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.     

Multigene‐based phylogeny of Urostylida (Ciliophora,Hypotrichia), with establishment of a novel family

The Urostylida is a major taxon of hypotrichs with many unresolved evolutionary relationships. Due to incomplete or inaccurate character states and a paucity of morphogenetic data, the phylogeny of several taxa within urostylids is unresolved. Molecular phylogeny studies based on single gene (SSU rDNA) data may lead to conflict between morphological classification and SSU rDNA tree. In this work, 20 new sequences (SSU rDNA, ITS1‐5.8S‐ITS2 and LSU rDNA) of five genera of urostylids are provided to further investigate the phylogenetic relationships of this group. The main findings are as follows: (i) the establishment of Hemicycliostylidae, a novel family presently including Hemicycliostyla and Australothrix, is supported by both single gene and concatenated phylogenies; (ii) all molecular data support the exclusion of Eschaneustyla from the family Epiclintidae; (iii) Australothrix, Bergeriella and Thigmokeronopsis are distinctly separated in all gene trees although they share the character that each posterior streak generates the ventral row together with the midventral pair; (iv) compared with closely related genera in all trees, that is Metaurostylopsis and Apourostylopsis, Neourostylopsis is characterized by having more than three frontal cirri arranged in distinct or indistinct corona rather than the length of the midventral complex; (v) Hemicycliostyla and Pseudourostyla, two morphologically similar genera, do not form a monophyletic group in all molecular trees, suggesting that the bicorona, multiple marginal cirral rows and high numbers of dorsal kineties may result from convergent evolution; (vi) species of Bakuella fall into three separate clades in all trees suggesting that this genus needs to be split.     

Molecular and morphological characterization of a poorly known marine ciliate, Myoschiston duplicatum precht 1935: implications for phylogenetic relationships between three morphologically similar genera -- Zoothamnium, Myoschiston, and Zoothamnopsis (Ciliophora, Peritrichia, Zoothamniidae)

We studied the morphology and molecular phylogeny of Myoschiston duplicatum, a peritrich ciliate that has been recorded as an epibiont of crustaceans, but which we also identified on marine algae from Korea. The important morphological characteristics revealed by silver staining of Myoschiston species have not been described because they are rarely collected. Using morphological methods, we redescribed the type species of the genus, Myoschiston duplicatum, and provided an improved diagnosis of Myoschiston. In addition, the coding regions for nuclear small subunit (SSU) rRNA and internal transcribed spacer 1‐5.8S‐internal transcribed spacer 2 sequences were sequenced. Phylogenetic analyses that included available SSU rDNA sequences of peritrichs from GenBank strongly supported a position of M. duplicatum within the family Zoothamniidae. In addition, phylogenetic analyses were performed with single datasets (ITS1‐5.8S‐ITS2) and combined datasets (SSU rDNA + ITS1‐5.8S‐ITS2) to explore further the phylogenetic relationship in the family Zoothamniidae between the three morphologically similar genera—Zoothamnium, Myoschiston, and Zoothamnopsis.     

Combined phylogenetic analysis of the subclass Marchantiidae (Marchantiophyta): towards a robustly diagnosed classification

