Additional data for nuclear DNA give new insights into the phylogenetic position of Sorex granarius within the Sorex araneus group

Many species contain genetic lineages that are phylogenetically intermixed with those of other species. In the Sorex araneus group, previous results based on mtDNA and Y chromosome sequence data showed an incongruent position of Sorex granarius within this group. In this study, we explored the relationship between species within the S. araneus group, aiming to resolve the particular position of S. granarius. In this context, we sequenced a total of 2447 base pairs (bp) of X-linked and nuclear genes from 47 individuals of the S. araneus group. The same taxa were also analyzed within a Bayesian framework with nine autosomal microsatellites. These analyses revealed that all markers apart from mtDNA showed similar patterns, suggesting that the problematic position of S. granarius is best explained by an incongruent behavior by mtDNA. Given their close phylogenetic relationship and their close geographic distribution, the most likely explanation for this pattern is past mtDNA introgression from S. araneus race Carlit to S. granarius.     

Additional insights into phylogenetic relationships of the Class Ophiuroidea (Echinodermata) from rRNA gene sequences

Ophiuroids are important benthic marine invertebrates with an unstable taxonomic history. Recent phylogenetic and morphological investigations have promoted major departures from established taxonomy. As such, additional insights into evolutionary relationships of ophiuroids are valuable. We analyzed ribosomal sequence data from a mitochondrial gene (16S rRNA) and a nuclear gene (18S rRNA) from 39 ophiuroids representing 14 of the 18 currently accepted families. Main findings from our study include support for a polyphyletic Ophiomyxidae, paraphyletic Amphiuridae, monophyletic Euryalida, a sister relationship between the Ophiactidae and Ophiotrichidae, and a clade comprised of the Amphiuridae and Amphilepididae. Relationships within the families Gorgonocephalidae, Ophiuridae, Ophiodermatidae and Ophiomyxidae are discussed.     

Exploring the potential of small RNA subunit and ITS sequences for resolving phylogenetic relationships within the phylum Ctenophora

Ctenophores are a phylum of non-bilaterian marine (mostly planktonic) animals, characterised by several unique synapomorphies (e.g., comb rows, apical organ). Relationships between and within the nine recognised ctenophore orders are far from understood, notably due to a paucity of phylogenetically informative anatomical characters. Previous attempts to address ctenophore phylogeny using molecular data (18S rRNA) led to poorly resolved trees but demonstrated the paraphyly of the order Cydippida. Here we compiled an updated 18S rRNA data set, notably including a few newly sequenced species representing previously unsampled families (Lampeidae, Euryhamphaeidae), and we constructed an additional more rapidly evolving ITS1 + 5.8S rRNA + ITS2 alignment. These data sets were analysed separately and in combination under a probabilistic framework, using different methods (maximum likelihood, Bayesian inference) and models (e.g., doublet model to accommodate secondary structure; data partitioning). An important lesson from our exploration of these datasets is that the fast-evolving internal transcribed spacer (ITS) regions are useful markers for reconstructing high-level relationships within ctenophores. Our results confirm the paraphyly of the order Cydippida (and thus a “cydippid-like” ctenophore common ancestor) and suggest that the family Mertensiidae could be the sister group of all other ctenophores. The family Lampeidae (also part of the former “Cydippida”) is probably the sister group of the order Platyctenida (benthic ctenophores). The order Beroida might not be monophyletic, due to the position of Beroe abyssicola outside of a clade grouping the other Beroe species and members of the “Cydippida” family Haeckeliidae. Many relationships (e.g. between Pleurobrachiidae, Beroida, Cestida, Lobata, Thalassocalycida) remain unresolved. Future progress in understanding ctenophore phylogeny will come from the use of additional rapidly evolving markers and improvement of taxonomic sampling.     

