PHYLOGENY OF ZYGNEMOPHYCEAE BASED ON COXIII GENE SEQUENCE DATA

Molecular phylogenetic analysis of the conjugating green algae (Class Zygnemophyceae) using nuclear (SSU rDNA) and chloroplast (rbcL) gene sequences has resolved hypotheses of relationship at the class, order, and family levels, but several key questions will require data from additional genes. Based on SSU and rbcL sequences, the Zygnemophyceae and Desmidiales are monophyletic, and families of placoderm desmids are distinct clades (Desmidiaceae, Peniaceae, Closteriaceae, and Gonatozygaceae). In contrast, the Zygnemataceae and Mesotaeniaceae are paraphyletic, although whether these two traditional families constitute a clade is uncertain. In addition, relationships of genera within families have proven resistant to resolution with these two oft‐used genes. We have sequenced the coxIII gene from the mitochondrial genome to address some of these ambiguous portions of the phylogeny of conjugating green algae. The coxIII gene is more variable than rbcL or SSU rDNA and offers greater resolving power for relationships of genera. We present preliminary analyses of coxIII sequences from each of the traditional families of Zygnemophyceae and contrast the resulting topologies with those derived from nuclear and chloroplast genes.     

CLONING AND CHARACTERIZATION OF NITRATE TRANSPORTER GENES FROM THE DIATOM CYLINDROTHECA FUSIFORMIS

Molecular phylogenetic analysis of the conjugating green algae (Class Zygnemophyceae) using nuclear (SSU rDNA) and chloroplast ( rbcL ) gene sequences has resolved hypotheses of relationship at the class, order, and family levels, but several key questions will require data from additional genes. Based on SSU and rbc L sequences, the Zygnemophyceae and Desmidiales are monophyletic, and families of placoderm desmids are distinct clades (Desmidiaceae, Peniaceae, Closteriaceae, and Gonatozygaceae). In contrast, the Zygnemataceae and Mesotaeniaceae are paraphyletic, although whether these two traditional families constitute a clade is uncertain. In addition, relationships of genera within families have proven resistant to resolution with these two oft-used genes. We have sequenced the cox III gene from the mitochondrial genome to address some of these ambiguous portions of the phylogeny of conjugating green algae. The cox III gene is more variable than rbc L or SSU rDNA and offers greater resolving power for relationships of genera. We present preliminary analyses of coxIII sequences from each of the traditional families of Zygnemophyceae and contrast the resulting topologies with those derived from nuclear and chloroplast genes.     

APPLICABILITY OF ITS DATA IN ROCCELLACEAE (ARTHONIALES,EUASCOMYCETES) PHYLOGENY

Abstract: The ability of the internal transcribed spacers (ITS regions) of ribosomal DNA to resolve phylogenetic relationships within the euascomycetous order Arthoniales, focusing on the family Roccellaceae was investigated. The effect of alignment on phylogenetic hypotheses was evaluated. A data matrix from the ITS regions was constructed from 33 specimens representing 14 genera, including the outgroup Arthothelium spectabile. Six different alignments were analysed cladistically using parsimony jackknifing. Most groups in the six trees were congruent and well supported under the different alignment settings. In a conservative analysis, where only unambiguously alignable regions were included, the resolution was low. These results indicate that the ITS regions contain phylogenetic structure, and all information, including the variable regions, should be utilised. A data matrix from the SSU rDNA sequences was constructed for the same taxa. The SSU rDNA tree was less resolved than the ITS trees. There were only minor conflicts between the two sources of data and an incongruence test confirmed that the ITS and SSU rDNA data matrices were not significantly incongruent. The six differently aligned data matrices generated from the ITS regions were each combined with the SSU rDNA data. Simultaneous analysis of the combined data sets is the best approach as it uses all available evidence. As with the ITS trees, most groups in the combined trees were congruent and well supported. The SSU rDNA provided resolution within one clade, otherwise the ITS sequences provided most of the signal in the combined analysis, both at the basal nodes and at the tips of the tree. Molecular data clearly indicates that the fruticose/crustose habits have evolved multiple times even in comparatively small groups as in the family Roccellaceae and that the characters such as fruticose-crustose may be overemphasized in morphological analyses.     

