Molecular phylogeny of gastrotricha based on 18S rRNA genes comparison: rejection of hypothesis of relatedness with nematodes

Gastrotrichs are meiobenthic free-living aquatic worms whose phylogenetic and intra-group relationships remain unclear despite some attempts to resolve them on the base of morphology or molecules. In this study we analysed complete sequences of the 18S rRNA gene of 15 taxa (8 new and 7 published) to test numerous hypotheses on gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data could be due to the short region available for analysis and the poor taxa sampling. Data were analysed using both maximum likelihood and Bayesian inference. Results obtained suggest that gastrotrichs, together with Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea and Lophotrochozoa, comprise a clade Spiralia. Statistical tests reject phylogenetic hypotheses regarding Gastrotricha as close relatives of Nematoda and other Ecdysozoa or placing them at the base of bilaterian tree close to acoels and nemertodermatides. Within Gastrotricha, Chaetonotida and Macrodasyida comprise two well supported clades. Our analysis confirmed the monophyly of the Chaetonotidae and Xenotrichulidae within Chaetonida as well as Turbanellidae and Thaumastodermatidae within Macrodasyida. Mesodasys is a sister group of the Turbanellidae, and Lepidodasyidae appears to be a polyphyletic group as Cephalodasys forms a separate lineage at the base of macrodasyids, whereas Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To infer a more reliable Gastrotricha phylogeny many species and additional genes should be involved in future analyses.     

Phylogeny of Gastrotricha: a morphology-based framework of gastrotrich relationships

Currently, the phylum Gastrotricha is divided into the orders Macrodasyida and Chaetonotida, with the structure of the myoepithelial pharynx being an important distinguishing feature. Macrodasyida currently has six recognized families, and Chaetonotida comprises seven families. However, within-group relationships are poorly understood. To arrive at a better understanding of gastrotrich systematics and phylogeny, we performed the first cladistic analysis of nearly all known gastrotrich genera using 71 morphological characters. Results suggest that the Gastrotricha is a monophyletic group (supported by 82% of bootstrap replications) with its most primitive taxa distributed among the families Dactylopodolidae and Neodasyidae. Monophyly of Macrodasyida and Chaetonotida was supported by 90% and 52% bootstrap replications, respectively. Within the Macrodasyida, the families Dactylopodolidae, Turbanellidae, Macrodasyidae, and Thaumastodermatidae all formed monophyletic clades. The families Planodasyidae and Lepidodasyidae were paraphyletic. Among the Chaetonotida, the marine family Xenotrichulidae was monophyletic, supported by 51% of bootstrap replications. A second clade containing all freshwater families was supported by 62% bootstrap values. However, Chaetonotidae were paraphyletic. Using this analysis as a framework, we now can explore possible patterns of evolution within it, and arrive at a consensus of the gastrotrich ground pattern. Moreover, in future molecular studies of metazoan phylogeny, we will be able to select gastrotrich species that are more appropriate representatives of the phylum.     

Phylogeny of Thaumastodermatidae (Gastrotricha: Macrodasyida) inferred from nuclear and mitochondrial sequence data

Background

Phylogenetic relationships within Gastrotricha are poorly known. Attempts to shed light on this subject using morphological traits have led to hypotheses lacking satisfactory statistical support; it seemed therefore that a different approach was needed.

Methodology/Principal Findings

In this paper we attempt to elucidate the relationships within the taxonomically vast family Thaumastodermatidae (Macrodasyida) using molecular sequence data. The study includes representatives of all the extant genera of the family and for the first time uses a multi-gene approach to infer evolutionary liaisons within Gastrotricha. The final data set comprises sequences of three genes (18S, 28S rDNA and COI mtDNA) from 41 species, including 29 thaumastodermatids, 11 non-thaumastodermatid macrodasyidans and a single chaetonotidan. Molecular data was analyzed as a combined set of 3 genes and as individual genes, using Bayesian and maximum likelihood approaches. Two different outgroups were used: Xenotrichula intermedia (Chaetonotida) and members of the putative basal Dactylopodola (Macrodasyida). Thaumastodermatidae and all other sampled macrodasyidan families were found monophyletic except for Cephalodasyidae. Within Thaumastodermatidae Diplodasyinae and Thaumastodermatinae are monophyletic and so are most genera. Oregodasys turns out to be the most basal group within Thaumastodermatinae in analyses of the concatenated data set as well as in analyses of the nuclear genes. Thaumastoderma appears as the sister taxon to the remaining species. Surprisingly, Tetranchyroderma is non-monophyletic in our analyses as one group of species clusters with Ptychostomella while another appears as the sister group of Pseudostomella.