New insights into relationships between active and dormant organisms,phylogenetic diversity and ecosystem productivity

Marine microbes make up a key part of ocean food webs and drive ocean chemistry through a range of metabolic processes. A fundamental question in ecology is whether the diversity of organisms in a community shapes the ecological functions of that community. While there is substantial evidence to support a positive link between diversity and ecological productivity for macro‐organisms in terrestrial environments, this relationship has not previously been verified for marine microbial communities. One factor complicating the understanding of this relationship is that many marine microbes are dormant and are easily dispersed by ocean currents, making it difficult to ensure that the organisms found in a given environmental sample accurately reflect processes occurring in that environment. Another complication is that, due to microbes great range of genotypic and phenotypic variability, communities with distantly related species may have greater range of metabolic functions than communities have the same richness and evenness, but in which the species present are more closely related to each other. In this issue of Molecular Ecology, Galand et al. (2015) provide compelling evidence that the most metabolically active communities are those in which the nondormant portion of the microbial community has the highest phylogenetic diversity. They also illustrate that focusing on the active portion of the community allows for detection of temporal patterns in community structure that would not be otherwise evident. The authors’ point out that the presence of many dormant organisms that do not contribute to ecosystem functioning is a feature that makes microbial ecosystems fundamentally different from macro‐ecosystems and that this difference needs to be accounted for in microbial ecology theory.     

Evolutionary relationships within the protostome phylum Sipuncula: a molecular analysis of ribosomal genes and histone H3 sequence data

The phylogenetic relationships of the members of the phylum Sipuncula are investigated by means of DNA sequence data from three nuclear markers, two ribosomal genes (18S rRNA and the D3 expansion fragment of 28S rRNA), and one protein-coding gene, histone H3. Phylogenetic analysis via direct optimization of DNA sequence data using parsimony as optimality criterion is executed for 12 combinations of parameter sets accounting for different indel costs and transversion/transition cost ratios in a sensitivity analysis framework. Alternative outgroup analyses are also performed to test whether they affected rooting of the sipunculan topology. Nodal support is measured by parsimony jackknifing and Bremer support values. Results from the different partitions are highly congruent, and the combined analysis for the parameter set that minimizes overall incongruence supports monophyly of Sipuncula, but nonmonophyly of several higher taxa recognized for the phylum. Mostly responsible for this is the split of the family Sipunculidae in three main lineages, with the genus Sipunculus being the sister group to the remaining sipunculans, the genus Phascolopsis nesting within the Golfingiiformes, and the genus Siphonosoma being associated to the Phascolosomatidea. Other interesting results are the position of Phascolion within Golfingiidae and the position of Antillesoma within Aspidosiphonidae. These results are not affected by the loci selected or by the outgroup chosen. The position of Apionsoma is discussed, although more data would be needed to better ascertain its phylogenetic affinities. Monophyly of the genera with multiple representatives (Themiste, Aspidosiphon, and Phascolosoma) is well supported, but not the monophyly of the genera Nephasoma or Golfingia. Interesting phylogeographic questions arise from analysis of multiple representatives of a few species.     

Two Urosoma species (Ciliophora,Hypotrichia): A multidisciplinary approach provides new insights into their ultrastructure and systematics

The general morphology and ultrastructure of two soil hypotrichous ciliates, Urosoma emarginata and U. salmastra, were investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. Phylogenetic analyses, based on the newly sequenced small subunit ribosomal (SSU) rRNA genes, were conducted on three U. emarginata populations and one U. salmastra population. Our findings support for the validity of Perilemmaphora Berger, 2008, a rankless taxon comprising spirotrich ciliates having a perilemma. The cortical granules of both species are extrusomes representing a new type of mucocyst in U. emarginata and possibly a new type of pigmentocyst in U. salmastra. Additionally, the lithosomes were revealed as subglobose structures composed of a low electron-dense, homogeneous inner part and an electron-dense outer part. The ultrastructural features of the cortical granules, together with ontogenetic and molecular phylogenetic data, suggest that the genus Urosoma might need to be divided. It is posited that ultrastructural features of hypotrichous ciliates in general may have important taxonomic value warranting further investigation.     

Reconstruction of phylogenetic relationships within Grapsidae (Crustacea: Brachyura) and comparison of trans-isthmian versus amphi-atlantic gene flow based on mtDNA

Following taxonomic revisions in recent years, the originally large family Grapsidae MacLeay, 1838 has become a relatively small and morphologically homogeneous family in terms of adult and larval morphology. Most available molecular studies including more than one genus of the family have also suggested monophyly of the corresponding taxa. However, no single phylogenetic study has ever included all constituent genera of the Grapsidae. In the current study, a molecular phylogeny based on sequences of the mitochondrial 16S rRNA gene from all eight grapsid genera and 34 species is presented and suggests that up to four genera are not monophyletic. This is mainly due to the polyphyletic nature of the genus Pachygrapsus which can be found in six different lineages of the phylogeny, suggesting that the genus currently does not represent a single evolutionary lineage and is in need of taxonomic revision. Amphi-atlantic and trans-isthmian species pairs or populations in four genera are compared and reveal relatively constant and pronounced divergences across the Panama Isthmus as opposed to moderate divergences across the Atlantic Ocean, thereby suggesting occurrence of gene flow across the Atlantic Ocean during the past three million years.     

