Biochemical genetics and taxonomy in Plumatella emarginata and P. repens (Bryozoa: Phylactolaemata)

SUMMARY. Although morphological distinctions are well documented, many authors have regarded the freshwater bryozoan Plumatella emarginata as a variety of P. repens. Differences between the two nominate species have been studied by the electrophoretic examination of genetic variation at enzyme loci. Genetic identity (Nei, 1972) was 0.286, a value well below that expected for conspecific allopatric populations. The results leave little doubt that specimens of the two species are from quite distinct and separate gene pools, with an unexpectedly high level of genetic differentiation. It is therefore concluded that P. emarginata is a valid species and is not merely a variety of P. repens. Morphological differences between the two species are also discussed.     

16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences analysis of the genus Myxococcus.

Phylogenetic relationships of the species belonging to the genus Myxococcus were elucidated based on the sequences of 16S rRNA genes and 16S-23S rRNA gene internal transcribed spacer (ITS) regions. The Myxococcus species were consequently classified into four distinct groups. The type strain of Myxococcus coralloides occupied an independent position (Group 1); it has been recently reclassified as Corallococcus coralloides. Group 2 comprised the type strains of both Myxococcus virescens and Myxococcus xanthus, and some strains assigned to Myxococcus flavescens. The type strain of M. flavescens was contained in Group 3 along with the strains of Myxococcus fulvus. Group 4 included the strains belonging to C. coralloides, M. fulvus, and M. stipitatus. The type strain of M. fulvus that was allocated outside Group 4 in the 16S rRNA gene tree belonged to Group 3 in the ITS tree. These results strongly suggest that the morphological characteristics of Myxococcus species are not consistent with the phylogenetic relationships. The Myxococcus species must therefore be redefined according to the phylogenetic relationships revealed in this study.     

Biochemical genetics and taxonomy in Plumatella fungosa and P. repens (Bryozoa: Phylactolaemata)

SUMMARY. Some authors have recently concluded that, using morphological criteria, the freshwater bryozoans Plumatella repens and P. fugosa cannot be separated and should be regarded as conspecific. To test this conclusion, electrophoretic techniques have been used to examine genetic differences between the two nominate species at several enzyme loci. Significant variation at a malate dehydrogenase locus and three aminopeptidase loci establish beyond doubt that P. repens and P. fugosa are separate but related species. No significant variation was found between two populations of P. repens . Morphological characters for the distinction of the two species are discussed and evaluated.     

Molecular evidence for the hybrid origin of species in the soft coral genus Alcyonium (Cnidaria: Anthozoa: Octocorallia)

Several recent studies have suggested that hybridization may play a previously unrecognized and important role in the evolution of corals. Our observations of polymorphic and recombinant sequences in the multicopy ribosomal internal transcribed spacer (ITS) region suggested the possible hybrid origin of two European soft coral species, Alcyonium hibernicum and Bellonella bocagei. To examine this possibility further we cloned and sequenced ITS-1 from multiple individuals and populations of these two species as well as two sympatric congeners, A. coralloides and A. sp. M2. Phylogenetic analyses separated the observed sequence variants into two distinct clades. All A. coralloides sequences belonged to clade A, while A. sp. M2 had only clade B sequences. A majority of A. hibernicum individuals, however, contained both clade A and B sequences that were identical to the predominant sequence variants found in A. coralloides and A. sp. M2, respectively. This pattern of additivity suggests that A. hibernicum originated from a hybrid cross between A. coralloides and A. sp. M2, a hypothesis that is supported by its unusual mode of reproduction (meiotic parthenogenesis). The predominant sequence variant found in B. bocagei was a unique, derived clade B sequence; in addition, however, most individuals of this species also had copies of a sequence identified as a recombinant between clade A and clade B sequence types. The presence of this recombinant sequence in the B. bocagei genome suggests that this species may also be the product of past hybridization events within the clade. Reticulate evolution may explain the failure of several previous studies to resolve the phylogeny of these four species.     