New genes for phylogenetic studies of lichenized fungi: glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin genes

Abstract:Primers for amplification and sequencing of partial glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were designed for lichenized fungi. The 5′ gpd primer is most probably fungal specific, since a BLAST search in GenBank found identical sequences only from ascomycetous taxa, whereas the 3′ gpd primer was more universal. Utility of the gpd primers and previously designed beta-tubulin primers was tested in nine lichen taxa. Both the gpd and beta-tubulin primer pairs amplified in most of the taxa examined: the gpd primers generated a c. 1100 nucleotide fragment, whereas the PCR product obtained from the beta-tubulin primers was c. 900 nucleotides long. The gpd amplification products of Cladonia arbuscula and C. rangiferina were sequenced and both were found to contain three introns, the length of which varied between 49 to 83 nucleotides. To examine the applicability of gpd sequences in resolving relationships within Ascomycota, trees were calculated from 22 fungal gpd sequences obtained from GenBank together with the twoCladonia sequences using parsimony jackknifing. The gpd tree was compared with the SSU rDNA tree of the respective species (or genera). A similar analysis of the beta-tubulin gene was not performed, because only a few beta-tublin sequences from the same taxa were available in GenBank. The gpd tree was well resolved but in conflict with the SSU rDNA tree. In contrast to the SSU rDNA tree, the gpd tree did not support the monophyly of the Ascomycota. Analysis of the combined data set produced a tree very similar to that of the SSU rDNA data. However, the relationship of Lecanorales to the other orders remained unresolved. Even though gpd and beta-tubulin are highly conserved proteins, the third codon positions and introns are variable and both genes have the potential for inferring phylogenetic relationships at the lower taxonomic levels in the lichenized fungi. The two genes may be useful even below species level, depending on the species investigated.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

PCR PRIMERS FOR THE AMPLIFICATION OF MITOCHONDRIAL SMALL SUBUNIT RIBOSOMAL DNA OF LICHEN-FORMING ASCOMYCETES

Abstract: Four primers for the amplification of mitochondrial DNA of lichen-forming ascomycetes are presented. The primers match the conserved regions U2, U4, and U6, respectively, of mitochondrial small subunit (SSU) ribosomal DNA (rDNA). Polymerase chain reaction using different combinations of the primers produced single amplification products from DNA of eight lichen-forming fungal species but did not amplify DNA of two axenic cultured algal species. The amplification product obtained from Lobaria pulmonaria was sequenced and the 894-bp sequence was compared with the mitochondrial SSU rDNA sequence of Podospora anserina. The two sequences revealed more than 76% identity in the conserved regions U3 to U5 demonstrating that we amplified mitochondrial DNA. The primers matching U2 and U6 yielded amplification products of 800–1000 bp depending on the species examined. The variation observed suggests that mitochondrial SSU rDNA may be useful for phylogenetic analyses of lichen-forming ascomycetes.     

Grellamoeba robusta gen. n., sp. n., a possible member of the family Acramoebidae Smirnov,Nassonova et Cavalier-Smith, 2008

A strain of naked amoeba isolated from pikeperch (Sander lucioperca (L.)) kidney tissue has been characterized using light- and transmission electron microscopy. Sequencing of SSU rDNA and phylogenetic analysis based on a broad dataset of sequences completed our study. All data obtained suggest that this strain belongs to a species that has not been described before. As none of the existing genera of amoebae is applicable to this organism, the new genus Grellamoeba is established and the type species Grellamoeba robusta is described. Although the phylogenetic position of the SSU rDNA sequence of the type strain of G. robusta is sensitive to the method of analysis applied, a tendency to group with Acramoeba dendroida Smirnov, Nassonova et Cavalier-Smith, 2008 is evident.     

The first survey of Cystobasidiomycete yeasts in the lichen genus Cladonia; with the description of Lichenozyma pisutiana gen. nov., sp. nov.

The view of lichens as a symbiosis only between a mycobiont and a photobiont has been challenged by discoveries of diverse associated organisms. Specific basidiomycete yeasts in the cortex of a range of macrolichens were hypothesized to influence the lichens' phenotype. The present study explores the occurrence and diversity of cystobasidiomycete yeasts in the lichen genus Cladonia. We obtained seven cultures and 56 additional sequences using specific primers from 27 Cladonia species from all over Europe and performed phylogenetic analyses based on ITS, LSU and SSU rDNA loci. We revealed yeast diversity distinct from any previously reported. Representatives of Cyphobasidiales, Microsporomycetaceae and of an unknown group related to Symmetrospora have been found. We present evidence that the Microsporomycetaceae contains mainly lichen-associated yeasts. Lichenozyma pisutiana is circumscribed here as a new genus and species. We report the first known associations between cystobasidiomycete yeasts and Cladonia (both corticate and ecorticate), and find that the association is geographically widespread in various habitats. Our results also suggest that a great diversity of lichen associated yeasts remains to be discovered.     