The most extensive combined phylogenetic analyses of the subclass Marchantiidae yet undertaken was conducted on the basis of morphological and molecular data. The morphological data comprised 126 characters and 56 species. Taxonomic sampling included 35 ingroup species with all genera and orders of Marchantiidae sampled, and 21 outgroup species with two genera of Blasiidae (Marchantiopsida), 15 species of Jungermanniopsida (the three subclasses represented) and the three genera of Haplomitriopsida. Takakia ceratophylla (Bryophyta) was employed to root the trees. Character sampling involved 92 gametophytic and 34 sporophytic traits, supplemented with ten continuous characters. Molecular data included 11 molecular markers: one nuclear ribosomal (26S), three mitochondrial genes (nad1, nad5, rps3) and seven chloroplast regions (atpB, psbT‐psbH, rbcL, ITS, rpoC1, rps4, psbA). Searches were performed under extended implied weighting, weighting the character blocks against the average homoplasy. Clade stability was assessed across three additional weighting schemes (implied weighting corrected for missing entries, standard implied weighting and equal weighting) in three datasets (molecular, morphological and combined). The contribution from different biological phases regarding node recovery and diagnosis was evaluated. Our results agree with many of the previous studies but cast doubt on some relationships, mainly at the family and interfamily level. The combined analyses underlined the fact that, by combining data, taxonomic enhancements could be achieved regarding taxon delimitation and quality of diagnosis. Support values for many clades of previous molecular studies were improved by the addition of morphological data. The long‐held assumption that morphology may render spurious or low‐quality results in this taxonomic group is challenged. The morphological trends previously proposed are re‐evaluated in light of the new phylogenetic scheme.     

Phylogenetic analysis of the genus Hexachlamys (Myrtaceae) based on plastid and nuclear DNA sequences and their taxonomic implications

Myrtaceae are one of the most species‐rich families of flowering plants in the Neotropics. They include several complex genera and species; Hexachlamys is one of the complex genera. It has not been recognized as a distinct genus and has been included in Eugenia, based on morphological grounds. Therefore, molecular systematic studies may be useful to understand and to help to solve these relationships. Here, we performed a molecular phylogenetic analysis using plastid and nuclear data in order to check the inclusion of Hexachlamys in Eugenia. Plastid (accD, rpoB, rpoC1, trnH‐psbA) and nuclear (ITS2) sequence data were analysed using Bayesian and maximum parsimony methods. The trees constructed using ITS2 and trnH‐psbA were the best able to resolve the relationships between species and genera, revealing the non‐monophyly of Hexachlamys. The molecular phylogenetic analyses were in agreement with previous morphological revisions that have included Hexachlamys in Eugenia. These results reinforce the importance of uniting knowledge and strategies to understand better issues of delimitation of genera and species in groups of plants with taxonomic problems. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 532–543.     

Evolution of the thaumastocheliform lobsters (Crustacea,Decapoda, Nephropidae)

Deep‐sea lobsters previously assigned to the family Thaumastochelidae Bate, 1888, the thaumastocheliforms, have very distinctive, greatly unequal first chelipeds, with the right side extremely elongate and pectinate, and in having short, quadrate pleonal pleura. Despite interesting morphology and a long taxonomic history, the phylogeny of the group has received little detailed analysis. Here, we conduct a species‐level phylogenetic analysis of the thaumastocheliforms based on morphological and molecular data (three mitochondrial genes: COI, 16S rDNA and 12S rDNA; two nuclear protein‐coding genes: H3 and NaK) to robustly reconstruct their evolutionary history and estimate divergence times. Separate and combined analyses of all data sources support thaumastocheliform monophyly, but as a clade deeply nested within the Nephropidae supporting recent synonymy of Thaumastochelidae with Nephropidae. Combined and molecular‐only analyses support generic monophyly of all three thaumastocheliform genera and Dinochelus as sister to Thaumastochelopsis, fully corroborating the current, morphology‐based taxonomy. In contrast, Thaumastocheles is recovered as paraphyletic in morphology‐only analyses owing to minimal character support. The Cretaceous–Paleogene Oncopareia was recovered as a stem‐lineage thaumastocheliform. The fossil record indicates that the thaumastocheliforms once lived in shallow, continental shelf depths, but moved into deeper water in the Cenozoic where they occur today. The thaumastocheliforms originated in northern Europe during the Mid‐Late Cretaceous and later dispersed westwards to the south‐eastern Pacific through the western Atlantic and eastwards to the western Pacific through the Indian Ocean. Thaumastochelopsis can be considered the most derived thaumastocheliform genus based on the degree of structural reduction relative to other thaumastocheliforms, its remote geographical occurrence (Australia) from the hypothesised place of origin (northern Europe) and its more recent estimated divergence than other genera (28 Mya for the MRCA of extant species of the genus).     