Cyanotypic frequencies in adjacent and mixed populations of Trifolium occidentale Coombe and Trifolium repens L. are regulated by different mechanisms

The cyanogenic polymorphism in Trifolium repens is caused by the variation in two genes, the interaction of which produces four distinct cyanotypes. Along the Atlantic coasts of Bretagne, T. repens is sometimes found in populations mixed with the related species Trifolium occidentale, although the latter species usually occurs only in a narrow fringe along the coast, whereas T. repens is a more inland species. No plants of T. occidentale have ever been reported to have linamarase activity. Indeed, of 763 T. occidentale plants studied, none contained linamarase activity. However, the variation in the proportion of cyanotypes in T. repens was enormous, even between sites less than 2km apart. Our results confirm the presumption that T. repens and T. occidentale are indeed separate species. Both the fact that T. occidentale plants never contain linamarase activity, and the difference in proportion of plants with cyanoglucosides in mixed stands show that gene flow between the species must be rare. These dissimilar distributions strongly indicate that cyanotypic frequencies in adjacent and mixed populations of the very closely related species T. occidentale and T. repens are regulated by different mechanisms     

Morphological and molecular analyses of the genera Peltidocotyle Diesing 1850 and Othinoscolex Woodland 1933, and a morphological study of Woodlandiella Freze, 1965 (Eucestoda,Proteocephalidea), parasites of South American siluriform fishes (Pimelodidae)

Morphological studies of type-species of the genera Peltidocotyle Diesing, 1850 and Othinoscolex Woodland, 1933 showed that these genera are synonyms. We therefore redescribe the type and fresh material of Peltidocotyle rugosa Diesing, 1850 and P. lenha (Woodland, 1933) [= Othinoscolex lenha Woodland, 1933], which appears to constitute two distinct species. They differ in the number and number of layers of testes, the diameter of the metascolex and the field occupied by the vitelline follicles. Phylogenetic analyses based on partial DNA sequences of 16S rDNA, 5.8S-ITS2 and 28S rDNA support these results. P. lenha and Woodlandiella myzofer (Woodland, 1933) are considered conspecific following a thorough morphological examination. With regard to host-parasite relationships, P. lenha has been reported from two host species, which is unusual as South American proteocephalideans generally display an oioxenous type of host-specificity.     

Gene sequence heterogeneity of Corallococcus coralloides strains isolated from geographically diverse locations

Thirty-three strains classified as Corallococcus coralloides isolated from mostly soil samples in 14 countries of four continents, were subjected to phylogenetic analyses. Based on 16S rDNA analyses the strains form a highly related cluster, sharing above 98.7% sequence similarity. Four groups were recognized within this cluster, only one of which, containing two strains from St. Lucia, Lower Antilles, was exclusively defined by strains from the same sample. The other groups contained members from different countries, even continents. The largest group embraced the type strains of C. coralloides DSM 2259(T) and Corallococcus exiguus 14696(T) which were almost indistinguishable in their 16S rRNA gene sequence. Corallococcus macrosporus DSM 14697(T) grouped outside the C. coralloides cluster, showing a higher relationship to a member of Myxococcus. The topology of the tree generated on the basis of the partial gyrase B (gyrB) gene sequence supports the rRNA gene tree, though some differences in the order of branching were observed. As judged by the binary similarity values the higher resolution power of gyrB sequences was confirmed. From a taxonomic standpoint, the size of myxospores is not a valuable taxonomic criterion, as small- and medium-sized myxospores are members of the same group. If the species status of C. coralloides and C. exiguus is verified by other methods (e.g. DNA-DNA hybridisation, RiboTyping), the genus Corallococcus may embrace a broad range of yet-to-be described novel species. The presence of strains within the same sample displaying higher relatedness to strains from other locations points towards an intensive dispersal of myxospores across continents.     

