Tuberculina-rusts: a unique basidiomycetous interfungal cellular interaction with horizontal nuclear transfer

Cellular interaction of the basidiomycete Tuberculina persicina with the haploid stages of two rusts Puccinia silvatica and Tranzschelia pruni-spinosae was analyzed by serial-section electron microscopy of chemically fixed samples and samples subjected to high-pressure freezing and freeze substitution. Tuberculina persicina is a contact parasite, forming neither haustoria nor other intracellular structures. However, at contact areas between T. persicina and its hosts, distinct interfungal interactions are present. At the beginning, a hyphal cell of T. persicina invades the host cell wall with a protuberance and the cell walls of both protuberance and host cell dissolve at the point of contact. Thus, the plasma membranes of the two organisms contact and fuse to form a pore that enlarges to a final diameter of approximately 1 μm. The membrane of the fusion pore is continuous with the plasma membranes of both cells, and Tuberculina nuclei and other organelles are transferred to the rust cells. Phylogenetic and functional aspects of this curious basidiomycetous interfungal interaction are discussed, and a hypothesis of the evolutionary derivation of the Tuberculina mycoparasitism from a sexual interaction is presented.     

Tuberculina-Helicobasidium: Host specificity of the Tuberculina-stage reveals unexpected diversity within the group

Tuberculina species are mitosporic parasites of rust fungi. It was demonstrated recently that Tuberculina represents the asexual life stage of the plant-parasitic genus Helicobasidium. Here we reveal the host specificities of Tuberculina and Helicobasidium species on rust fungal hosts by means of infection experiments and molecular analyses. We inoculated species of the rust genera Chrysomyxa, Coleosporium, Cronartium, Gymnosporangium, Puccinia, Tranzschelia and Uromyces with conidia and with basidiospores of Helicobasidium longisporum and H. purpureum and with conidia of Tuberculina maxima, T. persicina and T. sbrozzii. In addition we analyzed base sequences from the nuclear ITS region of 51 Tuberculina and Helicobasidium specimens collected in the field together with the sequences from the Tuberculina infections obtained by infection experiments. The resulting data show that at least six monophyletic lineages are within the Tuberculina/Helicobasidium-group that can be unambiguously distinguished by combining molecular and morphological characters and by specific host spectra of the Tuberculina-stage. This diversity opens up new vistas on the evolution of this exceptional mycoparasitic-phytoparasitic fungal group.     

基于28S rDNA D2基因片段与形态特征的优茧蜂亚科系统发育研究

本研究选取优茧蜂亚科Euphorinae(膜翅目Hymenoptera:茧蜂科Braconidae)的8族19属23种作为内群,茧蜂其它6个亚科的8属8种作外群,首次结合同源核糖体28S rDNA D2基因序列片段和41个形态学特征对该亚科进行了系统发育学研究。利用"圆口类"的内茧蜂亚科Rogadinae、茧蜂亚科Braconinae、矛茧蜂亚科Doryctinae的3个亚科为根,以PAUP*4.0和MrBayes3.0B4软件分别应用最大简约法(MP)和贝叶斯法对优茧蜂亚科的分子数据和分子数据与非分子数据的结合体进行了分析;并以PAUP*4.0对优茧蜂亚科的28S rDNA D2基因序列的片段的碱基组成与碱基替代情况进行了分析。结果表明:优茧蜂亚科的28S rDNA D2基因序列片段的GC%含量在40.00%~49.25%之间变动,而对于碱基替代情况来讲,优茧蜂亚科各个成员间序列变异位点上颠换(transversion)大于转换(transition);不同的分析和算法所产生的系统发育树都表明目前根据形态定义出的优茧蜂亚科Euphorinae不是一个单系群,而是一个与蚁茧蜂亚科Neoneurinae和高腹茧蜂亚科Cenocoelinae混杂在一起的并系群;在优茧蜂亚科内部,悬茧蜂族Meterorini和食甲茧蜂族Microctonini(排除猎户茧蜂属Orionis)为单系群,而宽鞘茧蜂族Centistini、大颚茧蜂族Cosmophorini、优茧蜂族Euphorini、瓢虫茧蜂族Dinocampini为并系群;悬茧蜂族Meterorini在优茧蜂亚科Euphorinae内位于基部位置的观点得到部分的支持,同时食甲茧蜂族Microctonini被判定为相对进化的类群。此外对于优茧蜂亚科内各属之间的相互亲缘关系,不同算法所得到的系统发育属的结果不完全一致,这表明优茧蜂亚科内(属及族)的系统发育关系还有待于进一步研究。     