Comparative analysis of the complete mitochondrial genomes of three rockfishes (Scorpaeniformes,Sebastiscus) and insights into the phylogenetic relationships of Sebastidae

Mitochondrial genome is a powerful molecule marker to provide information for phylogenetic relationships and revealing molecular evolution in ichthyological studies. Sebastiscus species, a marine rockfish, are of essential economic value. However, the taxonomic status and phylogenetic relationships of Sebastidae have been controversial so far. Here, the mitochondrial genomes (mitogenomes) of three species, S. tertius, S. albofasciatus, and S. marmoratus, were systemically investigated. The lengths of the mitogenomes’ sequences of S. tertius, S. albofasciatus, and S. marmoratus were 16910, 17056, and 17580 bp, respectively. It contained 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNA (tRNA) genes, and one identical control region (D-loop) among the three species. The genetic distance and K_a/K_s ratio analyses indicated 13 PCGs were suffering purifying selection and the selection pressures were different from certain deep-sea fishes, which were most likely due to the difference in their living environment. The phylogenetic tree was constructed by Bayesian Inference (BI) and Maximum Likelihood (ML). Most interestingly, the results indicated that Sebastidae and Scorpaenidae were grouped into a separate branch, so the taxonomic status of Sebastidae should be classified into subfamily Sebastinae. Our results may lead to a taxonomic revision of Scorpaenoidei.     

Ultrastructure and phylogenetic placement within Heterolobosea of the previously unclassified, extremely halophilic heterotrophic flagellate Pleurostomum flabellatum (Ruinen 1938)

Although Pleurostomum was described almost a century ago, flagellates assigned to this taxon have been recorded only in very occasional faunistic studies of highly saline habitats, and their phylogenetic position has remained uncertain. We report the cultivation, ultrastructure, and phylogenetic relationships of Pleurostomum flabellatum isolated from a Korean saltern pond of 313 per thousand salinity. This isolate is biflagellated with a cytostomal groove, and is not distinguishable from previous accounts of P. flabellatum from saturated brines in India and Australia. Pleurostomum flabellatum shows ultrastructural features characteristic of many Heterolobosea: (1) a striated rhizoplast, (2) an absence of stacked Golgi bodies, (3) parallel basal bodies and flagella, and (4) a large number of peripheral microtubules supporting a rostrum. 18S rRNA gene phylogenies strongly confirm the affinities of P. flabellatum within Heterolobosea. Furthermore, the 18S rRNA gene of P. flabellatum has the heterolobosean-specific helix 17_1, and a group I intron in the same position as in Acrasis rosea. Within Heterolobosea, the 'amoeboflagellate' genera Naegleria and Willaertia were its closest relatives with high bootstrap support and posterior probability. P. flabellatum was observed only as a flagellate, and never as an amoeba. Since light microscopy and electron microscopy observations indicate that P. flabellatum flagellates are capable both of feeding and division, there might be no amoeba stage. Being morphologically distinct from its closest relatives and phylogenetically distant from other flagellate-only Heterolobosea, P. flabellatum cannot be moved into any previously described heterolobosean genus. Instead, we move Pleurostomum into Heterolobosea, and assign as the type species Pleurostomum salinum Namyslowski 1913, a species that closely resembles P. flabellatum. The optimal temperature for growth of P. flabellatum is 40 degrees C. Interestingly, P. flabellatum grows optimally at 300 per thousand salinity and fails to grow below 200 per thousand salinity, indicating that it is an 'extreme halophile'. The optimal salinity for growth is the highest for any eukaryote examined to date.     