Infection of bryozoans by Tetracapsuloides bryosalmonae at sites endemic for salmonid proliferative kidney disease

Laboratory-reared colonies of the bryozoans Fredericella sultana and Plumatella fungosa were placed upstream of 2 fish farms endemic for salmonid proliferative kidney disease (PKD) to assess rates of infection of bryozoans by Tetracapsuloides bryosalmonae, the causative agent of PKD. Colonies were deployed in the field for 8 trial periods of 2 wk each throughout the summer of 2001. Following each trial, bryozoan colonies were maintained in laboratory culture for 28 d and were regularly monitored for infection by searching for sac stages of T. bryosalmonae. Infections were never identified by observations of sac stages, however positive PCR results and sequencing of cultured material confirmed that cryptic infections were present in colonies of both species deployed at one site. The possibility that PCR results reflected contamination of surfaces of bryozoans can be excluded, given the short period of spore viability of T. bryosalmonae. Highest rates of infection occurred when 4 of 23 colonies of F. sultana and 1 of 12 colonies of P. fungosa were infected during the period 10 to 24 July. No infections were detected from mid-August to late October at this site. None of the colonies at the other site became infected throughout the period of study. Our data provide the first estimates of infection rates of bryozoans by T. bryosalmonae. Additionally, they provide evidence that a cryptic stage can be maintained within bryozoan hosts for a period of 4 to 6 wk.     

Genome discrimination by in situ hybridization in Icelandic species of Elymus and Elytrigia (Poaceae: Triticeae).

The genome constitution of Icelandic Elymus caninus, E. alaskanus, and Elytrigia repens was examined by fluorescence in situ hybridization using genomic DNA and selected cloned sequences as probes. Genomic in situ hybridization (GISH) of Hordeum brachyantherum ssp. californicum (diploid, H genome) probe confirmed the presence of an H genome in the two tetraploid Elymus species and identified its presence in the hexaploid Elytrigia repens. The H chromosomes were painted uniformly except for some chromosomes of Elytrigia repens which showed extended unlabelled pericentromeric and subterminal regions. A mixture of genomic DNA from H. marinum ssp. marinum (diploid, Xa genome) and H. murinum ssp. leporinum (tetraploid, Xu genome) did not hybridize to chromosomes of the Elymus species or Elytrigia repens, confirming that these genomes were different from the H genome. The St genomic probe from Pseudoroegneria spicata (diploid) did not discriminate between the genomes of the Elymus species, whereas it produced dispersed and spotty hybridization signals most likely on the two St genomes of Elytrigia repens. Chromosomes of the two genera Elymus and Elytrigia showed different patterns of hybridization with clones pTa71 and pAes41, while clones pTa1 and pSc119.2 hybridized only to Elytrigia chromosomes. Based on FISH with these genomic and cloned probes, the two Elymus species are genomically similar, but they are evidently different from Elytrigia repens. Therefore the genomes of Icelandic Elymus caninus and E. alaskanus remain as StH, whereas the genomes of Elytrigia repens are proposed as XXH.     

Ancestors of white clover ( Trifolium repens L.), as revealed by isozyme polymorphisms

Isozymes were used to study the putative ancestors of white clover ( Trifolium repens L.). Ten enzymes were examined, and 18 loci were resolved via starch-gel electrophoresis for accessions representing T. repens, Trifolium isthmocarpum, Trifolium nigrescens, Trifolium occidentale and Trifolium uniflorum, in addition to two more distantly related species, Trifolium alpinum and Trifolium purseglovei. Nei's genetic identities indicate that T. uniflorum and T. nigrescens are the closest relatives of T. repens. The isozyme data thus support a hypothesis that the two genomes of the tetraploid T. repens could have been derived from hybridization between T. nigrescens and T. uniflorum. This conclusion is further supported by shared alleles between T. repens, T. nigrescens and T. uniflorum. However, the origin of T. repens is somewhat obscured by the presence of shared alleles between T. repens and both T. occidentale and T. isthmocarpum, suggesting that introgression of genes from the latter two species into T. repens may also have taken place. High values of genetic identity are shared between T. occidentale with T. nigrescens and T. uniflorum, also indicative of introgression. Alternatively the presence of shared alleles among the five species may reflect their recent common ancestry.     