Conclusions/Significance

Results in general agree with the current classification; however, a revision of the more derived thaumastodermatid taxa seems necessary. We also found that the ostensible COI sequences from several species do not conform to the general invertebrate or any other published mitochondrial genetic code; they may be mitochondrially derived nuclear genes (numts), or one or more modifications of the mitochondrial genetic code within Gastrotricha.     

Gastrotricha: a marine sister for a freshwater puzzle

Background

Within an evolutionary framework of Gastrotricha Marinellina flagellata and Redudasys fornerise bear special interest, as they are the only Macrodasyida that inhabit freshwater ecosystems. Notwithstanding, these rare animals are poorly known; found only once (Austria and Brazil), they are currently systematised as incertae sedis. Here we report on the rediscovery of Redudasys fornerise, provide an account on morphological novelties and present a hypothesis on its phylogenetic relationship based on molecular data.

Methodology/Principal Findings

Specimens were surveyed using DIC microscopy and SEM, and used to obtain the 18 S rRNA gene sequence; molecular data was analyzed cladistically in conjunction with data from 42 additional species belonging to the near complete Macrodasyida taxonomic spectrum. Morphological analysis, while providing new information on taxonomically relevant traits (adhesive tubes, protonephridia and sensorial bristles), failed to detect elements of the male system, thus stressing the parthenogenetic nature of the Brazilian species. Phylogenetic analysis, carried out with ML, MP and Bayesian approaches, yielded topologies with strong nodal support and highly congruent with each other. Among the supported groups is the previously undocumented clade showing the alliance between Redudasys fornerise and Dactylopodola agadasys; other strongly sustained clades include the densely sampled families Thaumastodermatidae and Turbanellidae and most genera.

Conclusions/Significance

A reconsideration of the morphological traits of Dactylopodola agadasys in light of the new information on Redudasys fornerise makes the alliance between these two taxa very likely. As a result, we create Anandrodasys gen. nov. to contain members of the previously described D. agadasys and erect Redudasyidae fam. nov. to reflect this novel relationship between Anandrodasys and Redudasys. From an ecological perspective, the derived position of Redudasys, which is deeply nested within the Macrodasyida clade, unequivocally demonstrates that invasion of freshwater by gastrotrichs has taken place at least twice, in contrast with the single event hypothesis recently put forward.     

Spermatology of the genus Lepidodasys Remane, 1926 (Gastrotricha, Macrodasyida): towards a revision of the family Lepidodasyidae Remane, 1927

The spermatozoa of Lepidodasys unicarenatus and Lepidodasys sp. are filiform and composed of a cork-screw shaped acrosome, a helical nucleus surrounding a mitochondrial axis, and a 9 × 2 + 2 flagellum as in the basic structural model of the macrodasyidan sperm. The genus Lepidodasys has a debated phylogenetic position and has been linked in turn to the family Lepidodasyidae and the family Thaumastodermatidae. The sperm features of the two Lepidodasys species examined are distinct from those typical of the two families: the absence of the periaxonemal cylinder, a character shared only with Turbanellidae among Macrodasyida, could be considered as a symplesiomorphy, suggesting a basal position of the genus along the Macrodasyida clade. Moreover, a comparison of the spermatogenic process of Lepidodasys sp. with those of Acanthodasys aculeatus (Thaumastodermatidae) and Cephalodasys maximus (Lepidodasyidae) has revealed that the process of acrosome formation and nuclear morphology during spermatogenesis are peculiar in Lepidodasys sp. and differences are evident especially in the late stages of spermatogenesis. Penetrated spermatozoa were observed in the oocytes at all maturation stages in L. unicarenatus.     

