Phylogeny of thrips (Insecta: Thysanoptera) based on five molecular loci

The order Thysanoptera (Paraneoptera), commonly known as thrips, displays a wide range of behaviours, and includes several pest species. The classification and suggested relationships among these insects remain morphologically based, and have never been evaluated formally with a comprehensive molecular phylogenetic analysis. We tested the monophyly of the suborders, included families and the recognized subfamilies, and investigated their relationships. Phylogenies were reconstructed based upon 5299 bp from five genetic loci: 18S ribosomal DNA, 28S ribosomal DNA, Histone 3, Tubulin‐alpha I and cytochrome oxidase c subunit I. Ninety‐nine thrips species from seven of the nine families, all six subfamilies and 70 genera were sequenced. Maximum parsimony, maximum likelihood and Bayesian analyses all strongly support a monophyletic Tubulifera and Terebrantia. The families Phlaeothripidae, Aeolothripidae, Melanthripidae and Thripidae are recovered as monophyletic. The relationship of Aeolothripidae and Merothripidae to the rest of Terebrantia is equivocal. Molecular data support previous suggestions that Aeolothripidae or Merothripidae could be a sister to the rest of Terebrantia. Four of the six subfamilies are recovered as monophyletic. The two largest subfamilies, Phlaeothripinae and Thripinae, are paraphyletic and require further study to understand their internal relationships.     

Phylogenetics of modern birds in the era of genomics

In the 14 years since the first higher-level bird phylogenies based on DNA sequence data, avian phylogenetics has witnessed the advent and maturation of the genomics era, the completion of the chicken genome and a suite of technologies that promise to add considerably to the agenda of avian phylogenetics. In this review, we summarize current approaches and data characteristics of recent higher-level bird studies and suggest a number of as yet untested molecular and analytical approaches for the unfolding tree of life for birds. A variety of comparative genomics strategies, including adoption of objective quality scores for sequence data, analysis of contiguous DNA sequences provided by large-insert genomic libraries, and the systematic use of retroposon insertions and other rare genomic changes all promise an integrated phylogenetics that is solidly grounded in genome evolution. The avian genome is an excellent testing ground for such approaches because of the more balanced representation of single-copy and repetitive DNA regions than in mammals. Although comparative genomics has a number of obvious uses in avian phylogenetics, its application to large numbers of taxa poses a number of methodological and infrastructural challenges, and can be greatly facilitated by a 'community genomics' approach in which the modest sequencing throughputs of single PI laboratories are pooled to produce larger, complementary datasets. Although the polymerase chain reaction era of avian phylogenetics is far from complete, the comparative genomics era-with its ability to vastly increase the number and type of molecular characters and to provide a genomic context for these characters-will usher in a host of new perspectives and opportunities for integrating genome evolution and avian phylogenetics.     

Chromosomal diversity and phylogenetic inferences concerning thrips (Insecta, Thysanoptera) in a semi-arid region of Brazil

The order Thysanoptera is composed of cosmopolitan phytophagous and predaceous insects with diverse life histories, behaviors and habits. This order is currently thought to form a trichotomy with Hemiptera and Psocodea; Hemiptera and Thysanoptera are considered to be sister groups. The interrelationships within Thysanoptera remain unclear and cytotaxonomic studies are scarce in thrips. We report, for the first time, chromosomal data on seven species of thrips collected from a semi-arid region in the States of Bahia and Pernambuco (Northeast Brazil). A distinctive chromosomal pattern was observed in Thysanoptera when compared to other members within the infraclass Paraneoptera. Considerable karyotypic differences were also found within genera and species of Thysanoptera. Based on these data, we suggest that Paraneoptera forms a polyphyletic group and that Terebrantia and Tubulifera should be regarded as sister groups. The high chromosomal variability observed in Thysanoptera indicates that chromosomal rearrangements have played a key role in their speciation pathways.     

The Jurassic thrips Liassothrips crassipes (Martynov, 1927) and its taxonomic position in the order Thysanoptera (Insecta)

The Late Jurassic thrips Liassothrips crassipes (Martynov) is redescribed, and its taxonomic position discussed. It is shown that the male genitalia of Liassothrips are similar to the terebrantian type, while the female genitalia resemble the tubuliferan type. Therefore, the Liassothripidae shows the possible evolutionary pathway of tubuliferan thrips. Liassothrips is treated as the oldest known member of the suborder Tubulifera (previously known beginning from the Eocene), the ancestors of which belonged to the family Aeolothripidae (Terebrantia) rather than Thripidae.     