Phylogenetic positions of rust fungi parasitic on ferns: Evidence from 18S rDNA sequence analysis

Molecular phylogenetic analyses of fern rusts were carried out based on 18S rDNA sequences. We sequenced the 18S rDNAs of fern rusts (Hyalopsora polypodii andUredinopsis intermedia) and non-fern rusts (Aecidium epimedii, Coleosporium asterum, Ochropsora kraunhiae, Puccinia suzutake andPhysopella ampelopsidis) and analyzed their phylogenetic relationships with other members of the Urediniomycetes. Our bootstrapped neighbor-joining tree obtained from these analyses showed that rust fungi were apparently monophyletic at high confidence level (100% bootstrap confidence). In this molecular phylogenetic tree, the two fern rusts did not occupy the basal position within the rust fungal lineage and did not form a monophyletic lineage. Two species of the Cronartiaceae (Peridermium harknessii, Cronartium ribicola) and one species of the Coleosporiaceae (Coleosporium asterum) grouped with the fern rusts. Therefore, our results suggested that the two fern rusts were not primitive. On the other hand,Mixia osmundae, which is parasitic on the primitive fernOsmunda, was phylogenetically far from the fern rusts.     

我国新记录海链藻属物种的形态学和系统学研究

为了廓清我国海链藻属的物种多样性, 并丰富其分子生物学信息, 为后续的系统学研究提供基础数据, 从我国沿海分离并建立了海链藻的单克隆培养株系, 利用光学显微镜和电镜技术进行了形态学研究, 同时还对其核糖体大亚基的高变区序列进行了扩增和测序分析。结合形态学和分子生物学数据, 鉴定了我国海链藻属的2个新记录种: 狭线形海链藻Thalassiosira anguste-lineata (Schmidt) Fryxell & Hasle和碟形海链藻T. minicosmica Lee & Park。对它们的形态学特征进行了较为详尽的描述, 并与相似种进行了比较研究。此外, 还基于核糖体大亚基高变区的碱基序列信息, 分析了它们的分子系统学位置。在分子系统树上, 狭线形海链藻和碟形海链藻均不与该属模式种——诺氏海链藻T. nordenskioeldii 聚在同一分支上, 显示它们与典型海链藻属物种之间具有较大的遗传差异。分子系统树还显示, 目前基于形态学建立的海链藻属并非自然类群, 而是被骨条藻属Skeletonema、小环藻属Cyclotella、漂流藻属Planktoniella等邻近属种分隔成多个分支, 这预示着现存的海链藻属应该是一个并系类群, 在后续的系统学研究中, 或许会有较大的系统学调整, 但目前有限的分子生物学信息是相关研究推进的重要限制。     

Ribosomal DNA-ITS sequence polymorphism in the sugarcane rust,Puccinia kuehnii

The two highly divergent ITS types previously observed in isolates ofPuccinia kuehnii were amplified from the same isolates through the use of ITS type-specific primers for PCR and are therefore considered as polymorphisms of ITS regions within the species. Although homology of sequences of one of the ITS types with otherPuccinia species was of expected levels, significantly high homology of sequences of the other type with those ofCronartium members indicates that abnormal genetic events led to the occurrence of these polymorphic regions. These results indicate that ITS gene tree phylogeny may not reflect true species phylogeny in this group of rust fungi. Rather, D1/D2 region tree phylogeny, which was concordant with the differences in morphology with and among related rusts, more correctly reflects phylogenetic relationships of sugarcane and related grass rusts. contribution No. 159, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Re-evaluation of the phylogenetic relationship among species of the genus Tricholoma

The internal transcribed spacer sequences spanning the regions between the 17S and 25S rRNAs (ITS1 and ITS2) and including the complete sequence of the 5.8S rRNA were used for phylogenetic analyses. This approach to define phylogenetic relationships within the genus Tricholoma was tested using different isolates of T. terreum. Fruitbodies identified in nature were analysed in order to allow use of morphology for taxonomy. The isolates from different locations were closely related as could be expected for one species. Thus, the method could be applied to different Tricholoma species. Three clusters within the genus Tricholoma can be distinguished with four additional species not included in any of these clusters. Molecular analyses of two Cortinarius species confirm a phylogenetically distinct genus.     