A phylogeny of Lophocoleaceae-Plagiochilaceae-Brevianthaceae and a revised classification of Plagiochilaceae

The Lophocoleaceae-Plagiochilaceae-Brevianthaceae clade is a largely terrestrial, subcosmopolitan lineage of jungermannialean leafy liverworts that may include significantly more than 1000 species. Here we present the most comprehensively sampled phylogeny available to date based on the nuclear ribosomal internal transcribed spacer region and the chloroplast markers rbcL and rps4 of 372 accessions. Brevianthaceae (consisting of Brevianthus and Tetracymbaliella) form a sister relationship with Lophocoleaceae; this lineage is in turn sister to Plagiochilaceae. Plagiochila is resolved monophyletic subsequent to exclusion of Plagiochila radiculosa; this species is placed in a new genus Cryptoplagiochila. Chiastocaulon and a polyphyletic Acrochila nest in Plagiochilion; these three genera are united under Chiastocaulon to include the Plagiochilaceae species with dominating or exclusively ventral branching. The generic classification of the Lophocoleaceae is still unresolved. We discuss alternative approaches to obtain strictly monophyletic genera by visualizing their consistence with the obtained consensus topology. The presented phylogeny will serve as a basis for follow-up studies including several thousand accessions. These studies will enable revision of current hypotheses on species diversity and distribution of Lophocoleaceae-Plagiochilaceae-Brevianthaceae and allow for a reconstruction of their evolution in time and space.     

Molecular phylogeny and morphology of <Emphasis Type="Italic">Ophiocordyceps unituberculata</Emphasis> sp. nov. (Ophiocordycipitaceae), a pathogen of caterpillars (Noctuidae,Lepidoptera) from Yunnan,China

Ophiocordyceps unituberculata, a new insect pathogenic fungus from southwestern China, is described using molecular phylogenetic and morphological data. This fungus differs from other Ophiocordyceps species by its enormously long monophialidic conidiogenous cells and a large periclinal protuberance often growing near the apex of conidiogenous cells, single conidia (lanceolate to fusiform) embedded in mucous sheath, and much larger conidial dimensions. Four-locus (nrSSU, nrLSU, tef-1α and rpb1) and ITS data phylogenetic analyses show that O. unituberculata belongs to the Hirsutella nodulosa clade within the genus Ophiocordyceps of Ophiocordycipitaceae and is a separate clade from other allied species. Molecular phylogeny and morphology both strongly support the distinctiveness of this taxon. The interspecific relationships in the H. nodulosa clade are discussed.     

<Emphasis Type="Italic">Thyridariella</Emphasis>, a novel marine fungal genus from India: morphological characterization and phylogeny inferred from multigene DNA sequence analyses

A novel saprobic fungal genus, Thyridariella (Thyridariaceae), is herein described to include Thyridariella mangrovei, the type species and T. mahakoshae spp. nov. Both species were collected as saprobes on decaying wood of Avicennia marina, a common mangrove species found near Kaveri River Delta, Tamil Nadu, on the east coast of India. Thyridariella is diagnosed by having an exclusive combination of characters, such as ascomata with ostiolar necks thickened laterally, hyaline, and centrally constricted muriform ascospores with a single longitudinal septum in each segment and surrounded by a mucilaginous sheath. These characters demarcate these taxa from morphologically similar genera such as Halojulella and Julella. In addition, the new genus also differs from Parathyridaria and Thyridaria in having hyaline, muriform ascospores with distinct mucilaginous sheaths. The monophyly of Thyridariella is well supported in the phylogenetic analysis based on a concatenated dataset from two proteins and three nuclear gene regions. The phylogeny also depicts a sister group relationship of our new genus to Parathyridaria and Thyridaria and hence confirms its position within Thyridariaceae.     