Phylogeny of cetrarioid lichens (Parmeliaceae) inferred from ITS and b-tubulin sequences,morphology, anatomy and secondary chemistry

Phylogenetic relationships within the family Parmeliaceae (lichenized ascomycetes) with emphasis on the heterogeneous group of cetrarioid lichens are reconstructed. The results are based on cladistic analyses of DNA-sequences, morphological and chemical data. Almost all currently recognized cetrarioid genera were included in the analyses together with parmelioid and alectorioid members of the presumably monophyletic family Parmeliaceae. We tried to sample taxonomic diversity of the family as widely as possible. The ITS1-5.8S-ITS2 region of the rDNA and a partial β-tubulin gene from 126 samples representing 82 species were analysed. Cetrarioid lichens were identified as a monophyletic group, supported by both ITS and β-tubulin characters. This group was reanalysed using 47 morphological, anatomical and secondary chemistry characters combined with the DNA data matrix. ITS and β-tubulin sequences provide congruent information, and a clear correlation between DNA-data and conidial shape is observed. The current taxonomy of the cetrarioid lichens is discussed and compared with the phylogenetic trees obtained here. A comprehensive study of the phylogeography of some bipolar or subcosmopolitic species with representatives from both hemispheres was performed. Cetraria aculeata is the only taxon where correlation between DNA-data and geographic origin is observed.     

PHYLOGENY OF THE HYDRODICTYACEAE (CHLOROPHYCEAE): INFERENCES FROM rDNA DATA1

The hydrodictyacean green algal lineage has been the focus of much research due to the fossil record of at least some members, their ornamented cell walls, and their distinctive reproductive strategies. The phylogeny of the family was, until recently, exclusively morphology based. This investigation examines hydrodictyacean isolates from several culture collections, focusing on sequences from ribosomal data: 18S rDNA, 26S rDNA (partial), and internal transcribed spacer (ITS)‐2 data. Results from phylogenetic analyses of independent and combined data matrices support the Hydrodictyaceae as a monophyletic lineage that includes isolates of Chlorotetraedron, Hydrodictyon, Pediastrum, Sorastrum, and Tetraedron. Phylogenetic analyses of rDNA data indicate that the three‐dimensional coenobium of Hydrodictyon is evolutionarily distinct from the three‐dimensional coenobium of Sorastrum. The more robust aspects of the ITS‐2 data corroborate the 18S+26S rDNA topology and provide a structural autapomorphy for the Hydrodictyaceae and Neochloridaceae, that is, an abridgment of helix IV in the secondary structure. The rDNA data do not support monophyly of Pediastrum but rather suggest the existence of four additional hydrodictyacean genera: Monactinus, Parapediastrum, Pseudopediastrum, and Stauridium.     

Ultrastructure and molecular phylogenetic position of a new marine sand-dwelling dinoflagellate from British Columbia,Canada: Pseudadenoides polypyrenoides sp. nov. (Dinophyceae)

Two monospecific genera of marine benthic dinoflagellates, Adenoides and Pseudadenoides, have unusual thecal tabulation patterns (lack of cingular plates in the former; and no precingular plates and a complete posterior intercalary plate series in the latter) and are thus difficult to place within a phylogenetic framework. Although both genera share morphological similarities, they have not formed sister taxa in previous molecular phylogenetic analyses. We discovered and characterized a new species of Pseudadenoides, P. polypyrenoides sp. nov., at both the ultrastructural and molecular phylogenetic levels. Molecular phylogenetic analyses of SSU and LSU rDNA sequences demonstrated a close relationship between P. polypyrenoides sp. nov. and Pseudadenoides kofoidii, and Adenoides and Pseudadenoides formed sister taxa in phylogenetic trees inferred from LSU rDNA sequences. Comparisons of morphological traits, such as the apical pore complex (APC), demonstrated similarities between Adenoides, Pseudadenoides and several planktonic genera (e.g. Heterocapsa, Azadinium and Amphidoma). Molecular phylogenetic analyses of SSU and LSU rDNA sequences also demonstrated an undescribed species within Adenoides.     