Comparative study of mycelial growth and basidiomata formation in seven different species of the edible mushroom genus Hericium

The potential of using several agricultural by-products as supplements of sawdust substrate for the production of edible mushroom Hericium was evaluated using seven Hericium species. All the tested supplements (rice bran, wheat bran, barley bran, Chinese cabbage, egg shell, and soybean powder) were found to be suitable for the mycelial growth of all the tested species. In mycelial growth, soybean powder was the best supplement for Hericium americanum, Hericium coralloides, and Hericium erinaceum while barley bran was the best for Hericium alpestre, Hericium laciniatum, and Hericium erinaceus. For Hericium abietis, rice bran and Chinese cabbage was the best. The possibility of mushroom production on oak sawdust substrate with 20% rice bran supplement was demonstrated with H. coralloides, H. americanum, H. erinaceus, and H. erinaceum which showed 26-70% biological efficiency. Our results also showed that strain selection is important to improve biological efficiency and mushroom yield in Hericium cultivation.     

Pyrene and pollen observations in the pantropical genus Geophila (Rubiaceae-Psychotrieae)

Pyrene and pollen morphology of several neo- and paleotropical Geophila species is documented and discussed. Pyrenes show distinct morphological variation. They have one central or several adaxial ribs and are straight or twisted. Neotropical species only have twisted pyrenes, whereas Madagascan species have straight ones. Both pyrene types occur on the tropical African mainland and in tropical Asia. The genus is eurypalynous, including inaperturate, colpate and pantoporate pollen. The paper includes the first record of operculate pollen in the Rubiaceae, viz. in Geophila repens var. repens . Pyrenes as well as pollen morphology indicate that the varieties of the pantropical species G. repens need recognition at the specific level.     

Reticulate evolution, introgression, and intertribal gene capture in an allohexaploid grass

Recent molecular phylogenetic studies of polyploid plants have successfully clarified complex patterns of reticulate evolution. In this study of Elymus repens, an allohexaploid member of the wheat tribe Triticeae, chloroplast and nuclear DNA data reveal an extreme reticulate pattern, revealing at least five distinct gene lineages coexisting within the species, acquired through a possible combination of allohexaploidy and introgression from both within and beyond the Triticeae. Earlier cytogenetic studies of E. repens suggested that Hordeum (genome H) and Pseudoroegneria (St) were genome donors to E. repens. Chloroplast DNA data presented here (from the rpoA gene and from the region between trnT and trnF) identify three potential maternal genome donors (Pseudoroegneria, Thinopyrum, and Dasypyrum), and information from previous molecular work suggests that, of these, Pseudoroegneria is the most likely maternal donor. Nuclear starch synthase gene data indicate that both Hordeum and Pseudoroegneria have contributed to the nuclear genome of E. repens, in agreement with cytogenetic data. However, these data also show unexpected contributions from Taeniatherum, and from two additional donors of unknown identity. One of the sequences of unknown origin falls within the Triticeae, but is not closely associated with any of the sampled diploid genera. The second falls outside of the clade containing Triticeae and its outgroup Bromus, suggesting the acquisition of genetic material from a surprisingly divergent source. Bias toward the amplification of certain starch synthase variants has complicated attempts to thoroughly sample from within individuals, but the data clearly indicate a complex pattern of reticulate evolution, consistent not only with allohexaploidy, but also with introgression from unexpectedly divergent sources.     

Phylogenetic studies on the Thamnocalamus group and its allies (Gramineae: Bambusoideae) based on its sequence data.

