A molecular phylogenetic assessment of the advanced Asiatic and Malesian didymocarpoid Gesneriaceae with focus on non-monophyletic and monotypic genera

Based on a considerably enlarged sampling, a phylogenetic analysis of the largest group of didymocarpoid Gesneriaceae, the ??advanced Asiatic and Malesian genera??, was performed, covering all but 3 of the 60 genera presently recognised in this group (20 of these, mostly from China, are monotypic). The results suggest that no fewer than 17 out of the 57 genera examined are poly- (or rarely para-)phyletic. Highly polyphyletic are Briggsia, Chirita, Henckelia and Raphiocarpus. Only a dozen of the non-monotypic genera (including the three species-richest genera, Cyrtandra, Aeschynanthus and Agalmyla) are confirmed as monophyletic entities, though some exhibit considerable genetic variation. For eight genera, no statement can be made, as only one (of two or several) species was included in the analysis. For a dozen of the (particularly Chinese) monotypic genera a close relationship (or possible congenerity) with other genera was found. In China, only Allostigma, Cathayanthe, Conandron and Metapetrocosmea seem to have no strong affinities to other genera, indicating that they represent phylogenetically isolated lineages or represent remnants of previously larger and earlier diversified groups. The present study forms the foundation for targeted molecular, morphological and phytogeographic studies of the polyphyletic and monotypic genera and particular of clades of genera with interrelations uncovered here for the first time.     

Phylogenetic reconstruction of character development in Physciaceae

Abstract:A cladistic analysis of the Physciaceae, based on morphological and chemical data, is presented. In the resulting phylogenetic reconstruction two major clades are formed, one containing the foliose genera (Anaptychia, Dirinaria, Heterodermia, Hyperphyscia, Physcia, Phaeophyscia,Physconia , Pyxine) and the fruticose Tornabea, and the other containing the remaining, mainly crustose genera. Rinodina appears as paraphyletic with representatives both at the base of the tree, at the same level as the two major clades and at the base of the crustose clade. Also Mobergia has a basal position. The characters used and their distribution in the phylogenetic trees are discussed as well as their significance for the identification of monophyletic groups. The history of the family is also briefly hinted at and characters of importance for the recognition of new genera are surveyed. Relevant publications and the variation in secondary chemistry are presented in tables.     

Publication of descriptions of novel bacterial taxa in <Emphasis Type="Italic">Antonie van Leeuwenhoek</Emphasis>

The species from the order Neisseriales are currently distinguished from other bacteria on the basis of branching in 16S rRNA gene trees. For this order containing a single family, Neisseriaceae, no distinctive molecular, biochemical, or phenotypic characters are presently known. We report here detailed phylogenetic and comparative analyses on the 27 genome sequenced species of the order Neisseriales. Our comparative genomic analyses have identified 54 conserved signature indels (CSIs) in widely distributed proteins that are specific for either all of the sequenced Neisseriales species or a number of clades within this order that are also supported by phylogenetic analyses. Of these CSIs, 11 are specifically present in all of the sequenced species from this order, but are not found in homologous proteins from any other bacteria. These CSIs provide novel molecular markers specific for, and delimiting, this order. Twenty-one CSIs in diverse proteins are specific for a group comprised of the genera Neisseria, Eikenella, Kingella, and Simonsiella (Clade I), which are obligate host-associated organisms, lacking flagella and exhibiting varied morphology. The species from these genera also formed a strongly supported clade in phylogenetic trees based upon concatenated protein sequences; a monophyletic grouping of these genera and other genera displaying similar morphological characteristics was also observed in the 16S rRNA gene tree. A second clade (Clade II), supported by seven of the identified CSIs and phylogenetic trees based upon concatenated protein sequences, grouped together species from the genera Chromobacterium, Laribacter, and Pseudogulbenkiania that are rod-shaped bacteria, which display flagella-based motility and are capable of free living. The remainder of the CSIs were uniquely shared by smaller groups within these two main clades. Our analyses also provide novel insights into the evolutionary history of the Neisseriales and suggest that the CSIs that are specific for the Clade I species may play an important role in the evolution of obligate host-association within this order. On the basis of phylogenetic analysis, the identified CSIs, and conserved phenotypic characteristics of different Neisseriales genera, we propose a division of this order into two families: an emended family Neisseriaceae (corresponding to Clade I) containing the genera Alysiella, Bergeriella, Conchiformibius, Eikenella, Kingella, Neisseria, Simonsiella, Stenoxybacter, Uruburuella and Vitreoscilla and a new family, Chromobacteriaceae fam. nov., harboring the remainder of the genera from this order (viz. Andreprevotia, Aquaspirillum, Aquitalea, Chitinibacter, Chitinilyticum, Chitiniphilus, Chromobacterium, Deefgea, Formivibrio, Gulbenkiania, Iodobacter, Jeongeupia, Laribacter, Leeia, Microvirgula, Paludibacterium, Pseudogulbenkiania, Silvimonas, and Vogesella).     