Three novel species of Stemphylium from Sinkiang, China: their morphological and molecular characterization

Three new species of Stemphylium were isolated from diseased leaves of Luffa cylindrica, Lycium chinense and Cucumis melo growing in the Sinkiang province of Northwest China. Stemphylium luffae, S. lycii and S. cucumis are described by morphological and molecular phylogenetic analyses. The principal morphological characteristics of these three species are congruent with those of Stemphylium. They are distinguished from morphologically similar Stemphylium species based on characteristics of their conidia and conidiophores. In order to establish the molecular phylogenetic status of these species, DNA loci including the internal transcribed spacer (ITS) nuclear rDNA region and the glyceraldehyde-3-phosphate dehydrogenase (gpd) genes were amplified and sequenced. Phylogenetic analyses using Maximum-Parsimony (MP) and Neighbor-Joining (NJ) were performed on the combined DNA sequences of ITS and gpd gene regions. Both morphological observations and molecular analyses support S. luffae, S. lycii and S. cucumis as three new taxa.     

New Insights in the Systematics of the Phyllophoraceae (Gigartinales,Rhodophyta)

The Phyllophoraceae, consisting of about 100 species worldwide, stands out in exhibiting a wide spectrum of unique life history types that makes it unusually interesting for assessing the phylogenetic importance of reproductive traits relative to classification criteria. Type of life history and position of the reproductive structures on the thallus have traditionally formed the basis for separating 11 genera in the Phyllophoraceae; however, phylogenetic analyses inferred from three sets of DNA sequences [chloroplast‐encoded rbcL, nuclear large‐subunit ribosomal RNA gene (LSU rDNA), and internal transcribed spacer regions (ITS) of nuclear ribosomal DNA], instead indicate a lack of correlation between type of life history and phylogenetic relationships among the established taxa. This lack of correlation dramatically challenges all of the traditional taxonomy, and we will present a revised classification for the family that downplays life‐history features. The study will answer the question which morphological features can be used as meaningful indicators of phylogenetic relationships in the Phyllophoraceae. The results will also be addressed in light of global biogeographic hypotheses for the family.     

Phylogenic Diversity and Taxonomic Problems of the Dictyosphaerium Morphotype within the Parachlorella Clade (Chlorellaceae,Trebouxiophyceae)

The Parachlorella clade was put forward as a group within the family Chlorellaceae in 2004. Recent molecular analyses have revealed that Dictyosphaerium morphotype algae form several independent lineages within the Parachlorella clade, and new genera and species have been established. In this study, we focus on the diversity of Dictyosphaerium morphotype algae within the Parachlorella clade, based on 42 strains from China. We used combined analyses of morphology and molecular data based on SSU and internal transcribed spacer region (ITS) rDNA sequences to characterize these algae. In addition, the secondary structure of ITS2 was compared to delineate new lineages. Our results revealed high phylogenic diversity of Dictyosphaerium morphotype algae, and we describe five distinct lineages. We examined the morphological features of these five lineages, and morphological differences are difficult to find compared with other Dictyosphaerium morphotype algae. The five distinct lineages were not described as new genera currently. We lastly discuss the taxonomic problems regarding the Dictyosphaerium morphotype within the Parachlorella clade, and possible solutions are considered.     