Estimation of phylogenetic relationships within the Ascomycota on the basis of 18S rDNA sequences and chemotaxonomy

Small subunit rRNA gene sequences (18S rDNA), cell wall carbohydrate composition and ubiquinone components were analysed within a larger number of ascomycetous yeasts and dimorphic fungi to validate their congruence in predicting phylogenetic relationships. The glucose-mannose pattern distinguishes the Hemiascomycetes from the Euascomycetes and the Protomycetes which are characterised with the glucose-mannose-galactose-rhamnose-(fucose) profile. The glucose-mannose-galactose pattern was found in the cell walls of all the three classes. Different coenzyme Q component (CoQ5 to CoQ10) were found within the representatives of the Hemiascomycetes. Whereas CoQ9, CoQ10 and CoQ10H2 predominate within the Euascomycetes, CoQ9 and CoQ10 characterise the Protomycetes. Chemotaxonomic studies coupled with additional molecular and co-evolution studies support the idea that the Hemiascomycetes occupy a basal position in the phylogeny of Ascomycota. These results are not in line with the phylogenetic studies based on the sequences of 18S rRNA encoding gene. The maximum parsimony analysis indicated that Hemiascomycetes and Protomycetes might represent sister groups, opposing to the earlier reported results, where the Archiascomycetes (Protomycetes) or the Hemiascomycetes had been considered to be the most primitive ascomycetous fungi. Instead of the class Archiascomycetes, the term Protomycetes was introduced reflecting much better the properties of the whole class.     

Phylogenetic relationships of Serpulidae (Annelida: Polychaeta) based on 18S rDNA sequence data, and implications for opercular evolution

Phylogenetic relationships of (19) serpulid taxa (including Spirorbinae) were reconstructed based on 18S rRNA gene sequence data. Maximum likelihood, Bayesian inference, and maximum parsimony methods were used in phylogenetic reconstruction. Regardless of the method used, monophyly of Serpulidae is confirmed and four monophyletic, well-supported major clades are recovered: the Spirorbinae and three groups hitherto referred to as the Protula-, Serpula-, and Pomatoceros-group. Contrary to the taxonomic literature and the hypothesis of opercular evolution, the Protula-clade contains non-operculate (Protula, Salmacina) and operculate taxa both with pinnulate and non-pinnulate peduncle (Filograna vs. Vermiliopsis), and most likely is the sister group to Spirorbinae. Operculate Serpulinae and poorly or non-operculate Filograninae are paraphyletic. It is likely that lack of opercula in some serpulid genera is not a plesiomorphic character state, but reflects a special adaptation.     

The phylogenetic position of the Aeolosomatidae and Parergodrilidae, two enigmatic oligochaete-like taxa of the 'Polychaeta', based on molecular data from 18S rDNA sequences

The Aeolosomatidae and the Parergodrilidae are meiofaunal Annelida showing different combinations of clitellate‐like and non‐clitellate character states. Their phylogenetic positions and their systematic status within the Annelida are still in debate. Here we attempt to infer their systematic position using 18S rDNA sequences of the aeolosomatid Aeolosoma sp. and the parergodrilid Stygocapitella subterranea and several other meiofaunal taxa such as the Dinophilidae, Polygordiidae and Saccocirridae. The data matrix was complemented by sequences from several annelid, arthropod and molluscan species. After evaluation of the phylogenetic signal the data set was analysed with maximum‐parsimony, distance and maximum‐likelihood algorithms. Sequences from selected arthropods or molluscs were chosen for outgroup comparison. The resolution of the resulting phylogenies is discussed in comparison to previous studies. The results do not unequivocally support a sister‐group relationship of Aeolosoma sp. and the Clitellata. Instead, depending on the algorithms applied, Aeolosoma clusters in various clades within the polychaetes, for instance, together with eunicidan species, the Dinophilidae, Harmothoë impar or Nereis limbata. The position of Aeolosoma sp. thus cannot be resolved on the basis of the data available. S. subterranea always falls close to a cluster comprising Scoloplos armiger, Questa paucibranchiata and Magelona mirabilis, all of which were resolved as not closely related to both Aeolosoma sp. and the Clitellata. Therefore, convergent evolution of clitellate‐like characters in S. subterranea and hence in the Parergodrilidae is suggested by our phylogenetic analysis. Moreover, the Clitellata form a monophyletic clade within the paraphyletic polychaetes.     