Phylogenetic Classification at Generic Level in the Absence of Distinct Phylogenetic Patterns of Phenotypical Variation: A Case Study in Graphidaceae (Ascomycota)

Molecular phylogenies often reveal that taxa circumscribed by phenotypical characters are not monophyletic. While re-examination of phenotypical characters often identifies the presence of characters characterizing clades, there is a growing number of studies that fail to identify diagnostic characters, especially in organismal groups lacking complex morphologies. Taxonomists then can either merge the groups or split taxa into smaller entities. Due to the nature of binomial nomenclature, this decision is of special importance at the generic level. Here we propose a new approach to choose among classification alternatives using a combination of morphology-based phylogenetic binning and a multiresponse permutation procedure to test for morphological differences among clades. We illustrate the use of this method in the tribe Thelotremateae focusing on the genus Chapsa, a group of lichenized fungi in which our phylogenetic estimate is in conflict with traditional classification and the morphological and chemical characters do not show a clear phylogenetic pattern. We generated 75 new DNA sequences of mitochondrial SSU rDNA, nuclear LSU rDNA and the protein-coding RPB2. This data set was used to infer phylogenetic estimates using maximum likelihood and Bayesian approaches. The genus Chapsa was found to be polyphyletic, forming four well-supported clades, three of which clustering into one unsupported clade, and the other, supported clade forming two supported subclades. While these clades cannot be readily separated morphologically, the combined binning/multiresponse permutation procedure showed that accepting the four clades as different genera each reflects the phenotypical pattern significantly better than accepting two genera (or five genera if splitting the first clade). Another species within the Thelotremateae, Thelotrema petractoides, a unique taxon with carbonized excipulum resembling Schizotrema, was shown to fall outside Thelotrema. Consequently, the new genera Astrochapsa, Crutarndina, Pseudochapsa, and Pseudotopeliopsis are described here and 39 new combinations are proposed.     

Placing the origin of two species-rich genera in the late cretaceous with later species divergence in the tertiary: a phylogenetic, biogeographic and molecular dating analysis of Piper and Peperomia (Piperaceae)

Nearly all of the species diversity in Piperaceae is encompassed within Piper and Peperomia. Both genera are pan-tropical with areas of diversification in the Neotropics and Southeast Asia. Piperaceae are less diverse in Africa with only two native species of Piper. This study examines the distribution of both Piper and Peperomia with representative samples from the Neotropics, Asia, Pacific Islands, and Africa. Molecular dating is used to place an age for the crown clades of Piper and Peperomia as well as ages for diversification within the clades. Both genera have origins in the late Cretaceous, but species level diversification occurred much later in the Tertiary. Biogeography of both genera are correlated with paleoclimate evidence to better explain the distribution and diversification of these large genera.     

Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily

To infer the phylogenetic relationships of Xylaria species associated with termite nests within the genus Xylaria and among genera of the subfamily Xylarioideae, β-tubulin, RPB2, and α-actin sequences of 131 cultures of 114 species from Xylaria and 11 other genera of the subfamily were analyzed. These 11 genera included Astrocystis, Amphirosellinia, Discoxylaria, Entoleuca, Euepixylon, Kretzschmaria, Nemania, Podosordaria, Poronia, Rosellinia, and Stilbohypoxylon. We showed that Xylaria species were distributed among three major clades, TE, HY, and PO, with clade TE—an equivalent of the subgenus Pseudoxylaria—encompassing exclusively those species associated with termite nests and the other two clades containing those associated with substrates other than termite nests. Xylaria appears to be a paraphyletic genus, with most of the 11 genera submerged within it. Podosordaria and Poronia, which formed a distinct clade, apparently diverged from Xylaria and the other genera early. Species of Entoleuca, Euepixylon, Nemania, and Rosellinia constituted clade NR, a major clade sister to clade PO, while those of Kretzschmaria were inserted within clade HY and those of Astrocystis, Amphirosellinia, Discoxylaria, and Stilbohypoxylon were within clade PO.     

Molecular systematics, character evolution, and pollen morphology of Cistus and Halimium (Cistaceae)

Pollen analysis and parsimony-based phylogenetic analyses of the genera Cistus and Halimium, two Mediterranean shrubs typical of Mediterranean vegetation, were undertaken, on the basis of cpDNA sequence data from the trnL-trnF, and trnS-trnG regions, to evaluate limits between the genera. Neither of the two genera examined formed a monophyletic group. Several monophyletic clades were recognized for the ingroup. (1) The ??white and whitish pink Cistus??, where most of the Cistus sections were present, with very diverse pollen ornamentations ranging from striato-reticulate to largely reticulate, sometimes with supratectal elements; (2) The ??purple pink Cistus?? clade grouping all the species with purple pink flowers belonging to the Macrostylia and Cistus sections, with rugulate or microreticulate pollen. Within this clade, the pink-flowered endemic Canarian species formed a monophyletic group, but with weak support. (3) Three Halimium clades were recovered, each with 100% bootstrap support; all Halimium species had striato-reticulate pollen. Two Halimium clades were characterized by yellow flowers, and the other by white flowers.     

Morphological evolution and ecological diversification of the forest-dwelling poppies (Papaveraceae: Chelidonioideae) as deduced from a molecular phylogeny of the ITS region

Sequences of the ITS region of nrDNA were analyzed for the seven genera of Papaveraceae subf. Chelidonioideae s.str. Three major clades can be recognized. These are 1.Chelidonium/Hylomecon/Stylophorum, 2.Eomecon/Sanguinaria, and 3.Bocconia/Macleaya. The monophyly of genera in the first of these three clades is doubtful, and clades two and three are sister to each other. Use of the ITS phylogeny of the subfamily to trace its morphological and ecological evolution shows that morphological change is concentrated in theBocconia/Macleaya clade, and probably related to the evolution of wind-pollination from insect-pollination in these two genera after habitat shift.     

Paraholcoglossum and Tsiorchis, two new orchid genera established by molecular and morphological analyses of the Holcoglossum alliance

Background

Holcoglossum is a small orchid genus of 12 species ranging from SW China to Thailand and NE India. Although molecular and morphological analyses have been performed to establish the phylogenetic relationships within this genus, the interspecific relations and its relations with allied genera, such as Rhynchostylis, Aerides and Vanda, remain unclear.

Methodology/Principal Findings

In addition to morphological analysis, maximum parsimony, maximum likelihood, and Bayesian inference analyses were performed based on fragments of the nuclear ITS and chloroplast trnL-F and matK genes of 31 taxa (15 Holcoglossum, 14 Aeridinae, 2 outgroups) representing all major clades of the Holcoglossum alliance. The results suggest that Holcoglossum is triphyletic, comprising three clades: the Holcoglossum clade, its sister clade, and a distant clade more closely related to Rhynchostylis, Aerides, and Vanda than to the Holcoglossum clade. The Holcoglossum clade is further divided into three subclades; the genetic distances between these three subclades also support this delimitation. The molecular conclusion is consistent with their distinct morphological characters.