Phylogeny of Impatiens (Balsaminaceae): integrating molecular and morphological evidence into a new classification

Impatiens L. is one of the largest angiosperm genera, containing over 1000 species, and is notorious for its taxonomic difficulty. Here, we present, to our knowledge, the most comprehensive phylogenetic analysis of the genus to date based on a total evidence approach. Forty‐six morphological characters, mainly obtained from our own investigations, are combined with sequence data from three genetic regions, including nuclear ribosomal ITS and plastid atpB‐rbcL and trnL‐F. We include 150 Impatiens species representing all clades recovered by previous phylogenetic analyses as well as three outgroups. Maximum‐parsimony and Bayesian inference methods were used to infer phylogenetic relationships. Our analyses concur with previous studies, but in most cases provide stronger support. Impatiens splits into two major clades. For the first time, we report that species with three‐colpate pollen and four carpels form a monophyletic group (clade I). Within clade II, seven well‐supported subclades are recognized. Within this phylogenetic framework, character evolution is reconstructed, and diagnostic morphological characters for different clades and subclades are identified and discussed. Based on both morphological and molecular evidence, a new classification outline is presented, in which Impatiens is divided into two subgenera, subgen. Clavicarpa and subgen. Impatiens; the latter is further subdivided into seven sections.     

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.     

Phylogenetic position and delimitation of the moss family Plagiotheciaceae in the order Hypnales

The phylogenetic position and generic composition of the moss family Plagiotheciaceae were explored using DNA sequence data from three genomes: plastid trnL‐F and rps4, mitochondrial nad5 intron and nuclear ITS1‐5.8S‐ITS2. Our phylogenetic analyses included 35 terminals from Plagiotheciaceae and 71 outgroup taxa from a representative set of hypnalean moss families. The family Plagiotheciaceae is resolved in the early‐diverging Hypnales grade, together with Fontinalaceae, Habrodontaceae and several genera which are mainly distributed in the area of the former Gondwanan supercontinent. However, monophyly of the family can only be attained if the three Southern Hemisphere genera, Acrocladium, Catagonium and Rhizofabronia, are excluded. Ancestral state reconstruction for eight morphological characters reveals that many characters used to delimit the family, such as a lack of pseudoparaphyllia and rhizoids inserted in the leaf axils, were already present in the ancestor of Hypnales. Dispersal–vicariance analysis suggests that Plagiotheciaceae and Fontinalaceae have their ancestral distributions in the area of the former Laurasian supercontinent. As the analyses also reveal a Gondwanan distribution for the ancestor of Hypnales in general, Plagiotheciaceae and Fontinalaceae represent the first diverging Laurasian lineages in the order. © 2013 The Linnean Society of London     

Are Prorocentrum hoffmannianum and Prorocentrum belizeanum (DINOPHYCEAE,PROROCENTRALES), the same species? An integration of morphological and molecular data

The taxonomic assignment of Prorocentrum species is based on morphological characteristics; however, morphological variability has been found for several taxa isolated from different geographical regions. In this study, we evaluated species boundaries of Prorocentrum hoffmannianum and Prorocentrum belizeanum based on morphological and molecular data. A detailed morphological analysis was done, concentrating on the periflagellar architecture. Molecular analyses were performed on partial Small Sub‐Unit (SSU) rDNA, partial Large Sub‐Unit (LSU) rDNA, complete Internal Transcribed Spacer Regions (ITS1‐5.8S‐ITS2), and partial cytochrome b (cob) sequences. We concatenated the SSU‐ITS‐LSU fragments and constructed a phylogenetic tree using Bayesian Inference (BI) and maximum likelihood (ML) methods. Morphological analyses indicated that the main characters, such as cell size and number of depressions per valve, normally used to distinguish P. hoffmannianum from P. belizeanum, overlapped. No clear differences were found in the periflagellar area architecture. Prorocentrum hoffmannianum and P. belizeanum were a highly supported monophyletic clade separated into three subclades, which broadly corresponded to the sample collection regions. Subtle morphological overlaps found in cell shape, size, and ornamentation lead us to conclude that P. hoffmanianum and P. belizeanum might be considered conspecific. The molecular data analyses did not separate P. hoffmannianum and P. belizeanum into two morphospecies, and thus, we considered them to be the P. hoffmannianum species complex because their clades are separated by their geographic origin. These geographic and genetically distinct clades could be referred to as ribotypes: (A) Belize, (B) Florida‐Cuba, (C1) India, and (C2) Australia.