Phylogenetic analyses, using the parsimony method and the neighbor-joining method, of 31 species of the Thamnocalamus group and its allies based on internal transcribed spacer (ITS) sequences in nuclear ribosomal DNA are presented. Two species from Arundinaria and Acidosasa were used as outgroups. The ITS phylogeny strongly supports the monophyly of the Thamnocalamus group and its allies, which have pachymorph rhizomes and semelauctant synflorescences with three stamens. Within this clade, Chimonocalamus pallens was resolved in the basal position. The Thamnocalamus group, including species placed in Thamnocalamus, Fargesia (Sinarundinaria, Borinda), and Yushania, may be polyphyletic/paraphyletic according to the ITS phylogeny, but internal support was relatively low. All three sampled species of Ampelocalamus were resolved as a monophyletic group and may be related to Drepanostachyum hookerianum. Two taxa of Thamnocalamus and the species Gaoligongshania megalothyrsa were resolved as monophyletic despite their different morphological characters, but again with a low bootstrap support. Within the Fargesia yunnanensis subclade, the sister relationship of Fargesia fungosa and Fargesia edulis was supported. Another subclade, the Fargesia communis clade, was also weakly supported as monophyletic. However, further resolution within the Thamnocalamus group has not been provided by this sequence data. The results indicate that reevaluation of relationships within this group is necessary.     

Nine more cases of human infections by Dirofilaria repens diagnosed in Italy by morphology and recombinant DNA technology

Dirofilariosis is a mosquito-borne disease of carnivors due, in Italy, to Dirofilaria repens and D. immitis species which are sympatric in many areas. Nine more cases of human infection are here reported, detected in Italian patients living in northern regions (where D. immitis and D. repens are largely present), in Tuscany (where D. repens is more frequent than D. immitis), and in Sicily (where D. immitis is present only sporadically). The nematodes surgically removed (7 female and 2 male specimens) have been identified by a PCR-based method and by morphology as D. repens. Morphological, clinical, and epidemiological data of these human infections are discussed.     

Description of Scalithrium n. gen. (Cestoda,Tetraphyllidea) with Scalithrium minimum (Van Beneden, 1850) n. comb., a parasite of Dasyatis pastinaca (Elasmobranchii,Dasyatidae), as type species

Scalithrium gen. n. (Cestoda, Tetraphyllidea) is proposed with Scalithrium minimum (Van Beneden, 1850) n. comb., parasite of Dasyatis pastinaca (Elasmobranchii, Dasyatidae) as type-species. The new genus Scalithrium (Tetraphyllidea, Phyllobothriidae, Rhinebothriinae) is erected for several species previously included in the genus Rhinebothrium. These species have a scolex with four bothridia, the distal surface of which is divided by transverse septa in a single row of loculi. Scalithrium minimum (Van Beneden, 1850) n. comb. is redescribed from specimens collected from the type-host Dasyatis pastinaca in Tunisia and becomes the type-species of the new genus. After Braun (1900) Echeneibothrium variabile Van Beneden, 1850 is considered as type-species of the genus Echeneibothrium. Species of Rhinebothriinae to be transferred into the genus Scalithrium are discussed and a key is proposed for the eight species.     

Mites are the most common vectors of the fungus Gondwanamyces proteae in Protea infructescences

Entomochoric spore dispersal is well-documented for most ophiostomatoid fungal genera, most of which are associated with bark or ambrosia beetles. Gondwanamyces spp. are unusual members of this group that were first discovered in the flower heads of the primitive angiosperm genus Protea, that is mostly restricted to the Cape Floristic region of Africa. In this study, we present the discovery of the vectors of Gondwanamyces proteae in Protea repens infructescences, which were identified using PCR, direct isolation, and light microscopy. Gondwanamyces proteae DNA and ascospores were identified on diverse lineages of arthropods including beetles (Euderes lineicolis and Genuchus hottentottus), bugs (Oxycarenus maculates), a psocopteran species and five mite (Acari) species. Based on isolation frequency, however, a mite species in the genus Trichouropoda appears to be the most common vector of G. proteae. Gondwanamyces spores were frequently observed within pit mycangia at the base of the legs of these mites. Manipulative experiments demonstrated the ability of mites to carry viable G. proteae spores whilst in transit on the beetle G. hottentottus and that these mites are able to transfer G. proteae spores to uncolonised substrates in vitro. Interestingly, this same mite species has also been implicated as vector of Ophiostoma spores on P. repens and belongs to the same genus of mites that vector Ophiostoma spp. associated with pine-infesting bark beetles in the Northern Hemisphere.     