Conclusions

We propose that the latter two clades comprise two new genera, Paraholcoglossum and Tsiorchis, and Holcoglossum clade divides into three sections. In addition, a new section, Holcoglossum sect. Nujiangensia, and a new species, Holcoglossum linearifolium, are proposed. Some new combinations are made, and a new scheme is provided for the classification of all species of Holcoglossum, Paraholcoglossum, and Tsiorchis.     

Molecular Systematics of Threatened Seed Plant Species Endemic in the Caribbean Islands

A review of available Caribbean Island red-lists species (CR and EN categories based on the IUCN guidelines from 2001, and E category established according to the IUCN guidelines from 1980) is presented. A database of over 1,300 endemic species that are either Critically Endangered or Endangered sensu IUCN was created. There are molecular systematic studies available for 112 of them. Six of these species (in six genera) are the only members of early divergent lineages that are sister to groups composed of a large number of clades. Seven of the species (in seven genera) belong to clades that have a small number of taxa but are sister to species/genus-rich clades. Ten of the species (in six genera) are sister to taxa restricted to South America or nested in clades endemic to this region. Fifty-seven of the species (in 35 genera) are sister to Caribbean Island endemic species. Erigeron belliastroides, an Endangered (EN) Cuban endemic, is sister to the Galapagos genus Darwiniothamnus. The phylogenetic placement of four of the threatened species resulted in changes in their taxonomic placement; they belong to polyphyletic or paraphyletic genera.     

Molecular genetic divergence of the centric diatom Cyclotella and Discostella (Bacillariophyceae) revealed by nuclear ribosomal DNA comparisons

The centric diatom Cyclotella, including the recently separated Discostella, is commonly present in freshwater and several species are important bio-indicators. Here, we describe molecular characteristics of the nuclear rDNA, spanning 18S to D1/D2 region of the 28S rDNA, of two genera Cyclotella and Discostella, particularly using Korean isolates of C. meneghiniana, Discostella sp. c.f. D. pseudostelligera. Molecular and morphological analyses showed that our isolates had nearly identical genotypes in rDNA and similar morphology as compared to presumably the same species from other geographical areas. Phylogenetic analyses of individual 18S and partial 28S rDNAs of Cyclotella sensu lato showed that all sequences were separated into two clades: one containing Cyclotella, the other Discostella including C. ocellata and C. bodanica. Statistical tests with pairwise genetic distance scores showed that the two genera were significantly different (one-factor ANOVA, p?<?0.01). In addition, divergence in the partial 28S rDNA was significantly high (p?<?0.01) as compared to 18S rDNA. This provides evidence that the two genera, Cyclotella and Discostella, belong to genetically well-separated groups. In addition, 28S rDNAs is a more suitable molecular marker for the discrimination of Cyclotella sensu lato.     

Phylogeny and classification of Ranunculales: Evidence from four molecular loci and morphological data

Previous phylogenetic analyses of Ranunculales, which have mostly been focused on an individual family and were based on molecular data alone, have recovered three main clades within the order. However, support for relationships among these three clades was weak. Earlier hypotheses were often hampered by limited taxon sampling; to date less than one-tenth of the genera in the order have been sampled. In this study, we used a greatly enlarged taxon sampling (105 species, representing 99 genera of all seven families in the order). Our study is, furthermore, the first to employ morphology (65 characters) in combination with sequence data from four genomic regions, including plastid rbcL, matK and trnL-F, and nuclear ribosomal 26S rDNA to reconstruct phylogenetic relationships within Ranunculales. Maximum parsimony and Bayesian inference were performed on the individual and combined data sets. Our analyses concur with those of previous studies, but in most cases provide stronger support and better resolution for relationships among the three main clades retrieved. The first, comprised solely of the monogeneric family Eupteleaceae, is the earliest-diverging lineage. The second clade is composed exclusively of taxa of Papaveraceae, which is sister to the third clade, the core Ranunculales, comprising the other five families of the order. Circaeasteraceae and Lardizabalaceae form a strongly supported clade. Pteridophyllum is supported as sister to Hypecoum, contradicting the viewpoint that the former is the earliest-diverging genus in Papaveraceae. Glaucidium is basalmost in Ranunculaceae. Within this phylogenetic framework, the evolution of selected characters is inferred and diagnostic morphological characters at different taxonomic levels are identified and discussed. Based on both morphological and molecular evidence, a classification outline for Ranunculales is presented, including the proposal of two new subfamilies, Menispermoideae and Tinosporoideae in Menispermaceae and a new tribe, Callianthemeae, for the genus Callianthemum (Ranunculaceae).     