Shifts from specialised to generalised pollination systems in Miconieae (Melastomataceae) and their relation with anther morphology and seed number

Most species in Melastomataceae have poricidal anthers related to specialised bee buzz‐pollination, while some have anthers with large openings associated to non‐bee pollination systems. We tracked the evolution of anther morphology and seed number on the Miconieae phylogenetic tree to understand the evolutionary shifts in such pollination systems. Anther morphometric data and seed number were recorded for 54 taxa. Pollinators (bees, flies, wasps) were recorded for 20 available species. Ancestral state reconstruction was made using Maximum Likelihood from nrITS sequences. We used phylogenetic eigenvector regressions to estimate phylogenetic signal and the adaptive component for these traits. Species pollinated by bees or bees and wasps tend to have smaller pores and fruits with more seeds. Species pollinated by flies or flies and bees and/or wasps tend to have larger pores and fruits with less seeds. Independent evolution occurred three times for anthers with large pores and twice for fruits with few seeds. We detected a phylogenetic signal in both traits, and negative correlated evolution between them. In actinomorphic small‐flowered Miconieae, changes in anther morphology can be related to generalisation in the pollination system incorporating flies and wasps as pollinators and lessening the importance of buzzing bees in such process. Differences in pollen removal and deposition may explain differences in anther morphology and seed number in Miconieae.     

基于28S rDNA D2基因片段与形态特征的矛茧蜂亚科系统发育研究（膜翅目：茧蜂科）

本研究选取矛茧蜂亚科Doryctinae（昆虫纲Insecta：膜翅目Hymenoptera：茧蜂科Braconidae）的6族15属18种做内群,茧蜂科其它7亚科11属11种做外群,首次结合同源核糖体28S rDNA D2基因序列片段和100个形态学和解剖学特征对该亚科进行了系统发育学研究。利用“非圆口类"的小腹茧蜂亚科Microgastrinae为根,以PAUP*4.0和MrBayes 3.0B4软件分别应用最大简约法（MP）和贝叶斯法对矛茧蜂亚科的分子数据和分子数据与非分子数据的结合体进行了运算分析;并以PAUP*4.0对矛茧蜂亚科的28S rDNA D2基因序列片段的碱基组成与碱基替代情况进行了分析。结果表明：矛茧蜂亚科的28S rDNA D2基因序列片段的GC含量在39.33%～48.28%之间变动,而对于碱基替代情况来讲,矛茧蜂亚科各成员间序列变异位点上颠换（transversion）大于转换（transition）。不同的分析算法所产生的系统发育树都表明矛茧蜂亚科是一个界限分明的单系群;在矛茧蜂亚科内,除了吉丁茧蜂族Siragrini为单系群外,其他族（矛茧蜂族Doryctini和方头茧蜂族Hecabolini）都是并系群。对于矛茧蜂亚科内各属之间的相互亲缘关系,不同算法所得的系统发育树的拓扑结构不完全一致,表明矛茧蜂亚科内（属及族）的系统发育关系还有待于进一步研究。     

Morphological and phylogenetic analyses of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> and <Emphasis Type="Italic">U. vignae</Emphasis> on legumes in Japan

Uromyces appendiculatus, inclusive of three varieties, is distinguished from U. vignae primarily by the position of urediniospore germ pores and putative host specificity. However, opinions concerning these morphological and physiological features as taxonomic characters have varied greatly, and distinction of these species has often been confused. To clarify the taxonomy of these two species, morphological features of urediniospores and teliospores of 225 rust fungus specimens on species of Phaseolus, Vigna, Apios, Lablab, and Dunbaria were examined by light microscopy and scanning electron microscopy. Forty-five specimens were subjected to molecular phylogenetic analyses. As a result, the position of germ pores in urediniospores and the teliospore-wall thickness were considered as good characters to separate three morphological groups. In molecular analyses, the specimens fell into two and three clades based on the nucleotide sequence at D1/D2 domain of LSU rDNA and ITS regions, respectively. One of the D1/D2 clades corresponded to one morphological group whereas another D1/D2 clade included two other morphological groups. In contrast, each of the three ITS clades corresponded to a separate morphological group. Neither morphological groups nor molecular clades were host limited. It is suggested that the three morphological groups that corresponded to three distinct ITS clades constitute distinct species.Contribution no. 186 from the Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Skeletal morphogenesis in some living collosphaerid Radiolaria

Two major patterns of shell morphogenesis occurring in varying proportions among species were identified by examining skeletons from five species of living collosphaerid, colonial Radiolaria: Collosphaera huxleyi, Acrosphaera cyrtodon, Acrosphaera spinosa, Siphonosphaera tubulosa and Siphonosphaera socialis. Skeletons possessing large open-lattice structures characterized by ellipsoidal to polygonal pores separated by narrow bars are produced by repeated subdivision of large pores into smaller pores. A bridge-like bar grows across the pore and subdivides it into two or more smaller pores. In skeletons with small, nearly circular pores and occasionally elongated tubular rims, the pores appear to be developed by rim thickening. Silica is deposited at the perimeter of the pore, thus decreasing its diameter and increasing the bar width between pores. In some species exhibiting intermediate types of skeletal morphology, there appears to be varying amounts of both bridge growth and rim growth. These data are used to explore possible phylogenetic pathways for a variety of collosphaerid species and to elucidate the processes of skeletal deposition in Radiolaria.     