Intraordinal phylogenetics of treeshrews (Mammalia: Scandentia) based on evidence from the mitochondrial 12S rRNA gene

Despite their traditional and continuing prominence in studies of interordinal mammalian phylogenetics, treeshrews (order Scandentia) remain relatively unstudied with respect to their intraordinal relationships. At the same time, significant morphological variation among living treeshrews has been shown to have direct relevance to higher-level interpretations of character state change as reconstructed in traditional interordinal studies, which have often included only a single species of treeshrew. Therefore, the importance of resolving relationships among treeshrews extends well beyond a better understanding of patterns of diversification within the order. A recent review highlighted several shortcomings in published studies of treeshrew phylogenetics based on morphology. Here we present the first investigation of treeshrew phylogenetics based on DNA sequences, utilizing previously published sequences from the mitochondrial 12S rRNA gene and combining them with newly generated sequence data from 15 species. Parsimony, likelihood, and Bayesian analyses all strongly support a sister relationship between Ptilocercus and the remaining species, further substantiating its recent elevation to familial status. Dendrogale is consistently recovered as the next taxon to diverge, but relationships among the remaining taxa are poorly supported by these data. We provide evidence for a relatively rapid radiation within the genera Tupaia and Urogale, but limited resolution precludes more than a cursory interpretation of biogeographic patterns.     

Phylogeny of the Hymenoptera (Insecta): The state of the art

The Hymenoptera form one of the major insect orders in terms of species diversity and they are also of great ecological and economic importance, notably as pollinators and natural enemies of pests. Phylogenies provide an essential framework for systematic, comparative and evolutionary research on hymenopterans. Here, I summarise what we know about the higher-level phylogeny of the Hymenoptera based on presentations given at a recent symposium on hymenopteran phylogenetics. I identify agreement and conflict among morphological and molecular analyses and suggest important priority areas for future research.     

Phylogenetic relationships between the families of recent Thysanoptera (Insecta)

One family, the Phlaeothripidae, is recognized in the suborder Tubulifera, whereas extant species of Terebrantia are classified into seven families: Uzelothripidae, Merothripidae, Aeolothripidae, Adiheterothripidae, Fauriellidae (stat. n.), Heterothripidae and Thripidae. A phylogenetic analysis of the relationships between these families is given, based on consideration of 35 imaginal characters; however, the relationships of Uzelothripidae and Phlaeothripidae to the rest of the Thysanoptera remain equivocal. The Phlaeothripidae are either derived independently from Protothysanoptera, or else are the sister-group of the Thripidae, the most specialized family of Terebrantia.
Diagnostic characters, diversity, distribution and relationships of each family are discussed. Keys to family and, in Fauriellidae, to genus are provided. Holarthrothrips Bagnall (= Adiheterothrips Ramakrishna, syn. n.) and Oligothrips Moulton are removed from Heterothripidae to Adiheterothripidae and Fauriella Hood, Opisthothrips Hood and Ropotamothrips Pelikan (= Osmanothrips Priesner, syn. n.) from Heteromripidae to Fauriellidae (stat. n.). These transfers leave Aulacothrips Hood, Heterothrips Hood and Scutothrips Stannard as the only genera in Heterothripidae.     

A Phylogeny and Timescale for Marsupial Evolution Based on Sequences for Five Nuclear Genes

Even though marsupials are taxonomically less diverse than placentals, they exhibit comparable morphological and ecological diversity. However, much of their fossil record is thought to be missing, particularly for the Australasian groups. The more than 330 living species of marsupials are grouped into three American (Didelphimorphia, Microbiotheria, and Paucituberculata) and four Australasian (Dasyuromorphia, Diprotodontia, Notoryctemorphia, and Peramelemorphia) orders. Interordinal relationships have been investigated using a wide range of methods that have often yielded contradictory results. Much of the controversy has focused on the placement of Dromiciops gliroides (Microbiotheria). Studies either support a sister-taxon relationship to a monophyletic Australasian clade or a nested position within the Australasian radiation. Familial relationships within the Diprotodontia have also proved difficult to resolve. Here, we examine higher-level marsupial relationships using a nuclear multigene molecular data set representing all living orders. Protein-coding portions of ApoB, BRCA1, IRBP, Rag1, and vWF were analyzed using maximum parsimony, maximum likelihood, and Bayesian methods. Two different Bayesian relaxed molecular clock methods were employed to construct a timescale for marsupial evolution and estimate the unrepresented basal branch length (UBBL). Maximum likelihood and Bayesian results suggest that the root of the marsupial tree is between Didelphimorphia and all other marsupials. All methods provide strong support for the monophyly of Australidelphia. Within Australidelphia, Dromiciops is the sister-taxon to a monophyletic Australasian clade. Within the Australasian clade, Diprotodontia is the sister taxon to a Notoryctemorphia + Dasyuromorphia + Peramelemorphia clade. Within the Diprotodontia, Vombatiformes (wombat + koala) is the sister taxon to a paraphyletic possum group (Phalangeriformes) with kangaroos nested inside. Molecular dating analyses suggest Late Cretaceous/Paleocene dates for all interordinal divergences. All intraordinal divergences were placed in the mid to late Cenozoic except for the deepest splits within the Diprotodontia. Our UBBL estimates of the marsupial fossil record indicate that the South American record is approximately as complete as the Australasian record. The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The spermatogenesis and the sperm structure of Terebrantia (Thysanoptera, Insecta)