Mycorrhizal acquisition of inorganic phosphorus by the green-leaved terrestrial orchid Goodyera repens

BACKGROUND AND AIMS: Mycorrhizal fungi play a vital role in providing a carbon subsidy to support the germination and establishment of orchids from tiny seeds, but their roles in adult orchids have not been adequately characterized. Recent evidence that carbon is supplied by Goodyera repens to its fungal partner in return for nitrogen has established the mutualistic nature of the symbiosis in this orchid. In this paper the role of the fungus in the capture and transfer of inorganic phosphorus (P) to the orchid is unequivocally demonstrated for the first time. METHODS: Mycorrhiza-mediated uptake of phosphorus in G. repens was investigated using spatially separated, two-dimensional agar-based microcosms. RESULTS: External mycelium growing from this green orchid is shown to be effective in assimilating and transporting the radiotracer (33)P orthophosphate into the plant. After 7 d of exposure, over 10 % of the P supplied was transported over a diffusion barrier by the fungus and to the plants, more than half of this to the shoots. CONCLUSIONS: Goodyera repens can obtain significant amounts of P from its mycorrhizal partner. These results provide further support for the view that mycorrhizal associations in some adult green orchids are mutualistic.     

Germination response to temperature,salinity, light and depth of sowing of ten tropical dune species

This study describes the germination responses of ten tropical dune species to several factors to which their seeds are exposed in the field. Species studied were: three sand dune endemics (Amaranthus greggii, Palafoxia lindenii, and Trachypogon gouini), three pantropical coastal species (Sesuvium portulacastrum, Sporobolus virginicus and Ipomoea stolonifera) and four cosmopolitan grasses also found inland (Panicum repens, Panicum maximum, Pappophorum vaginatum and Andropogon glomeratus). Six species germinated when exposed to different constant temperatures. Four required temperature fluctuation (S. portulacastrum, P. lindenii, S. virginicus, P. repens). Light promoted germination of three species (S. Portulacastrum, P. vaginatum, P. lindenii). Species varied in their degree of sensitivity to salinity. Seeds of T. gouini, I. stolonifera, S. portulacastrum, P. repens, P. Maximum and P. vaginatum were able to germinate under some of the salinity concentrations. Not all species were able to recover after being transferred to distilled water. Seedling emergence was inhibited when seeds were buried. This response was related to depth and to seed size. S. portulacastrum and S. virginicus were the most affected. Nitrates only affected germination response of two species. Seed age promoted germination under a wider range of conditions. P. lindenii showed very heterogeneous responses depending on seed cohort. No dormancy mechanisms were found, other than a thermoperiod and/or light requirement for some of the species. Cosmopolitan grasses tolerated both sand burial and salinity, although the endemics were the most successful in emerging from sand burial; coastal pantropicals were very tolerant to high saline concentrations and recovered completely.     

Phylogenetic relationships among Phialocephala species and other ascomycetes

Phialocephala was established for species in the Leptographium complex that produce conidia from phialides at the apices of dark mononematous conidiophores. Some species previously included in Phialocephala were re-allocated to Sporendocladia because they resembled Thielaviopsis in having ring-wall-building conidial development and conidia with two attachment points that emerge in false chains. Despite this significant realignment of the genus, a great deal of morphological heterogeneity remains in Phialocephala. The objective of this study was to consider the heterogeneity among Phialocephala spp. based on comparisons of sequence data derived from the large and small subunits (LSU and SSU) of the rRNA operon of species in Phialocephala. Phialocephala dimorphospora, the type species of the genus, and P. fortinii grouped with genera of the Helotiales in phylogenetic trees generated based on the LSU and SSU datasets. Phialocephala xalapensis and P. fusca clearly are unrelated to Phialocephala sensu stricto and should represent a new genus in the Ophiostomatales. Phialocephala compacta resides with representatives of the Hypocreales, and we believe that it represents a distinct genus. Phialocephala scopiformis and P. repens are not closely related to the other Phialocephala species and group within the Dothideales. The morphological heterogeneity among species of Phialocephala clearly is reflected by phylogenetic analysis of sequence data from two conserved rRNA gene regions. Appropriate genera now need to be found to accommodate these fungi.