The molecular phylogeny of aquatic hyphomycetes with affinity to the Leotiomycetes

Aquatic hyphomycetes play a key role in decomposition of submerged organic matter and stream ecosystem functioning. We examined the phylogenetic relationships among various genera of aquatic hyphomycetes belonging to the Leotiomycetes (Ascomycota) using sequences of internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA generated from 42 pure cultures including 19 ex-types. These new sequence data were analyzed together with additional sequences from 36 aquatic hyphomycetes and 60 related fungi obtained from GenBank. Aquatic hyphomycetes, characterized by their tetraradiate or sigmoid conidia, were scattered in nine supported clades within the Helotiales (Leotiomycetes). Tricladium, Lemonniera, Articulospora, Anguillospora, Varicosporium, Filosporella, and Flagellospora are not monophyletic, with species from the same genus distributed among several major clades. The Gyoerffyella clade and the Hymenoscyphus clade accommodated species from eight and six different genera, respectively. Thirteen aquatic hyphomycete taxa were grouped in the Leotia-Bulgaria clade while twelve species clustered within the Hymenoscyphus clade along with several amphibious ascomycetes. Species of Filosporella and some species from four other aquatic genera were placed in the Ascocoryne-Hydrocina clade. It is evident that many aquatic hyphomycetes have relatives of terrestrial origin. Adaptation to colonize the aquatic environment has evolved independently in multiple phylogenetic lineages within the Leotiomycetes.     

Resurrection and emendation of the Hypoxylaceae,recognised from a multigene phylogeny of the Xylariales

A multigene phylogeny was constructed, including a significant number of representative species of the main lineages in the Xylariaceae and four DNA loci the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, the second largest subunit of the RNA polymerase II (RPB2), and beta-tubulin (TUB2). Specimens were selected based on more than a decade of intensive morphological and chemotaxonomic work, and cautious taxon sampling was performed to cover the major lineages of the Xylariaceae; however, with emphasis on hypoxyloid species. The comprehensive phylogenetic analysis revealed a clear-cut segregation of the Xylariaceae into several major clades, which was well in accordance with previously established morphological and chemotaxonomic concepts. One of these clades contained Annulohypoxylon, Hypoxylon, Daldinia, and other related genera that have stromatal pigments and a nodulisporium-like anamorph. They are accommodated in the family Hypoxylaceae, which is resurrected and emended. Representatives of genera with a nodulisporium-like anamorph and bipartite stromata, lacking stromatal pigments (i.e. Biscogniauxia, Camillea, and Obolarina) appeared in a clade basal to the xylarioid taxa. As they clustered with Graphostroma platystomum, they are accommodated in the Graphostromataceae. The new genus Jackrogersella with J. multiformis as type species is segregated from Annulohypoxylon. The genus Pyrenopolyporus is resurrected for Hypoxylon polyporus and allied species. The genus Daldinia and its allies Entonaema, Rhopalostroma, Ruwenzoria, and Thamnomyces appeared in two separate subclades, which may warrant further splitting of Daldinia in the future, and even Hypoxylon was divided in several clades. However, more species of these genera need to be studied before a conclusive taxonomic rearrangement can be envisaged. Epitypes were designated for several important species in which living cultures and molecular data are available, in order to stabilise the taxonomy of the Xylariales.     