Spermatogenesis and the sperm structure of the terebrantian Aeolothrips intermedius Bagnall are described. Spermatogenesis consists of two mitotic divisions; the second is characterized by the loss of half of the spermatids, which have pyknotic nuclei. Early spermatids have two centrioles, but when spermiogenesis starts, a third centriole is produced. The three basal bodies give rise to three flagella; later these fuse into a single flagellum which contains three 9 + 0 axonemes. The basal bodies are surrounded by a large amount of centriole adjunct material. During spermiogenesis this material contributes to the shifting of the three axonemes towards the anterior sperm region parallel to the elongating nucleus, and it is transformed into a dense cylinder. In the mature spermatids the three axonemes amalgamate to create a bundle of 27 doublet microtubules. Near the end of spermiogenesis the dense cylinder of the centriole adjunct lies parallel to the nucleus and the axonemes. It ends where the mitochondrion appears at half-sperm length. We confirm that Terebrantia testes have a single sperm cyst; their sperm are characterized by a cylindrical nucleus, three axonemes fused into one, a small mitochondrion and a short cylindrical centriole adjunct which corresponds to the dense body described in a previous work. The acrosome is lacking. At the midpoint of the anterior half of the sperm the outline of the cross-section is bilobed, with the nucleus contained in a pocket evagination of the plasma membrane. These characters are discussed in light of a comparison between Tubulifera and Terebrantia.     

New findings on sperm ultrastructure in thrips (Thysanoptera, Insecta)

Sperm ultrastructure of several species in each of the two suborders of Thysanoptera Tubulifera and Terebrantia shows a distinctive and unusual architecture. Members of the whole order share a bizarre axoneme consisting of 27 microtubular elements derived from the amalgamation of 3 (9+0) axonemes present in each spermatid at the beginning of spermiogenesis. The reciprocal shifting of these axonemes along the length of the sperm, together with their possible shortening and overlapping for short distances, could explain why in some species it is never possible to observe the complete set of 27 microtubular elements in any one cross section. Tubuliferan sperm have a small elliptical (in cross section) acrosome extending the length of the sperm. In Bolothrips insularis and Compsothrips albosignatus this structure is larger and is associated with an external, flattened vesicle throughout its length. Terebrantian sperm lack an acrosome, but display for half their length a dense body running parallel to the nucleus. The sperm, in members of this suborder, are also characterized by possession of a small mitochondrion and by the unusual bilobed outline of cross sections through the anterior sperm region, with the nucleus located in one of the two lobes. Structures serving to anchor sperm to the inner surface of the cyst cell have been observed at their anterior tips in the testes of tubuliferans. In B. insularis, an anterior appendage is formed in immature sperm and is maintained in the mature spermatozoon parallel to its long axis in the most anterior region. Such an anchoring structure has not been observed in sperm of the terebrantian species examined, probably because the testis of terebrantians contains only a single cyst of developing gametes.     

