members of the ascomycete order Caliciales s.l. (Calicium adspersum, Cyphelium inquinans, Texosporium sancti-jacobi, and Thelomma mammosum (Caliciaceae), Chaenothecopsis savonica and Stenocybe pullatula (Mycocaliciaceae), and Sphinctrina turbinata (Sphinctrinaceae)), included in a matrix of 58 homologous ascomycete sequences and analysed with maximum parsimony analysis. The result shows the Caliciaceae to be a strongly supported monophyletic group within the Lecanorales s.l., although the jackknife support for a monophyletic Lecanorales is low. Mycocaliciaceae and Sphinctrinaceae form a well-supported monophyletic group, grouping with representatives of Eurotiales and Onygenales. This larger group is the sister group to Lecanorales, but the support for this sister-group relationship and the monophyly of the group consisting of Mycocaliciaceae–Sphinctrinaceae and Eurotiales–Onygenales is low. The evolution of the prototunicate ascus is discussed, and it is concluded that there is neither any evidence for a monophyletic Caliciales s.l., nor for the suggested group "Euascomycetideae" sensu Tehler, with the prototunicate Caliciales being the sister group to the rest of the true ascomycetes. Prototunicate asci are shown to have reappeared at least four times in the evolution of the ascomycetes. Key words: Ascomycetes, lichens, molecular evolution, phylogeny, ribosomal DNA, 18S rDNA.

Structural and evolutionary relationships among chitinases of flowering plants.

The analysis of nuclear-encoded chitinase sequences from various angiosperms has allowed the categorization of the chitinases into discrete classes. Nucleotide sequences of their catalytic domains were compared in this study to investigate the evolutionary relationships between chitinase classes. The functionally distinct class III chitinases appear to be more closely related to fungal enzymes involved in morphogenesis than to other plant chitinases. The ordering of other plant chitinases into additional classes mainly relied on the presence of auxiliary domains-namely, a chitin-binding domain and a carboxy-terminal extension-flanking the main catalytic domain. The results of our phylogenetic analyses showed that classes I and IV form discrete and well-supported monophyletic groups derived from a common ancestral sequence that predates the divergence of dicots and monocots. In contrast, other sequences included in classes I* and II, lacking one or both types of auxiliary domains, were nested within class I sequences, indicating that they have a polyphyletic origin. According to phylogenetic analyses and the calculation of evolutionary rates, these chitinases probably arose from different class I lineages by relatively recent deletion events. The occurrence of such evolutionary trends in cultivated plants and their potential involvement in host-pathogen interactions are discussed.

EVOLUTION OF THE BISEXUAL SPECIES OF CAUCASIAN ROCK LIZARDS: A PHYLOGENETIC EVALUATION OF ALLOZYME DATA

The phylogeny of 12 species of Caucasian rock lizards and ground lizards, genus Lacerta , subgenus Archaeolacerta , was determined from allozyme data. Three additional species of Lacerta formed the outgroup. Of the 35 loci resolved, 20 were phylogenetically informative. Permutation tail probabilities calculated using Hennig86 and RandomCladistics and g 1 statistics obtained in PAUP revealed significant pattern in the data. Our analysis resolved 12 most parsimonious trees, among which were three well-supported monophyletic groups: 1) ( Lacerta alpina , L. caucasica , L. daghestanica) [the L. caucasica group]; 2) ( L. mixta , L. saxicola , L. raddei-nairensis ) [the L. saxicola group]; and 3) ( L. parvula , L. portschinskii ( L. valentini , L. rudis )) [the L. rudis group]. The second and third groups formed a clade. The ground lizards L. praticola and L. derjugini were resolved as ingroup members, but their placement in the globally most parsimonious trees was ambiguous. A preferred tree was chosen by alternately pruning L. praticola and L. derjugini from the tree. In doing so, L. praticola was resolved as the sister taxon of the L. caucasica group, and L. derjugini was resolved as the sister taxon of the clade containing the L. saxicola and L. rudis groups. Our results are somewhat congruent with the morphology-based phylogeny of Darevsky (1967).

Characterization of Superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers

An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes, and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.

Characterization of Superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers

An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes, and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.

Phylogenetic analysis of ten black yeast species using nuclear small subunit rRNA gene sequences.

The nuclear small subunit rRNA genes of authentic strains of the black yeasts Exophiala dermatitidis, Wangiella dermatitidis, Sarcinomyces phaemuriformis, Capronia mansonii, Nadsoniella nigra var. hesuelica, Phaeoannellomyces elegans, Phaeococcomyces exophialae, Exophiala jeanselmei var. jeanselmei and E. castellanii were amplified by PCR and directly sequenced. A putative secondary structure of the nuclear small subunit rRNA of Exophiala dermatitidis was predicted from the sequence data. Alignment with corresponding sequences from Neurospora crassa and Aureobasidium pullulans was performed and a phylogenetic tree was constructed using the neighbor-joining method. The obtained topology of the tree was confirmed by bootstrap analysis. Based upon this analysis all fungi studied formed a well-supported monophyletic group clustering as a sister group to one group of the Plectomycetes (Trichocomaceae and Onygenales). The analysis confirmed the close relationship postulated between Exophiala dermatitidis, Wangiella dermatitidis and Sarcinomyces phaeomuriformis. This monophyletic clade also contains the telemorph species Capronia mansonii thus confirming the concept of a teleomorph connection of the genus Exophiala to a member of the herpotrichiellaceae. However, Exophiala castellanii did not belong to this clade. Therefore, this species is not the anamorph of Capronia mansonii as it was postulated.

A new dhfrVIII trimethoprim-resistance gene, flanked by IS26, whose product is remote from other dihydrofolate reductases in parsimony analysis

A new plasmid-borne gene, dhfrVIII, encoding high-level trimethoprim resistance (Tp R) was found in an intestinal Escherichia coli. It seems to be a widely occurring mediator of Tp R. Among 973 examined Tp R E. coli, the new resistance gene was found in 13 (1.3%) isolates from Sweden, Finland and Nigeria. The new gene was sequenced and found to code for a dihydrofolate reductase (DHFR) of 169 amino acids ( M r 19005). The dhfrVIII gene on the studied plasmid pLMO226 was observed to be flanked by IS26 elements. The dhfrVIII gene and a 3′ unidentified open reading frame (ORF) seem to be borne on a compound transposon with IS26 at its ends, since the configuration of two IS26 flanking dhfrVIII and the unidentified ORF was conserved among the isolates that were probe-positive for the gene. Phylogeny parsimony analysis showed the dhfrVIII-encoded enzyme to be only remotely related to other known plasmid-mediated, drug-resistant DHFR. Only a few of the latter form well-supported monophyletic groups.