The only known white blister rust on a basal angiosperm is a member of the genus <Emphasis Type="Italic">Albugo</Emphasis>

Rare pathogens on unusual hosts are often providing valuable insight into the evolution of the pathogen group concerned, but it is often challenging to obtain sequence data for these, as because only very few, often decades-old specimens are available. One such example is Albugo tropica, the white blister pathogen of a basal angiosperm in the genus Peperomia (Piperaceae). For this species, only two, more than 70 and over 120-year-old collections available. Here, sequence data for A. tropica are reported and phylogenetic reconstructions reveal it as the sister group to all other white blister rusts of the genus Albugo. Its isolated position is also reflected by several morphological differences to the other species of the genus, such as very thin-walled sporangia and almost smooth oospores. The isolated phylogenetic position of the pathogen and its host might indicate that it is a relict species trapped on its host. The sister-group relationship to all members of the genus Albugo s.str., which have been investigated using molecular phylogenetics, hints at the possibility, that Albugo might have originated in South America or Gondwana and has later radiated in the holarctic on members of the Brassicales.     

杜氏盐藻psaB基因cDNA克隆及系统进化分析

根据真核生物莱茵衣藻(Chlamydomonas reinhardtii)、Chlamydomonas moewusii及Chlorella vulgaris等光系统Ⅰ反应中心蛋白psaB基因的氨基酸高度保守序列,设计一对简并引物,利用TRIzol试剂提取杜氏盐藻(Dunaliella salina)细胞的总RNA,通过RT-PCR,得到的一段长为1.8kb左右的cDNA片段。PCR产物经T-A克隆并测序以及测序结果推导成氨基酸序列进行同源性比较,表明所克隆的1815bp序列为杜氏盐藻光系统Ⅰ反应中心psaB基因的cDNA片段,GenBank收录号为AY820754。根据已经得到的psaB的核苷酸序列推导成氨基酸序列与一些已知物种的psaB氨基酸序列相比较,同源性分别为Chlamydomonas reinhardtii 92%,Chlamydomonas moewusii 91%,Chlorella vulgaris 86%,Mesostigma viride 85%,Phy -scomitrella patenssubsp.Patens 85%,Nephroselmis olivacea 84%。此外,psaB密码子偏爱性分析表明:杜氏盐藻psaB基因第三位密码子A和T的组成分别为35.7%和39.17%,而G和C分别为7.27%和17.85%,即杜氏盐藻psaB基因密码子的组成大多为NNA和NNT。根据psaB基因的特征,作者对绿藻门的50个物种的psaB基因作了进化分析,结果表明:杜氏盐藻与Haematococcaceae中的大多数种类进化地位最为接近,这为更进一步弄清杜氏盐藻的遗传背景提供了理论依据。     

Molecular Phylogeny and Ultrastructure of Caliculium glossobalani n. gen. et sp. (Apicomplexa) from a Pacific Glossobalanus minutus (Hemichordata) Confounds the Relationships Between Marine and Terrestrial Gregarines

Gregarines are a diverse group of apicomplexan parasites with a conspicuous extracellular feeding stage, called a “trophozoite”, that infects the intestines and other body cavities of invertebrate hosts. Although the morphology of trophozoites is very diverse in gregarines as a whole, high degrees of intraspecific variation combined with relatively low degrees of interspecific variation make the delimitation of different species based on trophozoite morphology observed with light microscopy difficult. The coupling of molecular phylogenetic data with comparative morphology has shed considerable light onto the boundaries and interrelationships of different gregarine species. In this study, we isolated a novel marine gregarine from the hepatic region of a Pacific representative of the hemichordate Glossobalanus minutus, and report the first ultrastructural and molecular data from any gregarine infecting this distinctive group of hosts. Molecular phylogenetic analyses of an SSU rDNA sequence derived from two single‐cell isolates of this marine gregarine demonstrated a strong and unexpected affiliation with a clade of terrestrial gregarines (e.g. Gregarina). This molecular phylogenetic data combined with a comparison of the morphological features in previous reports of gregarines collected from Atlantic representatives of G. minutus justified the establishment of a new binomial for the new isolate, namely Caliculium glossobalani n. gen. et sp. The molecular phylogenetic analyses demonstrated a clade of terrestrial gregarines associated with a sequence acquired from a marine species, which suggest that different groups of terrestrial/freshwater gregarines evolved independently from marine ancestors.     