Utility of arginine kinase for resolution of phylogenetic relationships among Brachyuran genera and families

The molecular phylogenetics of decapod crustaceans has been based on sequence data from a limited number of genes. These have included rapidly evolving mitochondrial genes, which are most appropriate for studies of closely related species, and slowly evolving nuclear ribosomal RNA genes, which have been most useful for resolution of deep branches within the Decapoda. Here we examine the utility of the nuclear gene that encodes arginine kinase for phylogenetic reconstruction at intermediate levels (relationships among genera and families) within the decapod infraorder Brachyura (the true crabs). Analyses based on arginine kinase sequences were compared and combined with those for the mitochondrial cytochrome oxidase I gene. All of the genera in our taxon sample were resolved with high support with arginine kinase data alone. However, some of these genera were grouped into clades that are in conflict with recognized brachyuran families. A phylogeny based on cytochrome oxidase I was consistent with the arginine kinase phylogeny, but with weaker support. A recently proposed measure of phylogenetic informativeness indicated that arginine kinase was generally more informative than cytochrome oxidase I for relationships above the level of genus. Combined analysis of data from both genes provided strong support for clades that are in conflict with current assignments of genera to the families Epialtidae, Mithracidae, Pisidae, and Portunidae.     

Cytochrome b and Bayesian inference of whale phylogeny

In the mid 1990s cytochrome b and other mitochondrial DNA data reinvigorated cetacean phylogenetics by proposing many novel and provocative hypotheses of cetacean relationships. These results sparked a revision and reanalysis of morphological datasets, and the collection of new nuclear DNA data from numerous loci. Some of the most controversial mitochondrial hypotheses have now become benchmark clades, corroborated with nuclear DNA and morphological data; others have been resolved in favor of more traditional views. That major conflicts in cetacean phylogeny are disappearing is encouraging. However, most recent papers aim specifically to resolve higher-level conflicts by adding characters, at the cost of densely sampling taxa to resolve lower-level relationships. No molecular study to date has included more than 33 cetaceans. More detailed molecular phylogenies will provide better tools for evolutionary studies. Until more genes are available for a high number of taxa, can we rely on readily available single gene mitochondrial data? Here, we estimate the phylogeny of 66 cetacean taxa and 24 outgroups based on Cytb sequences. We judge the reliability of our phylogeny based on the recovery of several deep-level benchmark clades. A Bayesian phylogenetic analysis recovered all benchmark clades and for the first time supported Odontoceti monophyly based exclusively on analysis of a single mitochondrial gene. The results recover the monophyly of all but one family level taxa within Cetacea, and most recently proposed super- and subfamilies. In contrast, parsimony never recovered all benchmark clades and was sensitive to a priori weighting decisions. These results provide the most detailed phylogeny of Cetacea to date and highlight the utility of both Bayesian methodology in general, and of Cytb in cetacean phylogenetics. They furthermore suggest that dense taxon sampling, like dense character sampling, can overcome problems in phylogenetic reconstruction.     

The relative importance of body size and paleoclimatic change as explanatory variables influencing lineage diversification rate: an evolutionary analysis of bullhead catfishes (Siluriformes: Ictaluridae)

We applied Bayesian phylogenetics, divergence time estimation, diversification pattern analysis, and parsimony-based methods of ancestral state reconstruction to a combination of nucleotide sequences, maximum body sizes, fossils, and paleoclimate data to explore the influence of an extrinsic (climate change) and an intrinsic (maximum body size) factor on diversification rates in a North American clade of catfishes (Ictaluridae). We found diversification rate to have been significantly variable over time, with significant (or nearly significant) rate increases in the early history of Noturus. Though the latter coincided closely with a period of dramatic climate change at the Eocene-Oligocene boundary, we did not detect evidence for a general association between climate change and diversification rate during the entire history of Ictaluridae. Within Ictaluridae, small body size was found to be a near significant predictor of species richness. Morphological stasis of several species appears to be a consequence of a homoplastic increase in body size. We estimated the maximum standard length of the ictalurid ancestor to be approximately 50 cm, comparable to Eocene ictalurids (Astephus) and similar to modern sizes of Ameiurus and their Asian sister-taxon Cranoglanis. During the late Paleocene and early Eocene, the ictalurid ancestor diversified into the lineages represented by the modern epigean genera. The majority of modern species originated in the Oligocene and Miocene, most likely according to a peripheral isolates model of speciation. We discuss the difficulties of detecting macroevolutionary patterns within a lineage history and encourage the scrutiny of the terminal Eocene climatic event as a direct promoter of diversification.     