PHYLOGENETIC ANALYSIS OF THE LICHEN FAMILY UMBILICARIACEAE BASED ON NUCLEAR ITS1 AND ITS2 rDNA SEQUENCES

Abstract: The lichen family Umbilicariaceae is accepted by most lichenologists as consisting of two genera, Lasallia and Umbilicaria. The monophyly of these two genera was examined by phylogenetic analyses of nucleotide sequences of ITS1 and ITS2 rDNA. Sequences of these regions from three Lasallia and 17Umbilicaria species were aligned to those of seven representatives of the outgroup taxa including Eurotiales, Onygenales and Caliciales (Mycocaliciaceae) and subjected to maximum parsimony, maximum likelihood and neighbour-joining analyses. The resulting phylogenetic hypotheses supported the monophyly of the representative species of Lasallia. However, the species of Umbilicaria did not form a monophyletic sister-group to Lasallia due to the basal placement of otherUmbilicaria species in some analyses. Based on these analyses, if Lasallia is recognized as a separate genus thenUmbilicaria appears to be paraphyletic. Although further taxon sampling is required to resolve the monophyly ofUmbilicaria , for the present we recommend retaining the current treatment of Lasallia as separate fromUmbilicaria .     

地衣型真菌的I型内含子及其在系统发育中的应用

以蜈蚣衣属、黑蜈蚣衣属地衣样品为材料,结合GenBank中相关数据,对地衣型真菌核糖体小亚基 DNA上的I型内含子分布模式进行归纳,并探讨了其在地衣型真菌系统发育研究中的应用。结果表明在地衣型真菌核糖体小亚基 DNA上存在多个I型内含子插入位点,通过二级结构分析给出了天然状态下I型内含子发生转座的证据。分析显示,I型内含子作为分子标记,只适合用于种下单位的系统发育研究中。     

PHYLOGENY OF THE EUGLENOID LORICATE GENERA TRACHELOMONAS AND STROMBOMONAS (EUGLENOPHYTA) INFERRED FROM NUCLEAR SSU AND LSU rDNA1

Previous studies using the nuclear SSU rDNA and partial LSU rDNA have demonstrated that the euglenoid loricate taxa form a monophyletic clade within the photosynthetic euglenoid lineage. It was unclear, however, whether the loricate genera Trachelomonas and Strombomonas were monophyletic. In order to determine the relationships among the loricate taxa, SSU and LSU nuclear rDNA sequences were obtained for eight Strombomonas and 25 Trachelomonas strains and combined in a multigene phylogenetic analysis. Conserved regions of the aligned data set were used to generate maximum‐likelihood (ML) and Bayesian phylogenies. Both methods recovered a strongly supported monophyletic loricate clade with Strombomonas and Trachelomonas species separated into two sister clades. Taxa in the genus Strombomonas sorted into three subclades. Within the genus Trachelomonas, five strongly supported subclades were recovered in all analyses. Key morphological features could be attributed to each of the subclades, with the major separation being that all of the spine‐bearing taxa were located in two sister subclades, while the more rounded, spineless taxa formed the remaining three subclades. The separation of genera and subclades was supported by 42 distinct molecular signatures (33 in Trachelomonas and nine in Strombomonas). The morphological and molecular data supported the retention of Trachelomonas and Strombomonas as separate loricate genera.     

A RE-ASSESSMENT OF THE FAMILY ALECTORIACEAE

Abstract: The phylogenetic relationship between the Alectoriaceae and theParmeliaceae (lichenized ascomycetes) were studied using sequences of the small subunit of the nuclear ribosomal DNA (SSU rDNA). The Alectoriaceae was represented by specimens of Alectoria ochroleuca and A. sarmentosa, and theParmeliaceae by Bryoria capillaris, Cetraria islandica, Cornicularia normoerica, Evernia prunastri, Hypogymnia physodes, Parmelia saxatilis, Platismatia glauca, Pleurosticta acetabulum, Usnea florida, Vulpicida juniperina, V. pinastri and Xanthoparmelia conspersa. The results suggest that the Alectoriaceae is derived from within the Parmeliaceae, and it is suggested that the families should be treated as synonyms. Morphological and anatomical characters are discussed, and it is concluded that earlier investigations might have over- emphasized minor variations in ascus structure.     