ITS2 sequence–structure phylogeny reveals diverse endophytic <Emphasis Type="Italic">Pseudocercospora</Emphasis> fungi on poplars

For matching the new fungal nomenclature to abolish pleomorphic names for a fungus, a genus Pseudocercospora s. str. was suggested to host holomorphic Pseudocercosproa fungi. But the Pseudocercosproa fungi need extra phylogenetic loci to clarify their taxonomy and diversity for their existing and coming species. Internal transcribed spacer 2 (ITS2) secondary structures have been promising in charactering species phylogeny in plants, animals and fungi. In present study, a conserved model of ITS2 secondary structures was confirmed on fungi in Pseudocercospora s. str. genus using RNAshape program. The model has a typical eukaryotic four-helix ITS2 secondary structure. But a single U base occurred in conserved motif of U-U mismatch in Helix 2, and a UG emerged in UGGU motif in Helix 3 to Pseudocercospora fungi. The phylogeny analyses based on the ITS2 sequence–secondary structures with compensatory base change characterizations are able to delimit more species for Pseudocercospora s. str. than phylogenic inferences of traditional multi-loci alignments do. The model was employed to explore the diversity of endophytic Pseudocercospora fungi in poplar trees. The analysis results also showed that endophytic Pseudocercospora fungi were diverse in species and evolved a specific lineage in poplar trees. This work suggested that ITS2 sequence–structures could become as additionally significant loci for species phylogenetic and taxonomic studies on Pseudocerospora fungi, and that Pseudocercospora endophytes could be important roles to Pseudocercospora fungi’s evolution and function in ecology.     

Phylogeny of anaerobic fungi (phylum <Emphasis Type="Italic">Neocallimastigomycota</Emphasis>), with contributions from yak in China

The phylum Neocallimastigomycota contains eight genera (about 20 species) of strictly anaerobic fungi. The evolutionary relationships of these genera are uncertain due to insufficient sequence data to infer their phylogenies. Based on morphology and molecular phylogeny, thirteen isolates obtained from yak faeces and rumen digesta in China were assigned to Neocallimastix frontalis (nine isolates), Orpinomyces joyonii (two isolates) and Caecomyces sp. (two isolates), respectively. The phylogenetic relationships of the eight genera were evaluated using complete ITS and partial LSU sequences, compared to the ITS1 region which has been widely used in this phylum in the past. Five monophyletic lineages corresponding to six of the eight genera were statistically supported. Isolates of Caecomyces and Cyllamyces were present in a single lineage and could not be separated properly. Members of Neocallimastigomycota with uniflagellate zoospores represented by Piromyces were polyphyletic. The Piromyces-like genus Oontomyces was consistently closely related to the traditional Anaeromyces, and separated the latter genus into two clades. The phylogenetic position of the Piromyces-like genus Buwchfawromyces remained unresolved. Orpinomyces and Neocallimastix, sharing polyflagellate zoospores, were supported as sister genera in the LSU phylogeny. Apparently ITS, specifically ITS1 alone, is not a good marker to resolve the generic affinities of the studied fungi. The LSU sequences are easier to align and appear to work well to resolve generic relationships. This study provides a comparative phylogenetic revision of Neocallimastigomycota isolates known from culture and sequence data.     

Revision of the taxonomic status of the genus <Emphasis Type="Italic">Gloeoporus</Emphasis> (Polyporales,Basidiomycota) reveals two new species

Gloeoporus Mont. is characterized by an easily separated gelatinous hymenophore and a continuous hymenium over the pore mouth. Recent molecular taxonomic and phylogenetic research showed that morphological grouping of Gloeoporus is polyphyletic. The lack of comprehensive phylogenetic studies of Gloeoporus exacerbates confusion in determining the taxonomic position of the genus. To delimit the genus Gloeoporus, we performed multi-locus phylogenetic analysis using the internal transcribed spacer (ITS) region, the nuclear large subunit ribosomal DNA (LSU), and the second-largest subunit of RNA polymerase II (rpb2). The phylogenetic analyses revealed that current delimitation of Gloeoporus is not monophyletic. Gloeoporus s.s. includes mostly clamped species lacking cystidia. Some species of Gloeoporus featuring simple septa and cystidia are proposed to be renamed to Meruliopsis. Two new species of Gloeoporus were also observed and they are named Gloeoporus africanus and Gloeoporus orientalis.     

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.     