Phylogenetic utility of the major opsin in bees (Hymenoptera: Apoidea): a reassessment

Major opsin (LW Rh) DNA sequence has been reported to provide useful data for resolving phylogenetic relationships among tribes of corbiculate bees based on analyses of 502 bp of coding sequence. However, the corbiculate tribes are believed to be of Cretaceous age, and strong support for insect clades of this age from small data sets of nucleotide sequence data has rarely been demonstrated. To more critically assess opsin's phylogenetic utility we generated an expanded LW Rh data set by sequencing the same gene fragment from 52 additional bee species from 24 tribes and all six extant bee families. Analyses of this data set failed to provide substantial support for monophyly of corbiculate bees, for relationships among corbiculate tribes, or for most other well-established higher-level relationships among long-tongued bees. However, monophyly of nearly all genera and tribes is strongly supported, indicating that LW Rh provides useful phylogenetic signal at lower taxonomic levels. When our expanded LW Rh data set is combined with a morphological and behavioral data set for corbiculate bees, the results unambiguously support the traditional phylogeny of the corbiculate bee tribes: (Euglossini + (Bombini + (Meliponini + Apini))). This implies a single origin of advanced eusocial behavior among bees rather than dual origins, as proposed by several recent studies.     

Should phylogenetic models be trying to "fit an elephant"?

For the past two decades, there has been an ongoing debate within the plylogenetics community over whether model-based approaches for molecular systematics (such as maximum likelihood) should be preferred over the more traditional "maximum parsimony" approach. A recent simulation study by Kolaczkowski and Thornton has brought this debate into sharp focus. In this article, I discuss the significance of their findings and offer a prognosis on the implications for molecular phylogenetics. I believe that biochemistry and model selection have an important role in developing accurate phylogenetic approaches.     

Evolutionary relationships of the cup-fungus genus Peziza and Pezizaceae inferred from multiple nuclear genes: RPB2, beta-tubulin, and LSU rDNA

To provide a robust phylogeny of Pezizaceae, partial sequences from two nuclear protein-coding genes, RPB2 (encoding the second largest subunit of RNA polymerase II) and beta-tubulin, were obtained from 69 and 72 specimens, respectively, to analyze with nuclear ribosomal large subunit RNA gene sequences (LSU). The three-gene data set includes 32 species of Peziza, and 27 species from nine additional epigeous and six hypogeous (truffle) pezizaceous genera. Analyses of the combined LSU, RPB2, and beta-tubulin data set using parsimony, maximum likelihood, and Bayesian approaches identify 14 fine-scale lineages within Pezizaceae. Species of Peziza occur in eight of the lineages, spread among other genera of the family, confirming the non-monophyly of the genus. Although parsimony analyses of the three-gene data set produced a nearly completely resolved strict consensus tree, with increased confidence, relationships between the lineages are still resolved with mostly weak bootstrap support. Bayesian analyses of the three-gene data, however, show support for several more inclusive clades, mostly congruent with Bayesian analyses of RPB2. No strongly supported incongruence was found among phylogenies derived from the separate LSU, RPB2, and beta-tubulin data sets. The RPB2 region appeared to be the most informative single gene region based on resolution and clade support, and accounts for the greatest number of potentially parsimony informative characters within the combined data set, followed by the LSU and the beta-tubulin region. The results indicate that third codon positions in beta-tubulin are saturated, especially for sites that provide information about the deeper relationships. Nevertheless, almost all phylogenetic signal in beta-tubulin is due to third positions changes, with almost no signal in first and second codons, and contribute phylogenetic information at the "fine-scale" level within the Pezizaceae. The Pezizaceae is supported as monophyletic in analyses of the three-gene data set, but its sister-group relationships is not resolved with support. The results advocate the use of RPB2 as a marker for ascomycete phylogenetics at the inter-generic level, whereas the beta-tubulin gene appears less useful.     

The tempo of avian diversification: a comment on Johnson and Cicero

Johnson and Cicero (2004) claimed that inspection of a distribution of uncorrected mitochondrial DNA avian sister-taxon distances illustrated that the late Pleistocene was an important time for avian speciation. They believed this finding to be at odds with conclusions of Klicka and Zink (1997). However, both studies document recent speciation events. More germane to the discussion is what is meant by an "important" time for speciation, which we take to mean above some baseline diversification rate. We constructed a null distribution of sister-taxon distances based on a model of constant speciation and extinction rates. The empirical distribution of sister-taxon distances in Johnson and Cicero (2004) did not differ from the null model. Therefore, our analysis of Johnson and Cicero's data suggests that the late Pleistocene was no more important for avian speciation than any other time during this time period.     