Phylogenetic affinity of Mycochaetophora gentianae, the causal fungus of brown leaf spot on gentian (Gentiana triflora), to Pseudocercosporella-like hyphomycetes in Helotiales

Mycochaetophora gentianae, the causal agent of brown leaf spot on gentian (Gentiana scabra), is characterized by its hyaline besom-like sporophore, although its conidiogenesis and phylogenetic position have so far remained unknown. We isolated the causal fungus from a new host, G. triflora, in Iwate, Japan. Both the G. triflora isolate and the ex-type M. gentianae isolate produced symptoms on G. triflora but not on G. scabra. Microscopic observations of the diseased leaves indicated that conidiogenesis was blastic from short conidiophores, and schizolytic secession of conidia left unthickened and inconspicuous conidial scars on the conidiogenous cells. Conidia were catenate, in branched acropetalous chains; secondary conidia were blastically produced from the first or second cell at the base of primary conidium. The G. triflora isolate was identified as M. gentianae because of its identity to the ex-type in characteristics of culture, pathogenicity, and conidia. Phylogenetic analyses using three ribosomal DNA (rDNA) sequences combined [small subunit (SSU) + large subunit (LSU) + 5.8S rDNA] indicated that both isolates clustered with Rhexocercosporidium carotae, and the cluster was placed within Helotiales–Rhytismatales. Additional analyses using internal transcribed spacers including 5.8S rDNA sequences revealed that both isolates were monophyletic and that they were closely related to three helotialean Pseudocercosporella-like hyphomycetous genera: Helgardia, Rhexocercosporidium and Rhynchosporium.     

O-Methylated mannogalactan from the microalga Coccomyxa mucigena, symbiotic partner of the lichenized fungus Peltigera aphthosa

A structural study of the carbohydrates from Coccomyxa mucigena, the symbiotic algal partner of the lichenized fungus Peltigera aphthosa, was carried out. It produced an O-methylated mannogalactan, with a (1 → 6)-linked β-galactopyranose main-chain partially substituted at O-3 by β-Galp, 3-OMe-α-Manp or α-Manp units. There were no similarities with polysaccharides previously found in the lichen thallus of P. aphthosa. Moreover, the influence of lichenization in polysaccharide production by symbiotic microalgae and the nature of the photobiont in carbohydrate production in lichen symbiosis are also discussed.     

Evolution of helotialean fungi (Leotiomycetes, Pezizomycotina): a nuclear rDNA phylogeny

The highly divergent characters of morphology, ecology, and biology in the Helotiales make it one of the most problematic groups in traditional classification and molecular phylogeny. Sequences of three rDNA regions, SSU, LSU, and 5.8S rDNA, were generated for 50 helotialean fungi, representing 11 out of 13 families in the current classification. Data sets with different compositions were assembled, and parsimony and Bayesian analyses were performed. The phylogenetic distribution of lifestyle and ecological factors was assessed. Plant endophytism is distributed across multiple clades in the Leotiomycetes. Our results suggest that (1) the inclusion of LSU rDNA and a wider taxon sampling greatly improves resolution of the Helotiales phylogeny, however, the usefulness of rDNA in resolving the deep relationships within the Leotiomycetes is limited; (2) a new class Geoglossomycetes, including Geoglossum, Trichoglossum, and Sarcoleotia, is the basal lineage of the Leotiomyceta; (3) the Leotiomycetes, including the Helotiales, Erysiphales, Cyttariales, Rhytismatales, and Myxotrichaceae, is monophyletic; and (4) nine clades can be recognized within the Helotiales.     

Positions of multiple insertions in SSU rDNA of lichen-forming fungi

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.      

MOLECULAR PHYLOGENY OF PHYCOCYANIN‐CONTAINING CRYPTOPHYTES: EVOLUTION OF BILIPROTEINS AND GEOGRAPHICAL DISTRIBUTION1

Of 34 strains assigned to the cryptophyte genera Chroomonas Hansg., Hemiselmis Parke, and Komma D. R. A. Hill, distribution patterns of biliproteins, habitats, and sampling sites across a phylogenetic tree have been examined. The combined data set assembled from nuclear SSU rDNA, partial nuclear LSU rDNA, and nucleomorph SSU rDNA sequences comprised 4,083 positions and yielded an almost completely resolved tree. Spectrophotometry of the biliproteins and mapping of the different types of biliproteins onto the phylogenetic tree unveiled a complex evolutionary history. Different from other cryptophyte clades, the types of biliproteins were not generally congruent with clades or subclades of the genera Chroomonas (paraphyletic, phycocyanins [PCs] 645 or 630), Hemiselmis (PCs 612, 630 or phycoerythrin [PE] 555), and Komma (PC 645). At least one putative character reversal took place in the genus Chroomonas. Several changes in biliproteins have been found in the genus Hemiselmis, including two new biliprotein variants that probably originated by slight modifications from PC 612 and PE 555, respectively (PC 577 and PE 545/555). Freshwater and marine/brackish taxa were intermingled across the tree without displaying a specific pattern. In four terminal clades, genetically identical strains have been found to occur both in Europe and in the USA. The Chroomonas/Hemiselmis/Komma clade proved to be the most diverse of all cryptophyte clades concerning types of biliproteins and distribution of clades across marine or freshwater habitats.     