Phylogenetic relationships of Cranichidinae and Prescottiinae (Orchidaceae, Cranichideae) inferred from plastid and nuclear DNA sequences

Background and Aims

Phylogenetic relationships of subtribes Cranichidinae and Prescottiinae, two diverse groups of neotropical terrestrial orchids, are not satisfactorily understood. A previous molecular phylogenetic study supported monophyly for Cranichidinae, but Prescottiinae consisted of two clades not sister to one another. However, that analysis included only 11 species and eight genera of these subtribes. Here, plastid and nuclear DNA sequences are analysed for an enlarged sample of genera and species of Cranichidinae and Prescottiinae with the aim of clarifying their relationships, evaluating the phylogenetic position of the monospecific genera Exalaria, Ocampoa and Pseudocranichis and examining the value of various structural traits as taxonomic markers.

Methods

Approx. 6000 bp of nucleotide sequences from nuclear ribosomal (ITS) and plastid DNA (rbcL, matK-trnK and trnL-trnF) were analysed with cladistic parsimony and Bayesian inference for 45 species/14 genera of Cranichidinae and Prescottiinae (plus suitable outgroups). The utility of flower orientation, thickenings of velamen cell walls, hamular viscidium and pseudolabellum to mark clades recovered by the molecular analysis was assessed by tracing these characters on the molecular trees.

Key Results

Spiranthinae, Cranichidinae, paraphyletic Prescottia (with Pseudocranichis embedded), and a group of mainly Andean ‘prescottioid’ genera (the ‘Stenoptera clade’) were strongly supported. Relationships among these clades were unresolved by parsimony but the Bayesian tree provided moderately strong support for the resolution (Spiranthinae–(Stenoptera clade-(Prescottia/Pseudocranichis–Cranichidinae))). Three of the four structural characters mark clades on the molecular trees, but the possession of a pseudolabellum is variable in the polyphyletic Ponthieva.

Conclusions

No evidence was found for monophyly of Prescottiinae and the reinstatement of Cranichidinae s.l. (including the genera of ‘Prescottiinae’) is favoured. Cranichidinae s.l. are diagnosed by non-resupinate flowers. Lack of support from parsimony for relationships among the major clades of core spiranthids is suggestive of a rapid morphological radiation or a slow rate of molecular evolution.Key words: Cranichideae, Cranichidinae, matK-trnK, molecular phylogenetics, nrITS, Orchidaceae, Prescottiinae, resupination, trnL-trnF     

A New Orchid Genus,Danxiaorchis, and Phylogenetic Analysis of the Tribe Calypsoeae

Background

Orchids have numerous species, and their speciation rates are presumed to be exceptionally high, suggesting that orchids are continuously and actively evolving. The wide diversity of orchids has attracted the interest of evolutionary biologists. In this study, a new orchid was discovered on Danxia Mountain in Guangdong, China. However, the phylogenetic clarification of this new orchid requires further molecular, morphological, and phytogeographic analyses.

Methodology/Principal Findings

A new orchid possesses a labellum with a large Y-shaped callus and two sacs at the base, and cylindrical, fleshy seeds, which make it distinct from all known orchid genera. Phylogenetic methods were applied to a matrix of morphological and molecular characters based on the fragments of the nuclear internal transcribed spacer, chloroplast matK, and rbcL genes of Orchidaceae (74 genera) and Calypsoeae (13 genera). The strict consensus Bayesian inference phylogram strongly supports the division of the Calypsoeae alliance (not including Dactylostalix and Ephippianthus) into seven clades with 11 genera. The sequence data of each species and the morphological characters of each genus were combined into a single dataset. The inferred Bayesian phylogram supports the division of the 13 genera of Calypsoeae into four clades with 13 subclades (genera). Based on the results of our phylogenetic analyses, Calypsoeae, under which the new orchid is classified, represents an independent lineage in the Epidendroideae subfamily.

Conclusions

Analyses of the combined datasets using Bayesian methods revealed strong evidence that Calypsoeae is a monophyletic tribe consisting of eight well-supported clades with 13 subclades (genera), which are all in agreement with the phytogeography of Calypsoeae. The Danxia orchid represents an independent lineage under the tribe Calypsoeae of the subfamily Epidendroideae. This lineage should be treated as a new genus, which we have named Danxiaorchis, that is parallel to Yoania. Both genera are placed under the subtribe Yoaniinae.