New contributions to the taxonomy of urostylid ciliates (Ciliophora,Hypotrichia), with establishment of a new genus and new species

Hypotrichia, one of the most complex and highly differentiated groups in Ciliophora, has been the object of extensive studies, especially in recent years. Nevertheless, methodological difficulties and insufficient faunistic studies have limited our understanding of their biodiversity and phylogeny. In this study, one novel urostylid ciliate, Pseudoholosticha zhaoi nov. gen., nov. spec. and two populations of Anteholosticha monilata (Kahl, 1928) Berger, 2003, type species of the latter genus, are studied using an integrative approach (live observation, protargol impregnation, scanning electron microscopy, and phylogenetic analysis) to provide further insights into the diversity, classification, and phylogeny of this group of ciliates. Pseudoholosticha nov. gen. can be separated from other morphologically similar genera mainly by the absence of buccal and caudal cirri. A key to 12 morphologically similar genera and illustrations of their cirral patterns are provided. The validation of the new genus and new species is supported by both morphological and phylogenetic analyses. The first 18S rRNA gene sequence of A. monilata, with detailed morphological data, provided a reliable clarification of A. monilata populations and corroborated the phylogenetic position of the type species of the polyphyletic genus Anteholosticha.     

九种绢丝昆虫线粒体12S rRNA基因的序列特征和系统发育分析

为探讨鳞翅目中绢丝昆虫之间的系统发育关系和分子进化特征,本研究测定了中国柞蚕Antheraea pernyi野生型和放养型的线粒体12S rRNA基因的部分序列,结合来自GenBank数据库的17条序列,对总共9种绢丝昆虫（2科3属）的12S rRNA基因序列进行了分析。利用软件MEGA 3.1进行碱基组成、变异位点的统计和分子进化分析,分别用类平均聚类法（UPGMA）、邻接法（NJ）、最小进化法（ME）、最大简约法（MP）重建系统发生树。测定的中国柞蚕野生型的12S rRNA基因序列（427 bp）与放养型“豫早1号”的序列完全一致。序列对齐后共鉴定80个变异位点,50个简约信息位点。碱基组成分析显示在科属间具有明显差异,AT含量蚕蛾科高于大蚕蛾科;在A和T碱基的使用上,大蚕蛾科偏好使用T,而蚕蛾科则偏好使用A。与动物中常见的以转换为主的碱基替换模式不同,所分析的9种昆虫中除桑蚕属内部为转换与颠换基本一致外,其余物种间均是颠换多于转换。进化分析支持柞蚕属、樗蚕属和桑蚕属的单系。基于UPGMA法的进化树支持琥珀蚕是柞蚕属的较原始类型,而NJ、ME和MP法则支持印度柞蚕是较原始的类型,因此,柞蚕属种间的进化关系尚需进一步研究。     

New aspects of an old discussion - phylogenetic relationships of Ammotragus and Pseudois within the subfamily Caprinae based on comparison of the 12S rDNA sequences

A mitochondrial 12S rDNA fragment was amplified by PCR and directly sequenced from eight members of the Caprinae ( Ammotragus lervia, Capra aegagrus, Ovis ammon, O. musimon, O. nivicola, O. orientalis, O. vignei and Pseudois nayaur ). Multiple alignment of the sequences and genetic distance calculation with the'distance of Taijama and Nei and the algorithms of Saitou and Nei (neighbour-joining - NJ) showed that Pseudois is grouped together with Capra and Ammotragus . All members of the genus Oxis formed a second cluster. Phylogenetic analyses supported these results. NJ-analyses and maximum parsimony (MP) trees did not support the present phylogenetic classification. Bootstrap values of 95% (NJ) and 96% (MP) confirms the grouping of P. nayaur and C. aegagrus . Another clade, O. ammon and O. orientalis supported weakly by bootstrap values of 65% (MP) and 70% (NJ) and the clade O. musimonand O. vignei had bootstrap values of 50% (MP) and 60% (NJ). So the phylogenetic relationship within the genus Oxis is still unsafe. But the parsimony analyses lead to the conclusion that Ammotragus represents an old lineage that diverged from ancestral caprines together with true sheep a long time ago and that both true goats and blue sheep forms represent one evolutionary lineage.     