Independent terrestrial origins of the Halosphaeriales (marine Ascomycota)

A phylogenetic study of marine ascomycetes was initiated to test and refine evolutionary hypotheses of marine-terrestrial transitions among ascomycetes. Taxon sampling focused on the Halosphaeriales, the largest order of marine ascomycetes. Approximately 1050 base pairs (bp) of the gene that codes for the nuclear small subunit (SSU) and 600 bp of the gene that codes for the nuclear large subunit (LSU) ribosomal RNAs (rDNA) were sequenced for 15 halosphaerialean taxa and integrated into a data set of homologous sequences from terrestrial ascomycetes. An initial set of phylogenetic analyses of the SSU rDNA from 38 taxa representing 15 major orders of the phylum Ascomycota confirmed a close phylogenetic relationship of the halosphaerialean species with several other orders of perithecial ascomycetes. A second set of analyses, which involved more intensive taxon sampling of perithecial ascomycetes, was performed using the SSU and LSU rDNA data in combined analyses. These second analyses included 15 halosphaerialean taxa, 26 terrestrial perithecial fungi from eight orders, and five outgroup taxa from the Pezizales. In these analyses the Halosphaeriales were polyphyletic and comprised two distinct lineages. One clade of Halosphaeriales comprised 12 taxa from 11 genera and was most closely related to terrestrial fungi of the Microascales. The second clade of halosphaerialean fungi comprised taxa from the genera Lulworthia and Lindra and was an isolated lineage among the perithecial fungi. Both the main clade of Halosphaeriales and the Lulworthia/Lindra clade are supported by the data as being independently derived from terrestrial ancestors.     

Life cycle and molecular phylogeny of the dinoflagellates Chytriodinium and Dissodinium,ectoparasites of copepod eggs

The dinoflagellates Chytriodinium affine, C. roseum and Dissodinium pseudolunula are ectoparasites of crustacean eggs. Here, we present new observations regarding their life cycle based on coastal plankton samples and incubations and analyze their molecular phylogeny using the small subunit ribosomal RNA gene (SSU rDNA) as a marker. In contrast to the typical stages already documented for its life cycle, we observed that D. pseudolunula dinospores may exceptionally differentiate inside a globular cyst. Despite its parasitic life style, the cysts and dinospores of D. pseudolunula contain chlorophyll a. We obtained the first SSU rDNA sequences for the genera Chytriodinium (the type C. roseum and C. affine) and Dissodinium (D. pseudolunula). Classical taxonomical schemes have ascribed these genera to the order Blastodiniales. However, our SSU rDNA-based phylogenetic analysis shows that these ectoparasites form a clade in the Gymnodinium sensu stricto group, unarmored dinokaryotic dinoflagellates of the order Gymnodiniales. They branch in a subgroup composed of warnowiids, polykrikoids, the type of Gymnodinium, G. fuscum and G. aureolum. Although Chytriodinium and Dissodinium appear to be relatives based on SSU rDNA phylogeny, feeding and host specificity, their life cycles are substantially different. Based on these data we consider that the type of life cycle is a poor criterion for classification at the family level. We suggest that the morphology of the infective cell is probably the most reliable phenotypic characteristic to determine the systematic position of parasitic dinoflagellates.     

Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences

Phylogenetic relationships among nematodes of the strongylid superfamily Metastrongyloidea were analyzed using partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes. Regions of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction, directly sequenced, aligned, and phylogenies inferred using maximum parsimony. Phylogenetic hypotheses inferred from the SSU rRNA gene supported the monophyly of representative taxa from each of the 7 currently accepted metastrongyloid families. Metastrongyloid taxa formed the sister group to representative trichostrongyloid sequences based on SSU data. Sequences from either the SSU or LSU RNA regions alone provided poor resolution for relationships within the Metastrongyloidea. However, a combined analysis using sequences from all rDNA regions yielded 3 equally parsimonious trees that represented the abursate Filaroididae as polyphyletic, Parafilaroides decorus as the sister species to the monophyletic Pseudaliidae, and a sister group relationship between Oslerus osleri and Metastrongylus salmi. Relationships among 3 members of the Crenosomatidae, and 1 representative of the Skrjabingylidae (Skrjabingylus chitwoodorum) were not resolved by these combined data. However, members of both these groups were consistently resolved as the sister group to the other metastrongyloid families. These relationships are inconsistent with traditional classifications of the Metastrongyloidea and existing hypotheses for their evolution.