Formation of germ cell cluster in tubuliferan thrips (Thysanoptera)

The ovarian structure and oogenesis in the larval stages of 2 tubuliferan species, Bactrothrips brevitubus (Idolothripinae) and Holothrips yuasai (Phlaeothripinae) of the Thysanoptera were examined using ultrathin serial sections, with special reference to the cluster formation of germ cells. No cells identifiable as stem cells were found in the ovarian rudiments of the 1st and 2nd-instar larvae. The clusters of oogonial cells were observed frequently in the 1st-instar, but scarcely in the 2nd-instar larvae: all the oogonial clusters observed were composed of 2 cells. In the 2nd-instar larvae, the ovarian region posterior to the germarium, or the vitellarium, contained both solitary and clustered oocytes. The oocyte clusters were composed of less than 5 cells. The oocytes, located in the posterior region of the vitellarium, were all solitary and at the previtellogenic stages.A protuberance was found in some solitary germ cells. The structure may represent a remnant of the intercellular bridge, previously formed between the germ cells. The number of oocytes composing a cluster is small but does not always fit the 2ⁿ-rule. One possible explanation is the accelerated detachment process of oocytes from a cluster. The cluster formation of germ cells has been confirmed in the Tubulifera as well as in the Terebrantia, and this phenomenon can be recognized as a general feature of the panoistic ovaries of the Thysanoptera.     

Reconstructing ordinal relationships in the Demospongiae using mitochondrial genomic data

Class Demospongiae (phylum Porifera) encompasses most of sponges' morphological and species diversity. It also represents one of the most challenging and understudied groups in animal phylogenetics, with many higher-level relationships still being unresolved. Among the unanswered questions are the most fundamental, including those about the monophyly of the Demospongiae and the relationships among the 14 recognized orders within the class. The lack of resolved phylogeny hampers progress in studies of demosponge biology, evolution and biodiversity and may interfere with the efficient conservation and economic use of this group. We addressed the question of demosponge relationships using mitochondrial genomic data. We assembled a mitochondrial genomic dataset comprising all orders of demosponges that includes 17 new and five previously published complete demosponge mitochondrial genomes. To test for the congruence between mtDNA-based and nuclear rRNA-based phylogenies, we also determined and analyzed 18S rRNA sequences for the same set of species. Our results provide strong support for five major clades within the Demospongiae: Homoscleromorpha=G0 (order Homosclerophorida), Keratosa=G1 (orders Dendroceratida, Dictyoceratida, and Verticillitida), Myxospongiae=G2 (orders Chondrosida, Halisarcida, and Verongida), marine Haplosclerida=G3 and the rest of demosponges=G4 (orders Agelasida, Astrophorida, Hadromerida, Halichondrida, Poecilosclerida, Spirophorida, and freshwater Haploscerida), and for the (G0((G1+G2)(G3+G4)) relationships among these clades. Conversely, mitochondrial genomic data do not support the monophylies of traditional subclasses Ceractinomorpha and Tetractinomorpha as well as several currently recognized orders of demosponges. Furthermore, we demonstrate that mitochondrial gene arrangements can also be informative for the inference of order-level demosponge relationships and propose a modified method for the analysis of gene order data that works well when translocation of tRNA genes are more frequent than other rearrangements.     

Performance of four ribosomal DNA regions to infer higher-level phylogenetic relationships of inoperculate euascomycetes (Leotiomyceta)

The inoperculate euascomycetes are filamentous fungi that form saprobic, parasitic, and symbiotic associations with a wide variety of animals, plants, cyanobacteria, and other fungi. The higher-level relationships of this economically important group have been unsettled for over 100 years. A data set of 55 species was assembled including sequence data from nuclear and mitochondrial small and large subunit rDNAs for each taxon; 83 new sequences were obtained for this study. Parsimony and Bayesian analyses were performed using the four-region data set and all 14 possible subpartitions of the data. The mitochondrial LSU rDNA was used for the first time in a higher-level phylogenetic study of ascomycetes and its use in concatenated analyses is supported. The classes that were recognized in Leotiomyceta (=inoperculate euascomycetes) in a classification by Eriksson and Winka [Myconet 1 (1997) 1] are strongly supported as monophyletic. The following classes formed strongly supported sister-groups: Arthoniomycetes and Dothideomycetes, Chaetothyriomycetes and Eurotiomycetes, and Leotiomycetes and Sordariomycetes. Nevertheless, the backbone of the euascomycete phylogeny remains poorly resolved. Bayesian posterior probabilities were always higher than maximum parsimony bootstrap values, but converged with an increase in gene partitions analyzed in concatenated analyses. Comparison of five recent higher-level phylogenetic studies in ascomycetes demonstrates a high degree of uncertainty in the relationships between classes.