Relationship of the genusCordyceps and related genera, based on parsimony and spectral analysis of partial 18S and 28S ribosomal gene sequences

A molecular phylogenetic study of selected species of three sub-genera of the genusCordyceps was undertaken, along with representatives of the generaAkanthomyces, Aschersonia, Gibellula, Hymenostilbe, Hypocrella, Nomuraea andTorrubiella, to examine their inter-relationship. Phylogenetic analyses of the data indicated that the Clavicipitaceae form a monophyletic group within the Hypocreales, while the monophyly ofCordyceps was not supported. Four clades were identified:Cor. militaris/Cor. pseudomilitaris; Cor. iranginesis/Cor. sphecocephala; Cor. intermedia/Cor. capitata; andCor. cylindrica/Nom. atypicola. The sub-genusNeocordyceps was shown to be monophyletic while the sub-generaEucordyceps andOphiocordyceps do not form monophyletic groups. The genusHypocrella appeared monophyletic, and radiated after the formation of the generaCordyceps, andTorrubiella. Akanthomyces arachnophilus andGi. pulchra, anamorphs ofTorrubiella species, formed a distinct clade that was separate from one formed by the scale insect pathogens,To. luteorostrata andPaecilomyces cinnamomeus, suggesting that this genus may be polyphyletic.     

大头蛙和脆皮大头蛙线粒体3个基因的测定及两栖类亲缘关系研究

现存的两栖类系统发生关系一直存在争议,特别是3个目间的亲缘关系。本文设计了5对引物,扩增和测定了大头蛙和脆皮大头蛙线粒体12S和16S rRNA基因和Cytb基因的全序列。在对所测序列进行分析的同时,基于3个基因全序列的相加数据,运用MEGA 3.1和PHYLIP 3.64软件中的NJ法、MP法和ML法,对两爬类17个物种,以鱼类非洲肺鱼为外群,重建出3个树形完全一致的分子系统树。研究结果显示:现存两栖类中无尾目和有尾目为姐妹群关系,并推断有尾目内小鲵科和隐鳃鲵科亲缘关系较近。此外,在研究两栖类系统发生关系方面,作者分析前人研究中产生两种不同观点的可能原因,同时总结了在此类研究中产生偏差的几种影响因素。     

脂肪酶产生菌Aspergillus sp. FS132产酶条件的初步优化及其18S rRNA基因序列的比较分析

从新疆火焰山口土样中分离的一株脂肪酶产生菌FS132,菌落形态表明为霉菌。对其产酶条件的初步优化表明,产酶的最适培养时间为45h,培养基最适初始pH 7.0,最适培养温度36℃,最适摇瓶空气量25mL/250mL锥形瓶。为进一步鉴定该菌株,克隆测定了该菌18S rRNA基因序列。并对其进行系统进化树分析,结果表明该菌与已报道的Aspergillus tamarii具有最紧密亲缘关系。     

脂肪酶产生菌Aspergillus sp.FS132产酶条件的初步优化及其18S rRNA基因序列的比较分析

从新疆火焰山口土样中分离的一株脂肪酶产生菌FS132,菌落形态表明为霉菌。对其产酶条件的初步优化表明,产酶的最适培养时间为45h,培养基最适初始pH7．0,最适培养温度36℃,最适摇瓶空气量25mL／250mL锥形瓶。为进一步鉴定该菌株,克隆测定了该菌18SrRNA基因序列。并对其进行系统进化树分析,结果表明该菌与已报道的Aspergillus tamarii具有最紧密亲缘关系。     

Morphological and molecular characterization of the name-bearing type species Rimaleptus binucleatus (Kahl, 1931), with a phylogenetic re-analysis of dileptid evolutionary history (Ciliophora: Litostomatea: Rhynchostomatia)

We collected Rimaleptus binucleatus from soil in the surroundings of the town of Ulsan, South Korea. Its morphology and 18S rRNA gene were studied using standard methods. This possibly widely distributed species is characterized by: (i) a size of about 170–400 × 20–65 μm; (ii) a narrowly to cylindrically dileptid body with proboscis occupying about 30–65% of body length; (iii) two dorsal contractile vacuoles; (iv) two size groups of rod-shaped extrusomes; and (v) about 18–29 ciliary rows, 4–6 of them anteriorly differentiated into a staggered dorsal brush. Phylogenetic analyses of five new rhynchostomatian 18S rRNA gene sequences supported monophylies of the orders Tracheliida and Dileptida, but revealed that the genera Rimaleptus and Pseudomonilicaryon are polyphyletic. Monophyly of genera with two macronuclear nodules was consistently rejected, but monophylies of dileptids with many scattered macronuclear nodules and of dileptids with moniliform macronucleus and multi-rowed dorsal brush could not be excluded by statistical topology tests. Nevertheless, phylogenetic network analyses indicated considerable conflict in the phylogenetic signal provided by the 18S rRNA gene to resolve unambiguously relationships among dileptid genera.