The Lactate Dehydrogenase Gene <Emphasis Type="Italic">LDH-C1</Emphasis>, a New Molecular Marker for Phylogenetic Analysis of Salmonid Fishes (Salmoniformes: Salmonidae)

We tested the locus of the nuclear lactate dehydrogenase gene (LDH-C1) as a phylogenetic marker in specimens of 11 salmonid genera (Thymallus, Coregonus, Hucho, Brachymystax, Salmo, Salmothymus, Acantholingua, Parahucho, Salvelinus, Parasalmo, and Oncorhynchus). All the sequences were veraciously clustered according to their taxonomic affiliation at the species and genus levels. It is shown that used complex of characters contains a phylogenetic signal that represents specific information about the phylogenesis process. This allows us to recommend the LDH-C1 locus to specify the phylogeny of salmonids in the combined analysis of several independent nuclear genes and mitochondrial DNA.     

Multi-gene phylogenetic analyses reveals <Emphasis Type="Italic">Neohelicosporium</Emphasis> gen. nov. and five new species of helicosporous hyphomycetes from aquatic habitats

Helicosporous hyphomycetes are a morphologically allied group of Tubeufiales. We introduce a new helicosporous genus, Neohelicosporium, with five new species, Neohelicosporium aquaticum, N. guangxiense, N. hyalosporum, N. parvisporum, and N. thailandicum, based on morphological and phylogenetic evidence. The RPB2 protein gene data are provided to analyze their phylogeny in Tubeufiales. Phylogenetic analyses of combined ITS, LSU, RPB2, and TEF1α sequence data from 13 new isolates of Neohelicosporium provided evidence to support the establishment of the new taxa. The morphological characters of Neohelicosporium that differentiate it from other helicosporous species are compared and discussed.     

Macrodasyida (Gastrotricha): a cladistic analysis of morphology

Abstract. A cladistic analysis based on 33 morphological characters was performed for the 31 genera currently assigned to the order Macrodasyida (Gastrotricha). Outgroup analysis indicated that the order is monophyletic and that it is defined by the structure of the pharynx and the complex distribution of duo-gland adhesive organs. Of the 6 currently recognized families in Macrodasyida, our analysis confirmed that 4 families are monophyletic: Dactylopodolidae, Macrodasyidae, Thaumastodermatidae and Turbanellidae. Dactylopodolidae was further confirmed as the most basal family within the order based on the retention of several plesiomorphies. The other three families have well-defined autapomorphies but will require further investigation to increase inter- and intrafamilial phylogenetic resolution. Planodasyidae appeared to be a paraphyletic taxon with no obvious autapomorphies; genera clustered among members of a polyphyletic family, Lepidodasyidae. We recommend that future research on macrodasyidan phylogeny focus on issues of comparative morphology and ultrastructure in lesser-known taxa such as the Dactylopodolidae, and on the taxa Lepidodasyidae and Planodasyidae.     

Morphological variation in pollen grains of <Emphasis Type="Italic">Mucuna</Emphasis> (Leguminosae): new biogeographic and evolutionary patterns

Mucuna comprises 105 species with an overall pantropical distribution and is divided into three subgenera: M. subg. Mucuna, M. subg. Stizolobium and M. subg. Macrocarpa. Although phylogenetic studies have supported the occurrence of three main clades, evolutionary relationships among them are not fully resolved. The objective of this study was to examine pollen grain morphology from representatives of all three subgenera and map these onto the phylogenetic trees generated by analysis of other characters. Pollen grain surface, form, size, and aperture number were compared. A Bayesian inference tree using matK sequences was constructed. The results indicate that the representatives of M. subg. Macrocarpa have the smallest pollen grains in the genus (a synapomorphic character here identified for this subgenus) and that species of subgenus Mucuna (those with umbelliform inflorescences) have the largest pollen grains. Additional morphological diversity of the pollen grain surface was noted: reticulate and/or micro-reticulate (in all three subgenera), perforate, gemmate or verrucose (only in M. subg. Mucuna). For all studied taxa, the pollen grains are triaperturate, except for two species of M. subg. Mucuna, which have tetraperturate pollen. The phylogenetic tree obtained using the matK marker resolved M. subg. Stizolobium as the earliest diverging lineage in Mucuna. Based on this phylogeny, a reticulate ornamentation pattern of the pollen surface may represent the ancestral state for the genus, while the larger pollen size and the foraminate, gemmate, and verrucose ornamentations are derived characteristics within the genus. These putative derived ornamentations have been observed only in neotropical species.