Evolution of overwintering strategies in Eurasian species of the Drosophila obscura species group

The phylogenetic relationship of Eurasian species of the Drosophila obscura species group remains ambiguous in spite of intensive analyses based on morphology, allozymes and DNA sequences. The present analysis based on sequence data for cytochrome oxidase subunit I (COI) and a-glycerophosphate dehydrogenase (Gpdh) suggests that the phylogenetic position of D. alpina is also ambiguous. These ambiguities have been considered to be attributable to rapid phyletic radiation in this group at an early stage of its evolution. Overwintering strategies are diversified among these species: D. alpina and D. subsihestris pass the winter in pupal diapause, D. bifasciata and D. obscura in reproductive diapause, and D. subobscura and D. guanche without entering diapause. This diversity may also suggest rapid radiation at an early phase of adaptations to temperate climates. On the other hand, adult tolerance of cold was closely related to overwintering strategy and distribution: D. obscura and D. bifasciata with reproductive diapause were very tolerant; D. alpina and D. subsilvestris which pass the winter in pupal diapause were less tolerant; D. subobscura having no diapause was moderately tolerant and D. guanche occurring in the Canary Islands was rather susceptible. Tolerance of high temperature at the preimaginal stages seemed to be also associated with overwintering strategy; i.e. lower in the species with pupal diapause than in those with reproductive diapause or without diapause mechanism.     

Dinoflagellate,Euglenid, or Cercomonad? The Ultrastructure and Molecular Phylogenetic Position of Protaspis grandis n. sp.

Protaspis is an enigmatic genus of marine phagotrophic biflagellates that have been tentatively classified with several different groups of eukaryotes, including dinoflagellates, euglenids, and cercomonads. This uncertainty led us to investigate the phylogenetic position of Protaspis grandis n. sp. with ultrastructural and small subunit (SSU) rDNA sequence data. Our results demonstrated that the cells were dorsoventrally flattened, shaped like elongated ovals with parallel lateral sides, 32.5-55.0 mum long and 20.0-35.0 mum wide. Moreover, two heterodynamic flagella emerged through funnels that were positioned subapically, each within a depression and separated by a distinctive protrusion. A complex multilayered wall surrounded the cell. Like dinoflagellates and euglenids, the nucleus contained permanently condensed chromosomes and a large nucleolus throughout the cell cycle. Pseudopodia containing numerous mitochondria with tubular cristae emerged from a ventral furrow through a longitudinal slit that was positioned posterior to the protrusion and flagellar apparatus. Batteries of extrusomes were present within the cytoplasm and had ejection sites through pores in the cell wall. The SSU rDNA phylogeny demonstrated a very close relationship between the benthic P. grandis n. sp. and the planktonic Cryothecomonas longipes. These ultrastructural and molecular phylogenetic data for Protaspis indicated that the current taxonomy of Protaspis and Crythecomonas is in need of re-evaluation. The composition and identity of Protaspis is reviewed and suggestions for future taxonomic changes are presented. Problems within the genus Cryothecomonas are highlighted as well, and the missing data needed to resolve ambiguities between the two genera are clarified.     

Gene and species trees reveal mitochondrial and nuclear discordance in the Drosophila cardini group (Diptera: Drosophilidae)

Abstract. The Drosophila cardini group includes 15 species, which are subdivided into the D. cardini and D. dunni subgroups. Although many phylogenetic hypotheses have been proposed for this group during the last five decades (based on patterns of reproductive isolation, morphology of male genitalia, chromosomal inversions, isozyme variation, or molecular sequence data), these are mostly discordant with each other. We aimed to clarify some of the evolutionary patterns related to the origin of this incongruence, while also attempting to provide a better-supported phylogenetic hypothesis for the D. cardini subgroup. For this purpose, sequences from three mitochondrial and three nuclear loci were gathered for at least eight species, and both individual gene trees and joint species tree estimates were evaluated. Although there was concordance among gene trees within each of the nuclear and mitochondrial sets, considerable incongruence was revealed in the comparisons between these two data sets. The branching position of D. neocardini was the main source of incongruence, and species trees reconstructed using different approaches with and without this species were particularly incongruent. In addition to providing a better approximation of the evolutionary history of the D. cardini group, this study suggests that incomplete lineage sorting or introgression may be biasing previous species tree estimates. More generally, the results also suggest that the use of supermatrix methods can lead to an overestimation of support for the inferred relationships, and highlight the potential effects of different taxon sampling strategies in phylogenetic reconstruction.